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Sample records for a-kinase anchor protein

  1. A-kinase anchoring proteins as potential drug targets

    PubMed Central

    Tröger, Jessica; Moutty, Marie C; Skroblin, Philipp; Klussmann, Enno

    2012-01-01

    A-kinase anchoring proteins (AKAPs) crucially contribute to the spatial and temporal control of cellular signalling. They directly interact with a variety of protein binding partners and cellular constituents, thereby directing pools of signalling components to defined locales. In particular, AKAPs mediate compartmentalization of cAMP signalling. Alterations in AKAP expression and their interactions are associated with or cause diseases including chronic heart failure, various cancers and disorders of the immune system such as HIV. A number of cellular dysfunctions result from mutations of specific AKAPs. The link between malfunctions of single AKAP complexes and a disease makes AKAPs and their interactions interesting targets for the development of novel drugs. LINKED ARTICLES This article is part of a themed section on Novel cAMP Signalling Paradigms. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-2 PMID:22122509

  2. A-Kinase Anchoring Proteins: From protein complexes to physiology and disease

    PubMed Central

    Carnegie, Graeme K.; Means, Christopher K.; Scott, John D.

    2009-01-01

    Protein scaffold complexes are a key mechanism by which a common signaling pathway can serve many different functions. Sequestering a signaling enzyme to a specific subcellular environment not only ensures that the enzyme is near its relevant targets, but also segregates this activity to prevent indiscriminate phosphorylation of other substrates. One family of diverse, well-studied scaffolding proteins are the A-kinase anchoring proteins (AKAPs). These anchoring proteins form multi-protein complexes that integrate cAMP signaling with other pathways and signaling events. In this review we focus on recent advances in the elucidation of AKAP function. PMID:19319965

  3. A-kinase anchoring proteins: molecular regulators of the cardiac stress response.

    PubMed

    Diviani, Dario; Maric, Darko; Pérez López, Irene; Cavin, Sabrina; Del Vescovo, Cosmo D

    2013-04-01

    In response to stress or injury the heart undergoes a pathological remodeling process, associated with hypertrophy, cardiomyocyte death and fibrosis, that ultimately causes cardiac dysfunction and heart failure. It has become increasingly clear that signaling events associated with these pathological cardiac remodeling events are regulated by scaffolding and anchoring proteins, which allow coordination of pathological signals in space and time. A-kinase anchoring proteins (AKAPs) constitute a family of functionally related proteins that organize multiprotein signaling complexes that tether the cAMP-dependent protein kinase (PKA) as well as other signaling enzymes to ensure integration and processing of multiple signaling pathways. This review will discuss the role of AKAPs in the cardiac response to stress. Particular emphasis will be given to the adaptative process associated with cardiac hypoxia as well as the remodeling events linked to cardiac hypertrophy and heart failure. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction. PMID:22889610

  4. Glycogen Synthase Kinase 3β Interaction Protein Functions as an A-kinase Anchoring Protein*

    PubMed Central

    Hundsrucker, Christian; Skroblin, Philipp; Christian, Frank; Zenn, Hans-Michael; Popara, Viola; Joshi, Mangesh; Eichhorst, Jenny; Wiesner, Burkhard; Herberg, Friedrich W.; Reif, Bernd; Rosenthal, Walter; Klussmann, Enno

    2010-01-01

    A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3β interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3β (glycogen synthase kinase 3β). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3β by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3β and thereby provides a mechanism for the integration of PKA and GSK3β signaling pathways. PMID:20007971

  5. A-kinase anchoring proteins: cAMP compartmentalization in neurodegenerative and obstructive pulmonary diseases

    PubMed Central

    Poppinga, W J; Muñoz-Llancao, P; González-Billault, C; Schmidt, M

    2014-01-01

    The universal second messenger cAMP is generated upon stimulation of Gs protein-coupled receptors, such as the β2-adreneoceptor, and leads to the activation of PKA, the major cAMP effector protein. PKA oscillates between an on and off state and thereby regulates a plethora of distinct biological responses. The broad activation pattern of PKA and its contribution to several distinct cellular functions lead to the introduction of the concept of compartmentalization of cAMP. A-kinase anchoring proteins (AKAPs) are of central importance due to their unique ability to directly and/or indirectly interact with proteins that either determine the cellular content of cAMP, such as β2-adrenoceptors, ACs and PDEs, or are regulated by cAMP such as the exchange protein directly activated by cAMP. We report on lessons learned from neurons indicating that maintenance of cAMP compartmentalization by AKAP5 is linked to neurotransmission, learning and memory. Disturbance of cAMP compartments seem to be linked to neurodegenerative disease including Alzheimer's disease. We translate this knowledge to compartmentalized cAMP signalling in the lung. Next to AKAP5, we focus here on AKAP12 and Ezrin (AKAP78). These topics will be highlighted in the context of the development of novel pharmacological interventions to tackle AKAP-dependent compartmentalization. PMID:25132049

  6. Emerging roles of A-kinase anchoring proteins in cardiovascular pathophysiology.

    PubMed

    Diviani, Dario; Reggi, Erica; Arambasic, Miroslav; Caso, Stefania; Maric, Darko

    2016-07-01

    Heart and blood vessels ensure adequate perfusion of peripheral organs with blood and nutrients. Alteration of the homeostatic functions of the cardiovascular system can cause hypertension, atherosclerosis, and coronary artery disease leading to heart injury and failure. A-kinase anchoring proteins (AKAPs) constitute a family of scaffolding proteins that are crucially involved in modulating the function of the cardiovascular system both under physiological and pathological conditions. AKAPs assemble multifunctional signaling complexes that ensure correct targeting of the cAMP-dependent protein kinase (PKA) as well as other signaling enzymes to precise subcellular compartments. This allows local regulation of specific effector proteins that control the function of vascular and cardiac cells. This review will focus on recent advances illustrating the role of AKAPs in cardiovascular pathophysiology. The accent will be mainly placed on the molecular events linked to the control of vascular integrity and blood pressure as well as on the cardiac remodeling process associated with heart failure. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel. PMID:26643253

  7. A-kinase-anchoring proteins coordinate inflammatory responses to cigarette smoke in airway smooth muscle

    PubMed Central

    Heijink, Irene H.; Holtzer, Laura J.; Skroblin, Philipp; Klussmann, Enno; Halayko, Andrew J.; Timens, Wim; Maarsingh, Harm; Schmidt, Martina

    2015-01-01

    β2-Agonist inhibitors can relieve chronic obstructive pulmonary disease (COPD) symptoms by stimulating cyclic AMP (cAMP) signaling. A-kinase-anchoring proteins (AKAPs) compartmentalize cAMP signaling by establishing protein complexes. We previously reported that the β2-agonist fenoterol, direct activation of protein kinase A (PKA), and exchange factor directly activated by cAMP decrease cigarette smoke extract (CSE)-induced release of neutrophil attractant interleukin-8 (IL-8) from human airway smooth muscle (ASM) cells. In the present study, we tested the role of AKAPs in CSE-induced IL-8 release from ASM cells and assessed the effect of CSE on the expression levels of different AKAPs. We also studied mRNA and protein expression of AKAPs in lung tissue from patients with COPD. Our data show that CSE exposure of ASM cells decreases AKAP5 and AKAP12, both capable of interacting with β2-adrenoceptors. In lung tissue of patients with COPD, mRNA levels of AKAP5 and AKAP12 were decreased compared with lung tissue from controls. Using immunohistochemistry, we detected less AKAP5 protein in ASM of patients with COPD Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage II compared with control subjects. St-Ht31, which disrupts AKAP-PKA interactions, augmented CSE-induced IL-8 release from ASM cells and diminished its suppression by fenoterol, an effect mediated by disturbed ERK signaling. The modulatory role of AKAP-PKA interactions in the anti-inflammatory effects of fenoterol in ASM cells and the decrease in expression of AKAP5 and AKAP12 in response to cigarette smoke and in lungs of patients with COPD suggest that cigarette smoke-induced changes in AKAP5 and AKAP12 in patients with COPD may affect efficacy of pharmacotherapy. PMID:25637608

  8. A-kinase-anchoring proteins coordinate inflammatory responses to cigarette smoke in airway smooth muscle.

    PubMed

    Poppinga, Wilfred J; Heijink, Irene H; Holtzer, Laura J; Skroblin, Philipp; Klussmann, Enno; Halayko, Andrew J; Timens, Wim; Maarsingh, Harm; Schmidt, Martina

    2015-04-15

    β2-Agonist inhibitors can relieve chronic obstructive pulmonary disease (COPD) symptoms by stimulating cyclic AMP (cAMP) signaling. A-kinase-anchoring proteins (AKAPs) compartmentalize cAMP signaling by establishing protein complexes. We previously reported that the β2-agonist fenoterol, direct activation of protein kinase A (PKA), and exchange factor directly activated by cAMP decrease cigarette smoke extract (CSE)-induced release of neutrophil attractant interleukin-8 (IL-8) from human airway smooth muscle (ASM) cells. In the present study, we tested the role of AKAPs in CSE-induced IL-8 release from ASM cells and assessed the effect of CSE on the expression levels of different AKAPs. We also studied mRNA and protein expression of AKAPs in lung tissue from patients with COPD. Our data show that CSE exposure of ASM cells decreases AKAP5 and AKAP12, both capable of interacting with β2-adrenoceptors. In lung tissue of patients with COPD, mRNA levels of AKAP5 and AKAP12 were decreased compared with lung tissue from controls. Using immunohistochemistry, we detected less AKAP5 protein in ASM of patients with COPD Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage II compared with control subjects. St-Ht31, which disrupts AKAP-PKA interactions, augmented CSE-induced IL-8 release from ASM cells and diminished its suppression by fenoterol, an effect mediated by disturbed ERK signaling. The modulatory role of AKAP-PKA interactions in the anti-inflammatory effects of fenoterol in ASM cells and the decrease in expression of AKAP5 and AKAP12 in response to cigarette smoke and in lungs of patients with COPD suggest that cigarette smoke-induced changes in AKAP5 and AKAP12 in patients with COPD may affect efficacy of pharmacotherapy. PMID:25637608

  9. Myomegalin is a novel A-kinase anchoring protein involved in the phosphorylation of cardiac myosin binding protein C

    PubMed Central

    2011-01-01

    Background Cardiac contractility is regulated by dynamic phosphorylation of sarcomeric proteins by kinases such as cAMP-activated protein kinase A (PKA). Efficient phosphorylation requires that PKA be anchored close to its targets by A-kinase anchoring proteins (AKAPs). Cardiac Myosin Binding Protein-C (cMyBPC) and cardiac troponin I (cTNI) are hypertrophic cardiomyopathy (HCM)-causing sarcomeric proteins which regulate contractility in response to PKA phosphorylation. Results During a yeast 2-hybrid (Y2H) library screen using a trisphosphorylation mimic of the C1-C2 region of cMyBPC, we identified isoform 4 of myomegalin (MMGL) as an interactor of this N-terminal cMyBPC region. As MMGL has previously been shown to interact with phosphodiesterase 4D, we speculated that it may be a PKA-anchoring protein (AKAP). To investigate this possibility, we assessed the ability of MMGL isoform 4 to interact with PKA regulatory subunits R1A and R2A using Y2H-based direct protein-protein interaction assays. Additionally, to further elucidate the function of MMGL, we used it as bait to screen a cardiac cDNA library. Other PKA targets, viz. CARP, COMMD4, ENO1, ENO3 and cTNI were identified as putative interactors, with cTNI being the most frequent interactor. We further assessed and confirmed these interactions by fluorescent 3D-co-localization in differentiated H9C2 cells as well as by in vivo co-immunoprecipitation. We also showed that quantitatively more interaction occurs between MMGL and cTNI under β-adrenergic stress. Moreover, siRNA-mediated knockdown of MMGL leads to reduction of cMyBPC levels under conditions of adrenergic stress, indicating that MMGL-assisted phosphorylation is requisite for protection of cMyBPC against proteolytic cleavage. Conclusions This study ascribes a novel function to MMGL isoform 4: it meets all criteria for classification as an AKAP, and we show that is involved in the phosphorylation of cMyBPC as well as cTNI, hence MMGL is an important

  10. A- Kinase Anchoring Protein 150 Controls Protein Kinase C-mediated Phosphorylation and Sensitization of TRPV1

    PubMed Central

    Jeske, Nathaniel A.; Patwardhan, Amol M.; Ruparel, Nikita B.; Akopian, Armen N; Shapiro, Mark S.; Henry, Michael A.

    2009-01-01

    Post-translational modifications on various receptor proteins have significant effects on receptor activation. For the Transient Receptor Potential family V type 1 (TRPV1) receptor, phosphorylation of certain serine/threonine amino acid residues sensitizes the receptor to activation by capsaicin and heat. Although Protein Kinase C (PKC) phosphorylates TRPV1 on certain serine/threonine residues, it is not completely understood how PKC functionally associates with TRPV1. Recent studies have reported that the A-kinase Anchoring Protein 150 (AKAP150) mediates PKA phosphorylation of TRPV1 in several nociceptive models. Here, we demonstrate that AKAP150 also mediates PKC-directed phosphorylation and sensitization of TRPV1. In cultured rat trigeminal ganglia, immunocytochemical analyses demonstrate co-localization of AKAP150 and PKC isoforms α, δ, ε, and γ in TRPV1-positive neurons. Additional biochemical evidence supports immunocytochemical results, indicating that AKAP150 preferentially associates with certain PKC isoforms in rat trigeminal ganglia neurons. Employing siRNA-mediated knock-down of AKAP150 expression, we demonstrate that PKC-mediated phosphorylation of TRPV1 and sensitization to a capsaicin response is dependent upon functional expression of the AKAP150 scaffolding protein. Furthermore, PKC-induced sensitization to a thermal stimulus is abrogated in AKAP150 knock-out animals relative to wild-type. Collectively, results from these studies indicate that the AKAP150 scaffolding protein functionally modulates PKC-mediated phosphorylation and sensitization of the TRPV1 receptor in rat sensory neurons, suggesting the scaffolding protein to be an integral regulator of peripheral inflammatory hyperalgesia. PMID:19767149

  11. The A-kinase Anchoring Protein GSKIP Regulates GSK3β Activity and Controls Palatal Shelf Fusion in Mice.

    PubMed

    Deák, Veronika Anita; Skroblin, Philipp; Dittmayer, Carsten; Knobeloch, Klaus-Peter; Bachmann, Sebastian; Klussmann, Enno

    2016-01-01

    A-kinase anchoring proteins (AKAPs) represent a family of structurally diverse proteins, all of which bind PKA. A member of this family is glycogen synthase kinase 3β (GSK3β) interaction protein (GSKIP). GSKIP interacts with PKA and also directly interacts with GSK3β. The physiological function of the GSKIP protein in vivo is unknown. We developed and characterized a conditional knock-out mouse model and found that GSKIP deficiency caused lethality at birth. Embryos obtained through Caesarean section at embryonic day 18.5 were cyanotic, suffered from respiratory distress, and failed to initiate breathing properly. Additionally, all GSKIP-deficient embryos showed an incomplete closure of the palatal shelves accompanied by a delay in ossification along the fusion area of secondary palatal bones. On the molecular level, GSKIP deficiency resulted in decreased phosphorylation of GSK3β at Ser-9 starting early in development (embryonic day 10.5), leading to enhanced GSK3β activity. At embryonic day 18.5, GSK3β activity decreased to levels close to that of wild type. Our findings reveal a novel, crucial role for GSKIP in the coordination of GSK3β signaling in palatal shelf fusion. PMID:26582204

  12. A-Kinase Anchoring Protein 79/150 Coordinates Metabotropic Glutamate Receptor Sensitization of Peripheral Sensory Neurons

    PubMed Central

    Szteyn, Kalina; Rowan, Matthew P.; Gomez, Ruben; Du, Junhui; Carlton, Susan M.; Jeske, Nathaniel A.

    2016-01-01

    Glutamate serves as the primary excitatory neurotransmitter in the nervous system. Previous studies have identified a role for glutamate and group I metabotropic receptors as targets for study in peripheral inflammatory pain. However, the coordination of signaling events that transpire from receptor activation to afferent neuronal sensitization has not been explored. Herein, we identify that scaffolding protein A-Kinase Anchoring Protein 79/150 (AKAP150) coordinates increased peripheral thermal sensitivity following group I metabotropic receptor (mGluR5) activation. In both acute and persistent models of thermal somatosensory behavior, we report that mGluR5 sensitization requires AKAP150 expression. Furthermore, electrophysiological approaches designed to record afferent neuronal activity reveal that mGluR5 sensitization also requires functional AKAP150 expression. In dissociated primary afferent neurons, mGluR5 activation increases TRPV1 responses in an AKAP dependent manner through a mechanism that induces AKAP association with TRPV1. Experimental results presented herein identify a mechanism of receptor-driven scaffolding association with ion channel targets. Importantly, this mechanism could prove significant in the search for therapeutic targets that repress episodes of acute pain from becoming chronic in nature. PMID:26172554

  13. Protein Kinase A Opposes the Phosphorylation-dependent Recruitment of Glycogen Synthase Kinase 3β to A-kinase Anchoring Protein 220.

    PubMed

    Whiting, Jennifer L; Nygren, Patrick J; Tunquist, Brian J; Langeberg, Lorene K; Seternes, Ole-Morten; Scott, John D

    2015-08-01

    The proximity of an enzyme to its substrate can influence rate and magnitude of catalysis. A-kinase anchoring protein 220 (AKAP220) is a multivalent anchoring protein that can sequester a variety of signal transduction enzymes. These include protein kinase A (PKA) and glycogen synthase kinase 3β (GSK3β). Using a combination of molecular and cellular approaches we show that GSK3β phosphorylation of Thr-1132 on AKAP220 initiates recruitment of this kinase into the enzyme scaffold. We also find that AKAP220 anchors GSK3β and its substrate β-catenin in membrane ruffles. Interestingly, GSK3β can be released from the multienzyme complex in response to PKA phosphorylation on serine 9, which suppresses GSK3β activity. The signaling scaffold may enhance this regulatory mechanism, as AKAP220 has the capacity to anchor two PKA holoenzymes. Site 1 on AKAP220 (residues 610-623) preferentially interacts with RII, whereas site 2 (residues 1633-1646) exhibits a dual specificity for RI and RII. In vitro affinity measurements revealed that site 2 on AKAP220 binds RII with ∼10-fold higher affinity than site 1. Occupancy of both R subunit binding sites on AKAP220 could provide a mechanism to amplify local cAMP responses and enable cross-talk between PKA and GSK3β. PMID:26088133

  14. rugose (rg), a Drosophila A kinase anchor protein, is required for retinal pattern formation and interacts genetically with multiple signaling pathways.

    PubMed Central

    Shamloula, Hoda K; Mbogho, Mkajuma P; Pimentel, Angel C; Chrzanowska-Lightowlers, Zosia M A; Hyatt, Vanneta; Okano, Hideyuki; Venkatesh, Tadmiri R

    2002-01-01

    In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the EGFR- and Notch-mediated signaling pathways. Our results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the EGFR and Notch signaling pathways. PMID:12072466

  15. Cytoplasmic anchoring of cAMP-dependent protein kinase (PKA) by A-kinase anchor proteins (AKAPs) is required for meiotic arrest of porcine full-grown and growing oocytes.

    PubMed

    Nishimura, Takanori; Fujii, Wataru; Sugiura, Koji; Naito, Kunihiko

    2014-03-01

    Mammalian growing oocytes (GOs) lack the ability to resume meiosis, although the molecular mechanism of this limitation is not fully understood. We previously hypothesized that the meiotic incompetence of porcine GOs was attributed to complex spatial-temporal regulation of cAMP-dependent protein kinase (PKA) by A-kinase anchor proteins (AKAPs), but found that AKAP1 is not involved in the meiotic incompetence of porcine GOs. In the present study, we cloned porcine cDNAs of AKAP5 and AKAP7alpha, and found that inhibiting the expression of these AKAPs induced PKA translocation into the nucleus and promoted meiotic resumption of porcine GOs without affecting the total PKA activity of GOs, whereas overexpressing these AKAPs had no effect. Because AKAPs regulate PKA localization through binding with regulatory subunits of PKA (PKA-Rs), PKA-R binding with AKAPs was inhibited by AKAP-binding inhibition peptides or PKA-R expression inhibition by antisense RNAs. We found that the expression inhibition and binding inhibition of PRKAR1A, an isoform of mammalian PKA-R, promoted meiotic resumption of porcine GOs, whereas these inhibitions of PRKAR2A, another PKA-R isoform, had no effect. In contrast, the expression inhibition and binding inhibition of PRKAR2A had higher effects than those of PRKAR1A on meiotic resumption of porcine full-grown oocytes. These results suggest that cytoplasmic anchoring of PKA by AKAPs is required for meiotic arrest of oocytes and that the PKA-R isoform working for the maintenance of meiotic arrest changed from PRKAR1A to PRKAR2A during the acquisition of meiotic competence. PMID:24501172

  16. Short RNA Molecules with High Binding Affinity to the KH Motif of A-Kinase Anchoring Protein 1 (AKAP1): Implications for the Regulation of Steroidogenesis

    PubMed Central

    Grozdanov, Petar N.

    2012-01-01

    One of the key regulators of acute steroid hormone biosynthesis in steroidogenic tissues is the steroidogenic acute regulatory (STAR) protein. Acute regulation of STAR production on the transcriptional level is mainly achieved through a cAMP-dependent mechanism, which is well understood. However, less is known about the posttranscriptional regulation of STAR synthesis, specifically the factors influencing the destiny of the Star mRNA after it leaves the nucleus. Here, we show that the 3′-untranslated region of Star mRNA interacts with the heterogeneous nuclear ribonucleoprotein K-homology (KH) motif of the mitochondrial scaffold A-kinase anchoring protein 1 (AKAP1) in vitro with a moderate affinity as measured by EMSAs. A mutation that mimics the phosphorylation state of the KH motif at a specific serine either did not alter, or had a negative impact on, protein-RNA binding under these conditions. The KH motif of AKAP1 binds short pyrimidine-rich RNA molecules with a stable hairpin structure as demonstrated by in vitro selection. AKAP1 also interacts with STAR mRNA in a dibutyryl-cAMP-stimulated human steroidogenic adrenocortical carcinoma cell line in vivo. Therefore, we propose a model in which AKAP1 anchors Star mRNA at the mitochondria, thus stabilizing the translational complex at this organelle, a situation that might affect STAR production and steroidogenesis. In addition, we suggest that the last 216 amino acid residues of AKAP1 might participate in the degradation of STAR and other nuclear-encoded mitochondrial mRNAs through interaction with a RNA-induced silencing complex, specifically with the argonaute 2 protein. PMID:23077346

  17. Identification of cAMP-dependent protein kinase holoenzymes in preantral- and preovulatory-follicle-enriched ovaries, and their association with A-kinase-anchoring proteins.

    PubMed

    Carr, D W; Cutler, R E; Cottom, J E; Salvador, L M; Fraser, I D; Scott, J D; Hunzicker-Dunn, M

    1999-12-01

    Undifferentiated cells from preantral (PA) follicles respond to high levels of cAMP in a different manner than do differentiated cells from preovulatory (PO) follicles. We hypothesized that this differential response of PA and PO cells to cAMP could be due, in part, to either a difference in the profile of isoforms that comprise the cAMP-dependent protein kinase (PKA) holoenzymes and/or a difference in the interaction of PKA with A-kinase-anchoring proteins (AKAPs). To test these hypotheses, PKA activity, PKA holoenzymes, PKA subunits and AKAPs from PA and PO ovaries were compared. Soluble PKA holoenzymes and regulatory (R) subunits were separated by DEAE-cellulose chromatography and sucrose-density-gradient centrifugation. PKA R subunits were distinguished by photoaffinity labelling, autophosphorylation, size, isoelectric point and immunoreactivity. AKAPs were identified by RII subunit overlay assays and immunoreactivity. The results showed that extracts from PA and PO ovaries exhibited equivalent PKA holoenzyme profiles and activities, characterized by low levels of PKA type I (PKAI) holoenzyme and two distinct PKAII holoenzyme peaks, one containing only RIIbeta subunits (PKAIIbeta) and one containing both PKAIIbeta and PKAIIalpha holoenzymes. Both PA and PO ovarian extracts also contained PKA catalytic (C)-subunit-free RIalpha, while only PO ovaries exhibited C-subunit-free RIIbeta. Consistent with the elevated levels of C-subunit-free RIIbeta in PO cells, PKA activation in PO cells required higher concentrations of forskolin than that in PA cells. While extracts of PA and PO ovaries exhibited a number of similar AKAPs, including four prominent ones reactive with anti-AKAP-KL antisera (where AKAP-KL is an AKAP especially abundant in kidney and liver), cAMP-agarose affinity chromatography revealed two major differences in AKAP binding to purified R subunits. PO ovaries contained increased levels of AKAP80 (AKAP of 80 kDa) bound selectively to R subunits in DEAE

  18. Association of functional genetic variants of A-kinase anchoring protein 10 with QT interval length in full-term Polish newborns

    PubMed Central

    Kaczmarczyk, Mariusz; Clark, Jeremy Simon; Gorący, Iwona; Horodnicka-Józwa, Anita; Ciechanowicz, Andrzej

    2013-01-01

    Introduction A-Kinase Anchoring Proteins (AKAPs) coordinate the specificity of protein kinase A signaling by localizing the kinase to subcellular sites. The 1936G (V646) AKAP10 allele has been associated in adults with low cholinergic/vagus nerve sensitivity, shortened PR intervals in ECG recording and in newborns with increased blood pressure and higher cholesterol cord blood concentration. The aim of the study was to answer the question of whether 1936A > G AKAP10 polymorphism is associated with the newborn electrocardiographic variables. Material and methods Electrocardiograms were recorded from 114 consecutive healthy Polish newborns (55 females, 59 males), born after 37 gestational weeks to healthy women with uncomplicated pregnancies. All recordings were made between 3rd and 7th day of life to avoid QT variability. The heart rate per minute and duration of PR, QRS, RR and QT intervals were usually measured. The ECGs were evaluated independently by three observers. At birth, cord blood of neonates was obtained for isolation of genomic DNA. Results The distribution of anthropometric and electrocardiographic variables in our cohort approached normality (skewness < 2 for all variables). No significant differences in anthropometric variables and electrocardiographic traits with respect to AKAP10 genotype were found. Multiple regression analysis with adjustment for gender, gestational age and birth mass revealed that QTc interval in GG AKAP10 homozygotes was significantly longer, but in range, when compared with A alleles carriers (AA + AG, recessive mode of inheritance). No rhythm disturbances were observed. Conclusions Results demonstrate possible association between AKAP10 1936A > G variant and QTc interval in Polish newborns. PMID:25861302

  19. Confinement of β1- and β2-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae

    PubMed Central

    Valentine, Cathleen D.; Haggie, Peter M.

    2011-01-01

    The sympathetic nervous system regulates cardiac output by activating adrenergic receptors (ARs) in cardiac myocytes. The predominant cardiac ARs, β1- and β2AR, are structurally similar but mediate distinct signaling responses. Scaffold protein–mediated compartmentalization of ARs into discrete, multiprotein complexes has been proposed to dictate differential signaling responses. To test the hypothesis that βARs integrate into complexes in live cells, we measured receptor diffusion and interactions by single-particle tracking. Unstimulated β1- and β2AR were highly confined in the membrane of H9c2 cardiomyocyte-like cells, indicating that receptors are tethered and presumably integrated into protein complexes. Selective disruption of interactions with postsynaptic density protein 95/disks large/zonula occludens-1 (PDZ)–domain proteins and A-kinase anchoring proteins (AKAPs) increased receptor diffusion, indicating that these scaffold proteins participate in receptor confinement. In contrast, modulation of interactions between the putative scaffold caveolae and β2AR did not alter receptor dynamics, suggesting that these membrane domains are not involved in β2AR confinement. For both β1- and β2AR, the receptor carboxy-terminus was uniquely responsible for scaffold interactions. Our data formally demonstrate that distinct and stable protein complexes containing β1- or β2AR are formed in the plasma membrane of cardiomyocyte-like cells and that selective PDZ and AKAP interactions are responsible for the integration of receptors into complexes. PMID:21680711

  20. Cross-species sequence analysis reveals multiple charged residue-rich domains that regulate nuclear/cytoplasmic partitioning and membrane localization of a kinase anchoring protein 12 (SSeCKS/Gravin).

    PubMed

    Streb, Jeffrey W; Miano, Joseph M

    2005-07-29

    A kinase anchoring proteins (AKAPs) assemble and compartmentalize multiprotein signaling complexes at discrete subcellular locales and thus confer specificity to transduction cascades using ubiquitous signaling enzymes, such as protein kinase A. Intrinsic targeting domains in each AKAP determine the subcellular localization of these complexes and, along with protein-protein interaction domains, form the core of AKAP function. As a foundational step toward elucidating the relationship between location and function, we have used cross-species sequence analysis and deletion mapping to facilitate the identification of the targeting determinants of AKAP12 (also known as SSeCKS or Gravin). Three charged residue-rich regions were identified that regulate two aspects of AKAP12 localization, nuclear/cytoplasmic partitioning and perinuclear/cell periphery targeting. Using deletion mapping and green fluorescent protein chimeras, we uncovered a heretofore unrecognized nuclear localization potential. Five nuclear localization signals, including a novel class of this type of signal termed X2-NLS, are found in the central region of AKAP12 and are important for nuclear targeting. However, this nuclear localization is suppressed by the negatively charged C terminus that mediates nuclear exclusion. In this condition, the distribution of AKAP12 is regulated by an N-terminal targeting domain that simultaneously directs perinuclear and peripheral AKAP12 localization. Three basic residue-rich regions in the N-terminal targeting region have similarity to the MARCKS proteins and were found to control AKAP12 localization to ganglioside-rich regions at the cell periphery. Our data suggest that AKAP12 localization is regulated by a hierarchy of targeting domains and that the localization of AKAP12-assembled signaling complexes may be dynamically regulated. PMID:15923193

  1. cAMP-stimulated Protein Phosphatase 2A Activity Associated with Muscle A Kinase-anchoring Protein (mAKAP) Signaling Complexes Inhibits the Phosphorylation and Activity of the cAMP-specific Phosphodiesterase PDE4D3*

    PubMed Central

    Dodge-Kafka, Kimberly L.; Bauman, Andrea; Mayer, Nicole; Henson, Edward; Heredia, Lorena; Ahn, Jung; McAvoy, Thomas; Nairn, Angus C.; Kapiloff, Michael S.

    2010-01-01

    The concentration of the second messenger cAMP is tightly controlled in cells by the activity of phosphodiesterases. We have previously described how the protein kinase A-anchoring protein mAKAP serves as a scaffold for the cAMP-dependent protein kinase PKA and the cAMP-specific phosphodiesterase PDE4D3 in cardiac myocytes. PKA and PDE4D3 constitute a negative feedback loop whereby PKA-catalyzed phosphorylation and activation of PDE4D3 attenuate local cAMP levels. We now show that protein phosphatase 2A (PP2A) associated with mAKAP complexes is responsible for reversing the activation of PDE4D3 by catalyzing the dephosphorylation of PDE4D3 serine residue 54. Mapping studies reveal that a C-terminal mAKAP domain (residues 2085–2319) binds PP2A. Binding to mAKAP is required for PP2A function, such that deletion of the C-terminal domain enhances both base-line and forskolin-stimulated PDE4D3 activity. Interestingly, PP2A holoenzyme associated with mAKAP complexes in the heart contains the PP2A targeting subunit B56δ. Like PDE4D3, B56δ is a PKA substrate, and PKA phosphorylation of mAKAP-bound B56δ enhances phosphatase activity 2-fold in the complex. Accordingly, expression of a B56δ mutant that cannot be phosphorylated by PKA results in increased PDE4D3 phosphorylation. Taken together, our findings demonstrate that PP2A associated with mAKAP complexes promotes PDE4D3 dephosphorylation, serving both to inhibit PDE4D3 in unstimulated cells and also to mediate a cAMP-induced positive feedback loop following adenylyl cyclase activation and B56δ phosphorylation. In general, PKA·PP2A·mAKAP complexes exemplify how protein kinases and phosphatases may participate in molecular signaling complexes to dynamically regulate localized intracellular signaling. PMID:20106966

  2. cAMP-stimulated protein phosphatase 2A activity associated with muscle A kinase-anchoring protein (mAKAP) signaling complexes inhibits the phosphorylation and activity of the cAMP-specific phosphodiesterase PDE4D3.

    PubMed

    Dodge-Kafka, Kimberly L; Bauman, Andrea; Mayer, Nicole; Henson, Edward; Heredia, Lorena; Ahn, Jung; McAvoy, Thomas; Nairn, Angus C; Kapiloff, Michael S

    2010-04-01

    The concentration of the second messenger cAMP is tightly controlled in cells by the activity of phosphodiesterases. We have previously described how the protein kinase A-anchoring protein mAKAP serves as a scaffold for the cAMP-dependent protein kinase PKA and the cAMP-specific phosphodiesterase PDE4D3 in cardiac myocytes. PKA and PDE4D3 constitute a negative feedback loop whereby PKA-catalyzed phosphorylation and activation of PDE4D3 attenuate local cAMP levels. We now show that protein phosphatase 2A (PP2A) associated with mAKAP complexes is responsible for reversing the activation of PDE4D3 by catalyzing the dephosphorylation of PDE4D3 serine residue 54. Mapping studies reveal that a C-terminal mAKAP domain (residues 2085-2319) binds PP2A. Binding to mAKAP is required for PP2A function, such that deletion of the C-terminal domain enhances both base-line and forskolin-stimulated PDE4D3 activity. Interestingly, PP2A holoenzyme associated with mAKAP complexes in the heart contains the PP2A targeting subunit B56delta. Like PDE4D3, B56delta is a PKA substrate, and PKA phosphorylation of mAKAP-bound B56delta enhances phosphatase activity 2-fold in the complex. Accordingly, expression of a B56delta mutant that cannot be phosphorylated by PKA results in increased PDE4D3 phosphorylation. Taken together, our findings demonstrate that PP2A associated with mAKAP complexes promotes PDE4D3 dephosphorylation, serving both to inhibit PDE4D3 in unstimulated cells and also to mediate a cAMP-induced positive feedback loop following adenylyl cyclase activation and B56delta phosphorylation. In general, PKA.PP2A.mAKAP complexes exemplify how protein kinases and phosphatases may participate in molecular signaling complexes to dynamically regulate localized intracellular signaling. PMID:20106966

  3. Ion transport proteins anchor and regulate the cytoskeleton.

    PubMed

    Denker, Sheryl P; Barber, Diane L

    2002-04-01

    Structurally diverse ion transport proteins anchor the spectrin-actin cytoskeleton to the plasma membrane by binding directly to linker proteins of the ankyrin and protein 4.1 families. Cytoskeletal anchoring regulates cell shape and restricts the activity of ion transport proteins to specialised membrane domains. New directions are being forged by recent findings that localised anchoring by ion transport proteins regulates the ordered assembly of actin filaments and the actin-dependent processes of cell adhesion and motility. PMID:11891121

  4. D-AKAP1a is a signal-anchored protein in the mitochondrial outer membrane.

    PubMed

    Jun, Yong-Woo; Park, Heeju; Lee, You-Kyung; Kaang, Bong-Kiun; Lee, Jin-A; Jang, Deok-Jin

    2016-04-01

    Dual A-kinase anchoring protein 1a (D-AKAP1a, AKAP1) regulates cAMP signaling in mitochondria. However, it is not clear how D-AKAP1a is associated with mitochondria. In this study, we show that D-AKAP1a is a transmembrane protein in the mitochondrial outer membrane (MOM). We revealed that the N-terminus of D-AKAP1a is exposed to the intermembrane space of mitochondria and that its C-terminus is located on the cytoplasmic side of the MOM. Moderate hydrophobicity and the positively charged flanking residues of the transmembrane domain of D-AKAP1a were important for targeting. Taken together, D-AKAP1a can be classified as a signal-anchored protein in the MOM. Our topological study provides valuable information about the molecular and cellular mechanisms of mitochondrial targeting of AKAP1. PMID:26950402

  5. Functional and Structural Mimicry of Cellular Protein Kinase A Anchoring Proteins by a Viral Oncoprotein

    PubMed Central

    King, Cason R.; Cohen, Michael J.; Fonseca, Gregory J.; Dirk, Brennan S.; Dikeakos, Jimmy D.; Mymryk, Joe S.

    2016-01-01

    The oncoproteins of the small DNA tumor viruses interact with a plethora of cellular regulators to commandeer control of the infected cell. During infection, adenovirus E1A deregulates cAMP signalling and repurposes it for activation of viral gene expression. We show that E1A structurally and functionally mimics a cellular A-kinase anchoring protein (AKAP). E1A interacts with and relocalizes protein kinase A (PKA) to the nucleus, likely to virus replication centres, via an interaction with the regulatory subunits of PKA. Binding to PKA requires the N-terminus of E1A, which bears striking similarity to the amphipathic α-helical domain present in cellular AKAPs. E1A also targets the same docking-dimerization domain of PKA normally bound by cellular AKAPs. In addition, the AKAP like motif within E1A could restore PKA interaction to a cellular AKAP in which its normal interaction motif was deleted. During infection, E1A successfully competes with endogenous cellular AKAPs for PKA interaction. E1A’s role as a viral AKAP contributes to viral transcription, protein expression and progeny production. These data establish HAdV E1A as the first known viral AKAP. This represents a unique example of viral subversion of a crucial cellular regulatory pathway via structural mimicry of the PKA interaction domain of cellular AKAPs. PMID:27137912

  6. Anchored Clathrate Waters Bind Antifreeze Proteins to Ice

    SciTech Connect

    C Garnham; R Campbell; P Davies

    2011-12-31

    The mechanism by which antifreeze proteins (AFPs) irreversibly bind to ice has not yet been resolved. The ice-binding site of an AFP is relatively hydrophobic, but also contains many potential hydrogen bond donors/acceptors. The extent to which hydrogen bonding and the hydrophobic effect contribute to ice binding has been debated for over 30 years. Here we have elucidated the ice-binding mechanism through solving the first crystal structure of an Antarctic bacterial AFP. This 34-kDa domain, the largest AFP structure determined to date, folds as a Ca{sup 2+}-bound parallel beta-helix with an extensive array of ice-like surface waters that are anchored via hydrogen bonds directly to the polypeptide backbone and adjacent side chains. These bound waters make an excellent three-dimensional match to both the primary prism and basal planes of ice and in effect provide an extensive X-ray crystallographic picture of the AFP{vert_ellipsis}ice interaction. This unobstructed view, free from crystal-packing artefacts, shows the contributions of both the hydrophobic effect and hydrogen bonding during AFP adsorption to ice. We term this mode of binding the 'anchored clathrate' mechanism of AFP action.

  7. The association between glycosylphosphatidylinositol-anchored proteins and heterotrimeric G protein alpha subunits in lymphocytes.

    PubMed Central

    Solomon, K R; Rudd, C E; Finberg, R W

    1996-01-01

    Glycosylphosphatidylinositol (GPI)-anchored proteins are nonmembrane spanning cell surface proteins that have been demonstrated to be signal transduction molecules. Because these proteins do not extend into the cytoplasm, the mechanism by which cross-linking of these molecules leads to intracellular signal transduction events is obscure. Previous analysis has indicated that these proteins are associated with src family member tyrosine kinases; however, the role this interaction plays in the generation of intracellular signals is not clear. Here we show that GPI-anchored proteins are associated with alpha subunits of heterotrimeric GTP binding proteins (G proteins) in both human and murine lymphocytes. When the GPI-anchored proteins CD59, CD48, and Thy-1 were immunoprecipitated from various cell lines or freshly isolated lymphocytes, all were found to be associated with a 41-kDa phosphoprotein that we have identified, by using specific antisera, as a mixture of tyrosine phosphorylated G protein alpha subunits: a small amount of Gialpha1, and substantial amounts of Gialpha2 and Gialpha3. GTP binding assays performed with immunoprecipitations of CD59 indicated that there was GTP-binding activity associated with this molecule. Thus, we have shown by both immunochemical and functional criteria that GPI-anchored proteins are physically associated with G proteins. These experiments suggest a potential role of G proteins in the transduction of signals generated by GPI-anchored molecules expressed on lymphocytes of both mouse and human. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8650218

  8. Defining A-Kinase Anchoring Protein (AKAP) Specificity for the Protein Kinase A Subunit RI (PKA-RI).

    PubMed

    Autenrieth, Karolin; Bendzunas, N George; Bertinetti, Daniela; Herberg, Friedrich W; Kennedy, Eileen J

    2016-04-15

    A-Kinase anchoring proteins (AKAPs) act as spatial and temporal regulators of protein kinase A (PKA) by localizing PKA along with multiple proteins into discrete signaling complexes. AKAPs interact with the PKA holoenzyme through an α-helix that docks into a groove formed on the dimerization/docking domain of PKA-R in an isoform-dependent fashion. In an effort to understand isoform selectivity at the molecular level, a library of protein-protein interaction (PPI) disruptors was designed to systematically probe the significance of an aromatic residue on the AKAP docking sequence for RI selectivity. The stapled peptide library was designed based on a high affinity, RI-selective disruptor of AKAP binding, RI-STAD-2. Phe, Trp and Leu were all found to maintain RI selectivity, whereas multiple intermediate-sized hydrophobic substitutions at this position either resulted in loss of isoform selectivity (Ile) or a reversal of selectivity (Val). As a limited number of RI-selective sequences are currently known, this study aids in our understanding of isoform selectivity and establishing parameters for discovering additional RI-selective AKAPs. PMID:26611881

  9. The glycosylphosphatidylinositol-anchored protein repertoire of babesia bovis and its significance for erythrocyte invasion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glycosylphosphatidyl-anchored proteins are particularly abundant on the surface of pathogenic protozoans and might play an important role for parasite survival. In the present work the relevance of GPI-anchored proteins for erythrocyte invasion of Babesia bovis, one of the tick-transmitted causative...

  10. Insulin-induced decrease in protein phosphorylation in rat adipocytes not explained by decreased A-kinase activity

    SciTech Connect

    Egan, J.J.; Greenberg, A.S.; Chang, M.K.; Londos, C.

    1987-05-01

    In isolated rat adipocytes, insulin inhibits lipolysis to a greater extent than would be predicted by the decrease in (-/+)cAMP activity ratio of cAMP-dependent protein kinase (A-kinase), from which it was speculated that insulin promotes the dephosphorylation of hormone-sensitive lipase. They have examined the phosphorylation state of cellular proteins under conditions of varying A-kinase activities in the presence and absence of insulin. Protein phosphorylation was determined by SDS-PAGE electrophoresis of extracts from /sup 32/P-loaded cells; glycerol and A-kinase activity ratios were measured in the cytosolic extracts from control, non-radioactive cells. Increased protein phosphorylation in general occurred over the same range of A-kinase activity ratios, 0.1-0.3, associated with increased glycerol release. The insulin-induced decrease in lipolysis was associated with a decrease in the /sup 32/P content of several proteins, an effect not explained by the modest reduction in A-kinase activity by insulin. This effect of insulin on protein phosphorylation was lost as the A-kinase activity ratios exceeded 0.5. The results suggest that insulin promotes the dephosphorylation of those adipocyte proteins which are subject to phosphorylation by A-kinase.

  11. Activation of the motor protein upon attachment: Anchors weigh in on cytoplasmic dynein regulation.

    PubMed

    Ananthanarayanan, Vaishnavi

    2016-06-01

    Cytoplasmic dynein is the major minus-end-directed motor protein in eukaryotes, and has functions ranging from organelle and vesicle transport to spindle positioning and orientation. The mode of regulation of dynein in the cell remains elusive, but a tantalising possibility is that dynein is maintained in an inhibited, non-motile state until bound to cargo. In vivo, stable attachment of dynein to the cell membrane via anchor proteins enables dynein to produce force by pulling on microtubules and serves to organise the nuclear material. Anchor proteins of dynein assume diverse structures and functions and differ in their interaction with the membrane. In yeast, the anchor protein has come to the fore as one of the key mediators of dynein activity. In other systems, much is yet to be discovered about the anchors, but future work in this area will prove invaluable in understanding dynein regulation in the cell. PMID:27143631

  12. An accessory protein required for anchoring and assembly of amyloid fibres in B. subtilis biofilms.

    PubMed

    Romero, Diego; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

    2011-06-01

    Cells within Bacillus subtilis biofilms are held in place by an extracellular matrix that contains cell-anchored amyloid fibres, composed of the amyloidogenic protein TasA. As biofilms age they disassemble because the cells release the amyloid fibres. This release appears to be the consequence of incorporation of D-tyrosine, D-leucine, D-tryptophan and D-methionine into the cell wall. Here, we characterize the in vivo roles of an accessory protein TapA (TasA anchoring/assembly protein; previously YqxM) that serves both to anchor the fibres to the cell wall and to assemble TasA into fibres. TapA is found in discrete foci in the cell envelope and these foci disappear when cells are treated with a mixture of D-amino acids. Purified cell wall sacculi retain a functional form of this anchoring protein such that purified fibres can be anchored to the sacculi in vitro. In addition, we show that TapA is essential for the proper assembly of the fibres. Its absence results in a dramatic reduction in TasA levels and what little TasA is left produces only thin fibres that are not anchored to the cell. PMID:21477127

  13. The structural basis of tail-anchored membrane protein recognition by Get3

    SciTech Connect

    Mateja, Agnieszka; Szlachcic, Anna; Downing, Maureen E.; Dobosz, Malgorzata; Mariappan, Malaiyalam; Hegde, Ramanujan S.; Keenan, Robert J.

    2009-10-05

    Targeting of newly synthesized membrane proteins to the endoplasmic reticulum is an essential cellular process. Most membrane proteins are recognized and targeted co-translationally by the signal recognition particle. However, nearly 5% of membrane proteins are 'tail-anchored' by a single carboxy-terminal transmembrane domain that cannot access the co-translational pathway. Instead, tail-anchored proteins are targeted post-translationally by a conserved ATPase termed Get3. The mechanistic basis for tail-anchored protein recognition or targeting by Get3 is not known. Here we present crystal structures of yeast Get3 in 'open' (nucleotide-free) and 'closed' (ADP {center_dot} AlF{sub 4}{sup -}-bound) dimer states. In the closed state, the dimer interface of Get3 contains an enormous hydrophobic groove implicated by mutational analyses in tail-anchored protein binding. In the open state, Get3 undergoes a striking rearrangement that disrupts the groove and shields its hydrophobic surfaces. These data provide a molecular mechanism for nucleotide-regulated binding and release of tail-anchored proteins during their membrane targeting by Get3.

  14. Distinct Pathways Mediate the Sorting of Tail-anchored Mitochondrial Outer Membrane Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little is known about the biogenesis of tail-anchored (TA) proteins localized to the mitochondrial outer membrane in plant cells. To address this issue, we screened all of the (>600) known and predicted TA proteins in Arabidopsis thaliana for those annotated, based on Gene Ontology, to possess mitoc...

  15. Distinct Pathways Mediate the Sorting of Tail-anchored Mitochondrial Outer Membrane Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little is known about the biogenesis of tail-anchored (TA) proteins localized to the mitochondrial outer membrane in plant cells. To address this issue, we screened all of the (>500) known and predicted TA proteins in Arabidopsis for those annotated, based on Gene Ontology, to possess mitochondrial...

  16. Vacuolar SNARE Protein Transmembrane Domains Serve as Nonspecific Membrane Anchors with Unequal Roles in Lipid Mixing*

    PubMed Central

    Pieren, Michel; Desfougères, Yann; Michaillat, Lydie; Schmidt, Andrea; Mayer, Andreas

    2015-01-01

    Membrane fusion is induced by SNARE complexes that are anchored in both fusion partners. SNAREs zipper up from the N to C terminus bringing the two membranes into close apposition. Their transmembrane domains (TMDs) might be mere anchoring devices, deforming bilayers by mechanical force. Structural studies suggested that TMDs might also perturb lipid structure by undergoing conformational transitions or by zipping up into the bilayer. Here, we tested this latter hypothesis, which predicts that the activity of SNAREs should depend on the primary sequence of their TMDs. We replaced the TMDs of all vacuolar SNAREs (Nyv1, Vam3, and Vti1) by a lipid anchor, by a TMD from a protein unrelated to the membrane fusion machinery, or by artificial leucine-valine sequences. Individual exchange of the native SNARE TMDs against an unrelated transmembrane anchor or an artificial leucine-valine sequence yielded normal fusion activities. Fusion activity was also preserved upon pairwise exchange of the TMDs against unrelated peptides, which eliminates the possibility for specific TMD-TMD interactions. Thus, a specific primary sequence or zippering beyond the SNARE domains is not a prerequisite for fusion. Lipid-anchored Vti1 was fully active, and lipid-anchored Nyv1 permitted the reaction to proceed up to hemifusion, and lipid-anchored Vam3 interfered already before hemifusion. The unequal contribution of proteinaceous TMDs on Vam3 and Nyv1 suggests that Q- and R-SNAREs might make different contributions to the hemifusion intermediate and the opening of the fusion pore. Furthermore, our data support the view that SNARE TMDs serve as nonspecific membrane anchors in vacuole fusion. PMID:25817997

  17. Molecular Mechanisms for cAMP-Mediated Immunoregulation in T cells – Role of Anchored Protein Kinase A Signaling Units

    PubMed Central

    Wehbi, Vanessa L.; Taskén, Kjetil

    2016-01-01

    The cyclic AMP/protein kinase A (cAMP/PKA) pathway is one of the most common and versatile signal pathways in eukaryotic cells. A-kinase anchoring proteins (AKAPs) target PKA to specific substrates and distinct subcellular compartments providing spatial and temporal specificity for mediation of biological effects channeled through the cAMP/PKA pathway. In the immune system, cAMP is a potent negative regulator of T cell receptor-mediated activation of effector T cells (Teff) acting through a proximal PKA/Csk/Lck pathway anchored via a scaffold consisting of the AKAP Ezrin holding PKA, the linker protein EBP50, and the anchoring protein phosphoprotein associated with glycosphingolipid-enriched microdomains holding Csk. As PKA activates Csk and Csk inhibits Lck, this pathway in response to cAMP shuts down proximal T cell activation. This immunomodulating pathway in Teff mediates clinically important responses to regulatory T cell (Treg) suppression and inflammatory mediators, such as prostaglandins (PGs), adrenergic stimuli, adenosine, and a number of other ligands. A major inducer of T cell cAMP levels is PG E2 (PGE2) acting through EP2 and EP4 prostanoid receptors. PGE2 plays a crucial role in the normal physiological control of immune homeostasis as well as in inflammation and cancer immune evasion. Peripherally induced Tregs express cyclooxygenase-2, secrete PGE2, and elicit the immunosuppressive cAMP pathway in Teff as one tumor immune evasion mechanism. Moreover, a cAMP increase can also be induced by indirect mechanisms, such as intercellular transfer between T cells. Indeed, Treg, known to have elevated levels of intracellular cAMP, may mediate their suppressive function by transferring cAMP to Teff through gap junctions, which we speculate could also be regulated by PKA/AKAP complexes. In this review, we present an updated overview on the influence of cAMP-mediated immunoregulatory mechanisms acting through localized cAMP signaling and the therapeutical

  18. Molecular Mechanisms for cAMP-Mediated Immunoregulation in T cells - Role of Anchored Protein Kinase A Signaling Units.

    PubMed

    Wehbi, Vanessa L; Taskén, Kjetil

    2016-01-01

    The cyclic AMP/protein kinase A (cAMP/PKA) pathway is one of the most common and versatile signal pathways in eukaryotic cells. A-kinase anchoring proteins (AKAPs) target PKA to specific substrates and distinct subcellular compartments providing spatial and temporal specificity for mediation of biological effects channeled through the cAMP/PKA pathway. In the immune system, cAMP is a potent negative regulator of T cell receptor-mediated activation of effector T cells (Teff) acting through a proximal PKA/Csk/Lck pathway anchored via a scaffold consisting of the AKAP Ezrin holding PKA, the linker protein EBP50, and the anchoring protein phosphoprotein associated with glycosphingolipid-enriched microdomains holding Csk. As PKA activates Csk and Csk inhibits Lck, this pathway in response to cAMP shuts down proximal T cell activation. This immunomodulating pathway in Teff mediates clinically important responses to regulatory T cell (Treg) suppression and inflammatory mediators, such as prostaglandins (PGs), adrenergic stimuli, adenosine, and a number of other ligands. A major inducer of T cell cAMP levels is PG E2 (PGE2) acting through EP2 and EP4 prostanoid receptors. PGE2 plays a crucial role in the normal physiological control of immune homeostasis as well as in inflammation and cancer immune evasion. Peripherally induced Tregs express cyclooxygenase-2, secrete PGE2, and elicit the immunosuppressive cAMP pathway in Teff as one tumor immune evasion mechanism. Moreover, a cAMP increase can also be induced by indirect mechanisms, such as intercellular transfer between T cells. Indeed, Treg, known to have elevated levels of intracellular cAMP, may mediate their suppressive function by transferring cAMP to Teff through gap junctions, which we speculate could also be regulated by PKA/AKAP complexes. In this review, we present an updated overview on the influence of cAMP-mediated immunoregulatory mechanisms acting through localized cAMP signaling and the therapeutical

  19. Glycosylphosphatidylinositol-Anchored Proteins Are Required for Cell Wall Synthesis and Morphogenesis in Arabidopsis

    PubMed Central

    Gillmor, C. Stewart; Lukowitz, Wolfgang; Brininstool, Ginger; Sedbrook, John C.; Hamann, Thorsten; Poindexter, Patricia; Somerville, Chris

    2005-01-01

    Mutations at five loci named PEANUT1-5 (PNT) were identified in a genetic screen for radially swollen embryo mutants. pnt1 cell walls showed decreased crystalline cellulose, increased pectins, and irregular and ectopic deposition of pectins, xyloglucans, and callose. Furthermore, pnt1 pollen is less viable than the wild type, and pnt1 embryos were delayed in morphogenesis and showed defects in shoot and root meristems. The PNT1 gene encodes the Arabidopsis thaliana homolog of mammalian PIG-M, an endoplasmic reticulum–localized mannosyltransferase that is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor. All five pnt mutants showed strongly reduced accumulation of GPI-anchored proteins, suggesting that they all have defects in GPI anchor synthesis. Although the mutants are seedling lethal, pnt1 cells are able to proliferate for a limited time as undifferentiated callus and do not show the massive deposition of ectopic cell wall material seen in pnt1 embryos. The different phenotype of pnt1 cells in embryos and callus suggest a differential requirement for GPI-anchored proteins in cell wall synthesis in these two tissues and points to the importance of GPI anchoring in coordinated multicellular growth. PMID:15772281

  20. VIP21/caveolin, glycosphingolipid clusters and the sorting of glycosylphosphatidylinositol-anchored proteins in epithelial cells.

    PubMed Central

    Zurzolo, C; van't Hof, W; van Meer, G; Rodriguez-Boulan, E

    1994-01-01

    We studied the role of the association between glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipid (GSL) clusters in apical targeting using gD1-DAF, a GPI-anchored protein that is differentially sorted by three epithelial cell lines. Differently from MDCK cells, where both gD1-DAF and glucosylceramide (GlcCer) are sorted to the apical membrane, in MDCK Concanavalin A-resistant cells (MDCK-ConAr) gD1-DAF was mis-sorted to both surfaces, but GlcCer was still targeted to the apical surface. In both MDCK and MDCK-ConAr cells, gD1-DAF became associated with TX-100-insoluble GSL clusters during transport to the cell surface. In dramatic contrast with MDCK cells, the Fischer rat thyroid (FRT) cell line targeted both gD1-DAF and GlcCer basolaterally. The targeting differences for GSLs in FRT and MDCK cells cannot be accounted for by a differential ability to form clusters because, in spite of major differences in the GSL composition, both cell lines assembled GSLs into TX-100-insoluble complexes with identical isopycnic densities. Surprisingly, in FRT cells, gD1-DAF did not form clusters with GSLs and, therefore, remained completely soluble. This clustering defect in FRT cells correlated with the lack of expression of VIP21/caveolin, a protein localized to both the plasma membrane caveolae and the trans Golgi network. This suggests that VIP21/caveolin may have an important role in recruiting GPI-anchored proteins into GSL complexes necessary for their apical sorting. However, since MDCK-ConAr cells expressed caveolin and clustered GPI-anchored proteins normally, yet mis-sorted them, our results also indicate that clustering and caveolin are not sufficient for apical targeting, and that additional factors are required for the accurate apical sorting of GPI-anchored proteins. Images PMID:8306971

  1. Delivery of a secreted soluble protein to the vacuole via a membrane anchor

    SciTech Connect

    Barrieu, F.; Chrispeels, M.J.

    1999-08-01

    To further understand how membrane proteins are sorted in the secretory system, the authors devised a strategy that involves the expression of a membrane-anchored yeast invertase in transgenic plants. The construct consisted of a signal peptide followed by the coding region of yeast invertase and the transmembrane domain and cytoplasmic tail of calnexin. The substitution of a lysine near the C terminus of calnexin with a glutamic acid residue ensured progression through the secretory system rather than retention in or return to the endoplasmic reticulum. In the transformed plants, invertase activity and a 70-kD cross-reacting protein were found in the vacuoles. This yeast invertase had plant-specific complex glycans, indicating that transport to the vacuole was mediated by the Golgi apparatus. The microsomal fraction contained a membrane-anchored 90-kD cross-reacting polypeptide, but was devoid of invertase activity. Their results indicate that this membrane-anchored protein proceeds in the secretory system beyond the point where soluble proteins are sorted for secretion, and is detached from its membrane anchor either just before or just after delivery to the vacuole.

  2. A novel partner for D-type cyclins: protein kinase A-anchoring protein AKAP95.

    PubMed Central

    Arsenijevic, Tatjana; Degraef, Chantal; Dumont, Jacques E; Roger, Pierre P; Pirson, Isabelle

    2004-01-01

    Using a yeast interaction screen to search for proteins that interact with cyclin D3 in thyroid gland, we identified the cAMP-dependent AKAP95 (protein kinase A-anchoring protein 95). AKAP95 is a scaffolding protein that primarily co-fractionates with the nuclear matrix, whereas a minor fraction associates with chromatin in interphase cells. In co-transfected Chinese-hamster ovary cells, AKAP95 strongly interacted with the three D-type cyclins, but not with CDK4 (cyclin-dependent kinase 4) or with p27kip1. CDK4 displaced the interaction between cyclin D3 and AKAP95, suggesting that AKAP95 could not be the elusive bridging adaptor between D-type cyclins and CDK4 or play a role in the regulation of cyclin D3-CDK4 activity. Interaction between endogenous AKAP95 and cyclin D3 or cyclin D1 was detected in canine thyrocytes, human fibroblasts and NIH-3T3 cells. As both AKAP95 and cyclins D were recently reported to associate with minichromosome maintenance proteins [Eide, Tasken, Carlson, Williams, Jahnsen, Tasken and Collas (2003) J. Biol. Chem. 278, 26750-26756; Gladden and Diehl (2003) J. Biol. Chem. 278, 9754-9760], we hypothesize that the interaction between AKAP95 and D-type cyclins might serve to facilitate the emerging regulatory role of cyclin D-CDK4 in the formation of the prereplication complex at the DNA replication origins. PMID:14641107

  3. Selective and programmed cleavage of GPI-anchored proteins from the surface membrane by phospholipase C.

    PubMed

    Müller, Alexandra; Klöppel, Christine; Smith-Valentine, Megan; Van Houten, Judith; Simon, Martin

    2012-01-01

    Many surface proteins of eukaryotic cells are tethered to the membrane by a GPI-anchor which is enzymatically cleavable. Here, we investigate cleavage and release of different GPI-proteins by phospholipase C from the outer membrane of the ciliate Paramecium tetraurelia. Our data indicate that different GPI-proteins are not equally cleaved as proteins of the surface antigen family are preferentially released in vitro compared to several smaller GPI-proteins. Likewise, the analysis of culture medium indicates exclusive in vivo release of surface antigens by two phospholipase C isoforms (PLC2 and PLC6). This suggests that phospholipase C shows affinity for select groups of GPI-anchored proteins. Our data also reveal an up-regulation of PLC isoforms in GPI-anchored protein cleavage during antigenic switching. As a consequence, silencing of these PLCs leads to a drastic decrease of antigen concentration in the medium. These results suggest a higher order of GPI-regulation by phospholipase C as cleavage occurs programmed and specific for single GPI-proteins instead of an unspecific shedding of the entire surface membrane GPI-content. PMID:22024023

  4. Multifunctional Roles for the Protein Translocation Machinery in RNA Anchoring to the Endoplasmic Reticulum*

    PubMed Central

    Jagannathan, Sujatha; Hsu, Jack C.-C.; Reid, David W.; Chen, Qiang; Thompson, Will J.; Moseley, Arthur M.; Nicchitta, Christopher V.

    2014-01-01

    Signal sequence-encoding mRNAs undergo translation-dependent localization to the endoplasmic reticulum (ER) and at the ER are anchored via translation on Sec61-bound ribosomes. Recent investigations into the composition and membrane association characteristics of ER-associated mRNAs have, however, revealed both ribosome-dependent (indirect) and ribosome-independent (direct) modes of mRNA association with the ER. These findings raise important questions regarding our understanding of how mRNAs are selected, localized, and anchored to the ER. Using semi-intact tissue culture cells, we performed a polysome solubilization screen and identified conditions that distinguish polysomes engaged in the translation of distinct cohorts of mRNAs. To gain insight into the molecular basis of direct mRNA anchoring to the ER, we performed RNA-protein UV photocross-linking studies in rough microsomes and demonstrate that numerous ER integral membrane proteins display RNA binding activity. Quantitative proteomic analyses of HeLa cytosolic and ER-bound polysome fractions identified translocon components as selective polysome-interacting proteins. Notably, the Sec61 complex was highly enriched in polysomes engaged in the translation of endomembrane organelle proteins, whereas translocon accessory proteins, such as ribophorin I, were present in all subpopulations of ER-associated polysomes. Analyses of the protein composition of oligo(dT)-selected UV photocross-linked ER protein-RNA adducts identified Sec61α,β and ribophorin I as ER-poly(A) mRNA-binding proteins, suggesting unexpected roles for the protein translocation and modification machinery in mRNA anchoring to the ER. In summary, we propose that multiple mechanisms of mRNA and ribosome association with ER operate to enable an mRNA transcriptome-wide function for the ER in protein synthesis. PMID:25063809

  5. Protein-anchoring Strategy for Delivering Acetylcholinesterase to the Neuromuscular Junction

    PubMed Central

    Ito, Mikako; Suzuki, Yumi; Okada, Takashi; Fukudome, Takayasu; Yoshimura, Toshiro; Masuda, Akio; Takeda, Shin'ichi; Krejci, Eric; Ohno, Kinji

    2012-01-01

    Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ). Congenital defects of ColQ cause endplate AChE deficiency and myasthenic syndrome. A single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq−/− mice recovered motor functions, synaptic transmission, as well as the morphology of the NMJ. ColQ-tailed AChE was specifically anchored to NMJ and its amount was restored to 89% of the wild type. We next characterized the molecular basis of this efficient recovery. We first confirmed that ColQ-tailed AChE can be specifically targeted to NMJ by an in vitro overlay assay in Colq−/− mice muscle sections. We then injected AAV1-COLQ-IRES-EGFP into the left tibialis anterior and detected AChE in noninjected limbs. Furthermore, the in vivo injection of recombinant ColQ-tailed AChE protein complex into the gluteus maximus muscle of Colq−/− mice led to accumulation of AChE in noninjected forelimbs. We demonstrated for the first time in vivo that the ColQ protein contains a tissue-targeting signal that is sufficient for anchoring itself to the NMJ. We propose that the protein-anchoring strategy is potentially applicable to a broad spectrum of diseases affecting extracellular matrix molecules. PMID:22371845

  6. Angiotensin-converting enzyme is a GPI-anchored protein releasing factor crucial for fertilization.

    PubMed

    Kondoh, Gen; Tojo, Hiromasa; Nakatani, Yuka; Komazawa, Nobuyasu; Murata, Chie; Yamagata, Kazuo; Maeda, Yusuke; Kinoshita, Taroh; Okabe, Masaru; Taguchi, Ryo; Takeda, Junji

    2005-02-01

    The angiotensin-converting enzyme (ACE) is a key regulator of blood pressure. It is known to cleave small peptides, such as angiotensin I and bradykinin and changes their biological activities, leading to upregulation of blood pressure. Here we describe a new activity for ACE: a glycosylphosphatidylinositol (GPI)-anchored protein releasing activity (GPIase activity). Unlike its peptidase activity, GPIase activity is weakly inhibited by the tightly binding ACE inhibitor and not inactivated by substitutions of core amino acid residues for the peptidase activity, suggesting that the active site elements for GPIase differ from those for peptidase activity. ACE shed various GPI-anchored proteins from the cell surface, and the process was accelerated by the lipid raft disruptor filipin. The released products carried portions of the GPI anchor, indicating cleavage within the GPI moiety. Further analysis by high-performance liquid chromatography-mass spectrometry predicted the cleavage site at the mannose-mannose linkage. GPI-anchored proteins such as TESP5 and PH-20 were released from the sperm membrane of wild-type mice but not in Ace knockout sperm in vivo. Moreover, peptidase-inactivated E414D mutant ACE and also PI-PLC rescued the egg-binding deficiency of Ace knockout sperms, implying that ACE plays a crucial role in fertilization through this activity. PMID:15665832

  7. Peptide nanofibers modified with a protein by using designed anchor molecules bearing hydrophobic and functional moieties.

    PubMed

    Miyachi, Ayaka; Takahashi, Tsuyoshi; Matsumura, Sachiko; Mihara, Hisakazu

    2010-06-11

    Self-assembly of peptides and proteins is a key feature of biological functions. Short amphiphilic peptides designed with a beta-sheet structure can form sophisticated nanofiber structures, and the fibers are available as nanomaterials for arranging biomolecules. Peptide FI (H-PKFKIIEFEP-OH) self-assembles into nanofibers with a coiled fine structure, as reported in our previous work. We have constructed anchor molecules that have both a binding moiety for the fiber structure and a functional unit capable of capturing target molecules, with the purpose of arranging proteins on the designed peptide nanofibers. Designed anchors containing an alkyl chain as a binding unit and biotin as a functional moiety were found to bind to peptide fibers FI and F2i (H-ALEAKFAAFEAKLA-NH(2)). The surface-exposed biotin moiety on the fibers could capture an anti-biotin antibody. Moreover, hydrophobic dipeptide anchor units composed of iminodiacetate connected to Phe-Phe or Ile-Ile and a peptide composed of six histidine residues connected to biotin could also connect FI peptide fibers to the anti-biotin antibody through the chelation of Ni(2+) ions. This strategy of using designed anchors opens a novel approach to constructing nanoscale protein arrays on peptide nanomaterials. PMID:20419712

  8. Cleavage by signal peptide peptidase is required for the degradation of selected tail-anchored proteins

    PubMed Central

    Boname, Jessica M.; Bloor, Stuart; Wandel, Michal P.; Nathan, James A.; Antrobus, Robin; Dingwell, Kevin S.; Thurston, Teresa L.; Smith, Duncan L.; Smith, James C.; Randow, Felix

    2014-01-01

    The regulated turnover of endoplasmic reticulum (ER)–resident membrane proteins requires their extraction from the membrane lipid bilayer and subsequent proteasome-mediated degradation. Cleavage within the transmembrane domain provides an attractive mechanism to facilitate protein dislocation but has never been shown for endogenous substrates. To determine whether intramembrane proteolysis, specifically cleavage by the intramembrane-cleaving aspartyl protease signal peptide peptidase (SPP), is involved in this pathway, we generated an SPP-specific somatic cell knockout. In a stable isotope labeling by amino acids in cell culture–based proteomics screen, we identified HO-1 (heme oxygenase-1), the rate-limiting enzyme in the degradation of heme to biliverdin, as a novel SPP substrate. Intramembrane cleavage by catalytically active SPP provided the primary proteolytic step required for the extraction and subsequent proteasome-dependent degradation of HO-1, an ER-resident tail-anchored protein. SPP-mediated proteolysis was not limited to HO-1 but was required for the dislocation and degradation of additional tail-anchored ER-resident proteins. Our study identifies tail-anchored proteins as novel SPP substrates and a specific requirement for SPP-mediated intramembrane cleavage in protein turnover. PMID:24958774

  9. Diffusion of GPI-anchored proteins is influenced by the activity of dynamic cortical actin

    PubMed Central

    Saha, Suvrajit; Lee, Il-Hyung; Polley, Anirban; Groves, Jay T.; Rao, Madan; Mayor, Satyajit

    2015-01-01

    Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24–37°C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an “active actin-membrane composite” cell surface. PMID:26378258

  10. Lipid transfer proteins do their thing anchored at membrane contact sites… but what is their thing?

    PubMed

    Wong, Louise H; Levine, Tim P

    2016-04-15

    Membrane contact sites are structures where two organelles come close together to regulate flow of material and information between them. One type of inter-organelle communication is lipid exchange, which must occur for membrane maintenance and in response to environmental and cellular stimuli. Soluble lipid transfer proteins have been extensively studied, but additional families of transfer proteins have been identified that are anchored into membranes by transmembrane helices so that they cannot diffuse through the cytosol to deliver lipids. If such proteins target membrane contact sites they may be major players in lipid metabolism. The eukaryotic family of so-called Lipid transfer proteins Anchored at Membrane contact sites (LAMs) all contain both a sterol-specific lipid transfer domain in the StARkin superfamily (related to StART/Bet_v1), and one or more transmembrane helices anchoring them in the endoplasmic reticulum (ER), making them interesting subjects for study in relation to sterol metabolism. They target a variety of membrane contact sites, including newly described contacts between organelles that were already known to make contact by other means. Lam1-4p target punctate ER-plasma membrane contacts. Lam5p and Lam6p target multiple contacts including a new category: vacuolar non-NVJ cytoplasmic ER (VancE) contacts. These developments confirm previous observations on tubular lipid-binding proteins (TULIPs) that established the importance of membrane anchored proteins for lipid traffic. However, the question remaining to be solved is the most difficult of all: are LAMs transporters, or alternately are they regulators that affect traffic more indirectly? PMID:27068964

  11. A robust protein host for anchoring chelating ligands and organocatalysts.

    PubMed

    Reetz, Manfred T; Rentzsch, Martin; Pletsch, Andreas; Taglieber, Andreas; Hollmann, Frank; Mondière, Régis J G; Dickmann, Norbert; Höcker, Birte; Cerrone, Simona; Haeger, Michaela C; Sterner, Reinhard

    2008-03-01

    In order to put the previously proposed concept of directed evolution of hybrid catalysts (proteins that harbor synthetic transition-metal catalysts or organocatalysts) into practice, several prerequisites must be met. The availability of a robust host protein that can be expressed in sufficiently large amounts, and that can be purified in a simple manner is crucial. The thermostable enzyme tHisF from Thermotoga maritima, which constitutes the synthase subunit of a bi-enzyme complex that is instrumental in the biosynthesis of histidine, fulfills these requirements. In the present study, fermentation has been miniaturized and parallelized, as has purification of the protein by simple heat treatment. Several mutants with strategically placed cysteines for subsequent bioconjugation have been produced. One of the tHisF mutants, Cys9Ala/Asp11Cys, was subjected to bioconjugation by the introduction of a variety of ligands for potential metal ligation, of a ligand/metal moiety, and of several organocatalytic entities that comprise a flavin or thiazolium salts. Characterization by mass spectrometry and tryptic digestion was achieved. As a result of this study, a platform for performing future directed evolution of these hybrid catalysts is now available. PMID:18273849

  12. MyRIP anchors protein kinase A to the exocyst complex.

    PubMed

    Goehring, April S; Pedroja, Benjamin S; Hinke, Simon A; Langeberg, Lorene K; Scott, John D

    2007-11-01

    The movement of signal transduction enzymes in and out of multi-protein complexes coordinates the spatial and temporal resolution of cellular events. Anchoring and scaffolding proteins are key to this process because they sequester protein kinases and phosphatases with a subset of their preferred substrates. The protein kinase A-anchoring family of proteins (AKAPs), which target the cAMP-dependent protein kinase (PKA) and other enzymes to defined subcellular microenvironments, represent a well studied group of these signal-organizing molecules. In this report we demonstrate that the Rab27a GTPase effector protein MyRIP is a member of the AKAP family. The zebrafish homolog of MyRIP (Ze-AKAP2) was initially detected in a two-hybrid screen for AKAPs. A combination of biochemical, cell-based, and immunofluorescence approaches demonstrate that the mouse MyRIP ortholog targets the type II PKA holoenzyme via an atypical mechanism to a specific perinuclear region of insulin-secreting cells. Similar approaches show that MyRIP interacts with the Sec6 and Sec8 components of the exocyst complex, an evolutionarily conserved protein unit that controls protein trafficking and exocytosis. These data indicate that MyRIP functions as a scaffolding protein that links PKA to components of the exocytosis machinery. PMID:17827149

  13. Radial Spoke Protein 3 Is a Mammalian Protein Kinase A-anchoring Protein That Binds ERK1/2*

    PubMed Central

    Jivan, Arif; Earnest, Svetlana; Juang, Yu-Chi; Cobb, Melanie H.

    2009-01-01

    Initially identified in Chlamydomonas, RSP3 (radial spoke protein 3) is 1 of more than 20 identified radial spoke structural components of motile cilia and is required for axonemal sliding and flagellar motility. The mammalian orthologs for this and other radial spoke proteins, however, remain to be characterized. We found mammalian RSP3 to bind to the MAPK ERK2 through a yeast two-hybrid screen designed to identify interacting proteins that have a higher affinity for the phosphorylated, active form of the protein kinase. Consistent with the screening result, the human homolog, RSPH3, interacts with and is a substrate for ERK1/2. Moreover, RSPH3 is a protein kinase A-anchoring protein (AKAP) that scaffolds the cAMP-dependent protein kinase holoenzyme. The binding of RSPH3 to the regulatory subunits of cAMP-dependent protein kinase, RIIα and RIIβ, is regulated by ERK1/2 activity and phosphorylation. Here we describe an ERK1/2-interacting AKAP and suggest a mechanism by which cAMP-dependent protein kinase-AKAP binding can be modulated by the activity of other enzymes. PMID:19684019

  14. Surface display of heterologous proteins in Bacillus thuringiensis using a peptidoglycan hydrolase anchor

    PubMed Central

    Shao, Xiaohu; Jiang, Mengtian; Yu, Ziniu; Cai, Hao; Li, Lin

    2009-01-01

    Background Previous studies have revealed that the lysin motif (LysM) domains of bacterial cell wall-degrading enzymes are able to bind to peptidoglycan moieties of the cell wall. This suggests an approach for a cell surface display system in Gram-positive bacteria using a LysM-containing protein as the anchoring motif. In this study, we developed a new surface display system in B. thuringiensis using a LysM-containing peptidoglycan hydrolase, endo-β-N-acetylglucosaminidase (Mbg), as the anchor protein. Results Homology searching in the B. thuringiensis YBT-1520 genome revealed a putative peptidoglycan hydrolase gene. The encoded protein, Mbg, exhibited substantial cell-wall binding capacity. The deduced amino acid sequence of Mbg was structurally distinguished as an N-terminal domain with two tandemly aligned LysMs and a C-terminal catalytic domain. A GFP-fusion protein was expressed and used to verify the surface localization by Western blot, flow cytometry, protease accessibility, SDS sensitivity, immunofluorescence, and electron microscopy assays. Low-level constitutive expression of Mbg was elevated by introducing a sporulation-independent promoter of cry3Aa. Truncated Mbg domains with separate N-terminus (Mbgn), C-terminus (Mbgc), LysM1, or LysM2 were further compared for their cell-wall displaying efficiencies. The Mbgn moiety contributed to cell-wall anchoring, while LysM1 was the active domain. Two tandemly repeated Mbgns exhibited the highest display activity, while the activity of three repeated Mbgns was decreased. A heterologous bacterial multicopper oxidase (WlacD) was successfully displayed onto the surface of B. thuringiensis target cells using the optimum (Mbgn)2 anchor, without radically altering its catalytic activity. Conclusion Mbg can be a functional anchor protein to target different heterologous proteins onto the surface of B. thuringiensis cells. Since the LysM domain appears to be universal in Gram-positive bacteria, the strategy

  15. GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane

    PubMed Central

    Sevcsik, Eva; Brameshuber, Mario; Fölser, Martin; Weghuber, Julian; Honigmann, Alf; Schütz, Gerhard J.

    2015-01-01

    The organization of proteins and lipids in the plasma membrane has been subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here, we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment. Intriguingly, this treatment does neither nucleate the formation of an ordered membrane phase, nor result in any enrichment of nanoscopic ordered domains within the micropatterned regions. In contrast, we find that immobilized mGFP-GPIs behave as inert obstacles to the diffusion of other membrane constituents without influencing their membrane environment over distances beyond their physical size. Our results indicate that phase partitioning is not a fundamental element of protein organization in the plasma membrane. PMID:25897971

  16. GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane.

    PubMed

    Sevcsik, Eva; Brameshuber, Mario; Fölser, Martin; Weghuber, Julian; Honigmann, Alf; Schütz, Gerhard J

    2015-01-01

    The organization of proteins and lipids in the plasma membrane has been the subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored-mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment. Intriguingly, this treatment does neither nucleate the formation of an ordered membrane phase nor result in any enrichment of nanoscopic-ordered domains within the micropatterned regions. In contrast, we find that immobilized mGFP-GPIs behave as inert obstacles to the diffusion of other membrane constituents without influencing their membrane environment over distances beyond their physical size. Our results indicate that phase partitioning is not a fundamental element of protein organization in the plasma membrane. PMID:25897971

  17. Sialic Acid within the Glycosylphosphatidylinositol Anchor Targets the Cellular Prion Protein to Synapses.

    PubMed

    Bate, Clive; Nolan, William; McHale-Owen, Harriet; Williams, Alun

    2016-08-12

    Although the cellular prion protein (PrP(C)) is concentrated at synapses, the factors that target PrP(C) to synapses are not understood. Here we demonstrate that exogenous PrP(C) was rapidly targeted to synapses in recipient neurons derived from Prnp knock-out((0/0)) mice. The targeting of PrP(C) to synapses was dependent upon both neuronal cholesterol concentrations and the lipid and glycan composition of its glycosylphosphatidylinositol (GPI) anchor. Thus, the removal of either an acyl chain or sialic acid from the GPI anchor reduced the targeting of PrP(C) to synapses. Isolated GPIs (derived from PrP(C)) were also targeted to synapses, as was IgG conjugated to these GPIs. The removal of sialic acid from GPIs prevented the targeting of either the isolated GPIs or the IgG-GPI conjugate to synapses. Competition studies showed that pretreatment with sialylated GPIs prevented the targeting of PrP(C) to synapses. These results are consistent with the hypothesis that the sialylated GPI anchor attached to PrP(C) acts as a synapse homing signal. PMID:27325697

  18. High sensitivity of mouse neuronal cells to tetanus toxin requires a GPI-anchored protein.

    PubMed

    Munro, P; Kojima, H; Dupont, J L; Bossu, J L; Poulain, B; Boquet, P

    2001-11-30

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani specifically cleaves VAMP/synaptobrevin (VAMP) in central neurons, thereby causing inhibition of neurotransmitter release and ensuing spastic paralysis. Although polysialogangliosides act as components of the neurotoxin binding sites on neurons, evidence has accumulated indicating that a protein moiety is implicated as a receptor of TeNT. We have observed that treatment of cultured mouse neuronal cells with the phosphatidylinositol-specific phospholipase C (PIPLC) inhibited TeNT-induced cleavage of VAMP. Also, we have shown that the blocking effects of TeNT on neuroexocytosis can be prevented by incubation of Purkinje cell preparation with PIPLC. In addition, treatment of cultured mouse neuronal cells with cholesterol sequestrating agents such as nystatin and filipin, which disrupt clustering of GPI-anchored proteins in lipid rafts, prevented intraneuronal VAMP cleavage by TeNT. Our results demonstrate that high sensitivity of neurons to TeNT requires rafts and one or more GPI-anchored protein(s) which act(s) as a pivotal receptor for the neurotoxin. PMID:11716521

  19. Clostridium difficile surface proteins are anchored to the cell wall using CWB2 motifs that recognise the anionic polymer PSII.

    PubMed

    Willing, Stephanie E; Candela, Thomas; Shaw, Helen Alexandra; Seager, Zoe; Mesnage, Stéphane; Fagan, Robert P; Fairweather, Neil F

    2015-05-01

    Gram-positive surface proteins can be covalently or non-covalently anchored to the cell wall and can impart important properties on the bacterium in respect of cell envelope organisation and interaction with the environment. We describe here a mechanism of protein anchoring involving tandem CWB2 motifs found in a large number of cell wall proteins in the Firmicutes. In the Clostridium difficile cell wall protein family, we show the three tandem repeats of the CWB2 motif are essential for correct anchoring to the cell wall. CWB2 repeats are non-identical and cannot substitute for each other, as shown by the secretion into the culture supernatant of proteins containing variations in the patterns of repeats. A conserved Ile Leu Leu sequence within the CWB2 repeats is essential for correct anchoring, although a preceding proline residue is dispensable. We propose a likely genetic locus encoding synthesis of the anionic polymer PSII and, using RNA knock-down of key genes, reveal subtle effects on cell wall composition. We show that the anionic polymer PSII binds two cell wall proteins, SlpA and Cwp2, and these interactions require the CWB2 repeats, defining a new mechanism of protein anchoring in Gram-positive bacteria. PMID:25649385

  20. A conserved archaeal pathway for tail-anchored membrane protein insertion

    PubMed Central

    Sherrill, John; Mariappan, Malaiyalam; Dominik, Pawel; Hegde, Ramanujan S.; Keenan, Robert J.

    2011-01-01

    Eukaryotic tail-anchored (TA) membrane proteins are inserted into the endoplasmic reticulum by a post-translational TRC40 pathway, but no comparable pathway is known in other domains of life. The crystal structure of an archaebacterial TRC40 sequence homolog bound to ADP•AlF4− reveals characteristic features of eukaryotic TRC40 including a zinc-mediated dimer and a large hydrophobic groove. Moreover, archaeal TRC40 interacts with the transmembrane domain of TA substrates and directs their membrane insertion. Thus, the TRC40 pathway is more broadly conserved than previously recognized. PMID:21658170

  1. Comparison between the behavior of different hydrophobic peptides allowing membrane anchoring of proteins

    PubMed Central

    Lhor, Mustapha; Bernier, Sarah; Horchani, Habib; Bussières, Sylvain; Cantin, Line; Desbat, Bernard; Salesse, Christian

    2014-01-01

    Membrane binding of proteins such as short chain dehydrogenases reductases or tail-anchored proteins relies on their N- and/or C-terminal hydrophobic transmembrane segment. In this review, we propose guidelines to characterize such hydrophobic peptide segments using spectroscopic and biophysical measurements. The secondary structure content of the C-terminal peptides of retinol dehydrogenase 8, RGS9-1 anchor protein, lecithin retinol acyl transferase, and of the N-terminal peptide of retinol dehydrogenase 11 has been deduced by prediction tools from their primary sequence as well as by using infrared or circular dichroism analyses. Depending on the solvent and the solubilization method, significant structural differences were observed, often involving α-helices. The helical structure of these peptides was found to be consistent with their presumed membrane binding. Langmuir monolayers have been used as membrane models to study lipid-peptide interactions. The values of maximum insertion pressure obtained for all peptides using a monolayer of 1,2-dioleoyl-sn-glycero-3-phospho-ethanolamine (DOPE) are larger than the estimated lateral pressure of membranes, thus suggesting that they bind membranes. Polarization modulation infrared reflection absorption spectroscopy has been used to determine the structure and orientation of these peptides in the absence and in the presence of a DOPE monolayer. This lipid induced an increase or a decrease in the organization of the peptide secondary structure. Further measurements are necessary using other lipids to better understand the membrane interactions of these peptides. PMID:24560216

  2. Comparison between the behavior of different hydrophobic peptides allowing membrane anchoring of proteins.

    PubMed

    Lhor, Mustapha; Bernier, Sarah C; Horchani, Habib; Bussières, Sylvain; Cantin, Line; Desbat, Bernard; Salesse, Christian

    2014-05-01

    Membrane binding of proteins such as short chain dehydrogenase reductases or tail-anchored proteins relies on their N- and/or C-terminal hydrophobic transmembrane segment. In this review, we propose guidelines to characterize such hydrophobic peptide segments using spectroscopic and biophysical measurements. The secondary structure content of the C-terminal peptides of retinol dehydrogenase 8, RGS9-1 anchor protein, lecithin retinol acyl transferase, and of the N-terminal peptide of retinol dehydrogenase 11 has been deduced by prediction tools from their primary sequence as well as by using infrared or circular dichroism analyses. Depending on the solvent and the solubilization method, significant structural differences were observed, often involving α-helices. The helical structure of these peptides was found to be consistent with their presumed membrane binding. Langmuir monolayers have been used as membrane models to study lipid-peptide interactions. The values of maximum insertion pressure obtained for all peptides using a monolayer of 1,2-dioleoyl-sn-glycero-3-phospho-ethanolamine (DOPE) are larger than the estimated lateral pressure of membranes, thus suggesting that they bind membranes. Polarization modulation infrared reflection absorption spectroscopy has been used to determine the structure and orientation of these peptides in the absence and in the presence of a DOPE monolayer. This lipid induced an increase or a decrease in the organization of the peptide secondary structure. Further measurements are necessary using other lipids to better understand the membrane interactions of these peptides. PMID:24560216

  3. GPI transamidase and GPI anchored proteins: oncogenes and biomarkers for cancer.

    PubMed

    Gamage, Dilani G; Hendrickson, Tamara L

    2013-01-01

    Cancer is second only to heart disease as a cause of death in the US, with a further negative economic impact on society. Over the past decade, details have emerged which suggest that different glycosylphosphatidylinositol (GPI)-anchored proteins are fundamentally involved in a range of cancers. This post-translational glycolipid modification is introduced into proteins via the action of the enzyme GPI transamidase (GPI-T). In 2004, PIG-U, one of the subunits of GPI-T, was identified as an oncogene in bladder cancer, offering a direct connection between GPI-T and cancer. GPI-T is a membrane-bound, multi-subunit enzyme that is poorly understood, due to its structural complexity and membrane solubility. This review is divided into three sections. First, we describe our current understanding of GPI-T, including what is known about each subunit and their roles in the GPI-T reaction. Next, we review the literature connecting GPI-T to different cancers with an emphasis on the variations in GPI-T subunit over-expression. Finally, we discuss some of the GPI-anchored proteins known to be involved in cancer onset and progression and that serve as potential biomarkers for disease-selective therapies. Given that functions for only one of GPI-T's subunits have been robustly assigned, the separation between healthy and malignant GPI-T activity is poorly defined. PMID:23978072

  4. Hypomorphic Mutations in PGAP2, Encoding a GPI-Anchor-Remodeling Protein, Cause Autosomal-Recessive Intellectual Disability

    PubMed Central

    Hansen, Lars; Tawamie, Hasan; Murakami, Yoshiko; Mang, Yuan; ur Rehman, Shoaib; Buchert, Rebecca; Schaffer, Stefanie; Muhammad, Safia; Bak, Mads; Nöthen, Markus M.; Bennett, Eric P.; Maeda, Yusuke; Aigner, Michael; Reis, André; Kinoshita, Taroh; Tommerup, Niels; Baig, Shahid Mahmood; Abou Jamra, Rami

    2013-01-01

    PGAP2 encodes a protein involved in remodeling the glycosylphosphatidylinositol (GPI) anchor in the Golgi apparatus. After synthesis in the endoplasmic reticulum (ER), GPI anchors are transferred to the proteins and are remodeled while transported through the Golgi to the cell membrane. Germline mutations in six genes (PIGA, PIGL, PIGM, PIGV, PIGN, and PIGO) in the ER-located part of the GPI-anchor-biosynthesis pathway have been reported, and all are associated with phenotypes extending from malformation and lethality to severe intellectual disability, epilepsy, minor dysmorphisms, and elevated alkaline phosphatase (ALP). We performed autozygosity mapping and ultra-deep sequencing followed by stringent filtering and identified two homozygous PGAP2 alterations, p.Tyr99Cys and p.Arg177Pro, in seven offspring with nonspecific autosomal-recessive intellectual disability from two consanguineous families. Rescue experiments with the altered proteins in PGAP2-deficient Chinese hamster ovary cell lines showed less expression of cell-surface GPI-anchored proteins DAF and CD59 than of the wild-type protein, substantiating the pathogenicity of the identified alterations. Furthermore, we observed a full rescue when we used strong promoters before the mutant cDNAs, suggesting a hypomorphic effect of the mutations. We report on alterations in the Golgi-located part of the GPI-anchor-biosynthesis pathway and extend the phenotypic spectrum of the GPI-anchor deficiencies to isolated intellectual disability with elevated ALP. GPI-anchor deficiencies can be interpreted within the concept of a disease family, and we propose that the severity of the phenotype is dependent on the location of the altered protein in the biosynthesis chain. PMID:23561846

  5. cAMP signaling in neurons: patterns of neuronal expression and intracellular localization for a novel protein, AKAP 150, that anchors the regulatory subunit of cAMP-dependent protein kinase II beta.

    PubMed Central

    Glantz, S B; Amat, J A; Rubin, C S

    1992-01-01

    In mammalian brain, physiological signals carried by cyclic AMP (cAMP) seem to be targeted to effector sites via the tethering of cAMP-dependent protein kinase II beta (PKAII beta) to intracellular structures. Recently characterized A kinase anchor proteins (AKAPs) are probable mediators of the sequestration of PKAII beta because they contain a high-affinity binding site for the regulatory subunit (RII beta) of the kinase and a distinct intracellular targeting domain. To establish a cellular basis for this targeting mechanism, we have employed immunocytochemistry to 1) identify the types of neurons that are enriched in AKAPs, 2) determine the primary intracellular location of the anchor protein, and 3) demonstrate that an AKAP and RII beta are coenriched and colocalized in neurons that utilize the adenylate cyclase-cyclic AMP-dependent protein kinase (PKA) signaling pathway. Antibodies directed against rat brain AKAP 150 were used to elucidate the regional, cellular and intracellular distribution of a prototypic anchor protein in the CNS. AKAP 150 is abundant in Purkinje cells and in neurons of the olfactory bulb, basal ganglia, cerebral cortex, and other forebrain regions. In contrast, little AKAP 150 is detected in neurons of the thalamus, hypothalamus, midbrain, and hindbrain. A high proportion of total AKAP 150 is concentrated in primary branches of dendrites, where it is associated with microtubules. We also discovered that the patterns of accumulation and localization of RII beta (and PKAII beta) in brain are similar to those of AKAP 150. The results suggest that bifunctional AKAP 150 tethers PKAII beta to the dendritic cytoskeleton, thereby creating a discrete target site for the reception and propagation of signals carried by cAMP. Images PMID:1333841

  6. Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous proteins.

    PubMed Central

    Van der Vaart, J M; te Biesebeke, R; Chapman, J W; Toschka, H Y; Klis, F M; Verrips, C T

    1997-01-01

    The carboxyl-terminal regions of five cell wall proteins (Cwp1p, Cwp2p, Ag alpha 1p, Tip1p, and Flo1p) and three potential cell wall proteins (Sed1p, YCR89w, and Tir1p) all proved capable of immobilizing alpha-galactosidase in the cell wall of Saccharomyces cerevisiae. The fraction of the total amount of fusion protein that was localized to the cell wall varied depending on the anchor domain used. The highest proportion of cell wall incorporation was achieved with Cwp2p, Ag alpha 1p, or Sed1p as an anchor. Although 80% of these fusion proteins were incorporated in the cell wall, the total production of alpha-galactosidase-Ag alpha 1p was sixfold lower than that of alpha-galactosidase-Cwp2p and eightfold lower than that of alpha-galactosidase-Sed1p. Differences in mRNA levels were not responsible for this discrepancy, nor was an intracellular accumulation of alpha-galactosidase-Ag alpha 1p detectable. A lower translation efficiency of the alpha-galactosidase-AG alpha 1 fusion construct is most likely to be responsible for the low level of protein production. alpha-Galactosidase immobilized by the carboxyl-terminal 67 amino acids of Cwp2p was most effective in the hydrolysis of the high-molecular-weight substrate guar gum from Cyamopsis tetragonoloba. This indicates that the use of a large anchoring domain does not necessarily result in a better exposure of the immobilized enzyme to the exterior of the yeast cell. PMID:9023939

  7. A HaloTag Anchored Ruler for Week-Long Studies of Protein Dynamics.

    PubMed

    Popa, Ionel; Rivas-Pardo, Jaime Andrés; Eckels, Edward C; Echelman, Daniel J; Badilla, Carmen L; Valle-Orero, Jessica; Fernández, Julio M

    2016-08-24

    Under physiological conditions, protein oxidation and misfolding occur with very low probability and on long times scales. Single-molecule techniques provide the ability to distinguish between properly folded and damaged proteins that are otherwise masked in ensemble measurements. However, at physiological conditions these rare events occur with a time constant of several hours, inaccessible to current single-molecule approaches. Here we present a magnetic-tweezers-based technique that allows, for the first time, the study of folding of single proteins during week-long experiments. This technique combines HaloTag anchoring, sub-micrometer positioning of magnets, and an active correction of the focal drift. Using this technique and protein L as a molecular template, we generate a magnet law by correlating the distance between the magnet and the measuring paramagnetic bead with unfolding/folding steps. We demonstrate that, using this magnet law, we can accurately measure the dynamics of proteins over a wide range of forces, with minimal dispersion from bead to bead. We also show that the force calibration remains invariant over week-long experiments applied to the same single proteins. The approach demonstrated in this Article opens new, exciting ways to examine proteins on the "human" time scale and establishes magnetic tweezers as a valuable technique to study low-probability events that occur during protein folding under force. PMID:27409974

  8. Ergosterol content specifies targeting of tail-anchored proteins to mitochondrial outer membranes

    PubMed Central

    Krumpe, Katrin; Frumkin, Idan; Herzig, Yonatan; Rimon, Nitzan; Özbalci, Cagakan; Brügger, Britta; Rapaport, Doron; Schuldiner, Maya

    2012-01-01

    Tail-anchored (TA) proteins have a single C-terminal transmembrane domain, making their biogenesis dependent on posttranslational translocation. Despite their importance, no dedicated insertion machinery has been uncovered for mitochondrial outer membrane (MOM) TA proteins. To decipher the molecular mechanisms guiding MOM TA protein insertion, we performed two independent systematic microscopic screens in which we visualized the localization of model MOM TA proteins on the background of mutants in all yeast genes. We could find no mutant in which insertion was completely blocked. However, both screens demonstrated that MOM TA proteins were partially localized to the endoplasmic reticulum (ER) in ∆spf1 cells. Spf1, an ER ATPase with unknown function, is the first protein shown to affect MOM TA protein insertion. We found that ER membranes in ∆spf1 cells become similar in their ergosterol content to mitochondrial membranes. Indeed, when we visualized MOM TA protein distribution in yeast strains with reduced ergosterol content, they phenocopied the loss of Spf1. We therefore suggest that the inherent differences in membrane composition between organelle membranes are sufficient to determine membrane integration specificity in a eukaryotic cell. PMID:22918956

  9. The mechanism of membrane-associated steps in tail-anchored protein insertion

    SciTech Connect

    Mariappan, Malaiyalam; Mateja, Agnieszka; Dobosz, Malgorzata; Bove, Elia; Hegde, Ramanujan S.; Keenan, Robert J.

    2012-06-19

    Tail-anchored (TA) membrane proteins destined for the endoplasmic reticulum are chaperoned by cytosolic targeting factors that deliver them to a membrane receptor for insertion. Although a basic framework for TA protein recognition is now emerging, the decisive targeting and membrane insertion steps are not understood. Here we reconstitute the TA protein insertion cycle with purified components, present crystal structures of key complexes between these components and perform mutational analyses based on the structures. We show that a committed targeting complex, formed by a TA protein bound to the chaperone ATPase Get3, is initially recruited to the membrane through an interaction with Get2. Once the targeting complex has been recruited, Get1 interacts with Get3 to drive TA protein release in an ATPase-dependent reaction. After releasing its TA protein cargo, the now-vacant Get3 recycles back to the cytosol concomitant with ATP binding. This work provides a detailed structural and mechanistic framework for the minimal TA protein insertion cycle.

  10. The role of Listeria monocytogenes cell wall surface anchor protein LapB in virulence, adherence, and intracellular replication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lmof2365_2117 is a Listeria monocytogenes putative cell wall surface anchor protein with a conserved domain found in collagen binding proteins. We constructed a deletion mutation in lmof2365_2117 in serotype 4b strain F2365, evaluated its virulence, and determined its ability to adhere and invade co...

  11. New insights into the targeting of a sub-set of tail-anchored proteins to the outer mitochondrial membrane

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tail-anchored (TA) proteins are a unique class of functionally diverse membrane proteins that are defined by their single C-terminal membrane-spanning domain and their ability to insert post-translationally into specific organelles with an Nout-Cin orientation. The molecular mechanisms by which TA p...

  12. Cold acclimation is accompanied by complex responses of glycosylphosphatidylinositol (GPI)-anchored proteins in Arabidopsis

    PubMed Central

    Takahashi, Daisuke; Kawamura, Yukio; Uemura, Matsuo

    2016-01-01

    Cold acclimation results in changes of the plasma membrane (PM) composition. The PM is considered to contain specific lipid/protein-enriched microdomains which can be extracted as detergent-resistant plasma membrane (DRM). Previous studies in animal cells have demonstrated that glycosylphosphatidylinositol-anchored proteins (GPI-APs) can be targeted to microdomains and/or the apoplast. However, the functional significance of GPI-APs during cold acclimation in plants is not yet fully understood. In this study, we aimed to investigate the responsiveness of GPI-APs to cold acclimation treatment in Arabidopsis. We isolated the PM, DRM, and apoplast fractions separately and, in addition, GPI-AP-enriched fractions were prepared from the PM preparation. Label-free quantitative shotgun proteomics identified a number of GPI-APs (163 proteins). Among them, some GPI-APs such as fasciclin-like arabinogalactan proteins and glycerophosphoryldiester phosphodiesterase-like proteins predominantly increased in PM- and GPI-AP-enriched fractions while the changes of GPI-APs in the DRM and apoplast fractions during cold acclimation were considerably different from those of other fractions. These proteins are thought to be associated with cell wall structure and properties. Therefore, this study demonstrated that each GPI-AP responded to cold acclimation in a different manner, suggesting that these changes during cold acclimation are involved in rearrangement of the extracellular matrix including the cell wall towards acquisition of freezing tolerance. PMID:27471282

  13. Cold acclimation is accompanied by complex responses of glycosylphosphatidylinositol (GPI)-anchored proteins in Arabidopsis.

    PubMed

    Takahashi, Daisuke; Kawamura, Yukio; Uemura, Matsuo

    2016-09-01

    Cold acclimation results in changes of the plasma membrane (PM) composition. The PM is considered to contain specific lipid/protein-enriched microdomains which can be extracted as detergent-resistant plasma membrane (DRM). Previous studies in animal cells have demonstrated that glycosylphosphatidylinositol-anchored proteins (GPI-APs) can be targeted to microdomains and/or the apoplast. However, the functional significance of GPI-APs during cold acclimation in plants is not yet fully understood. In this study, we aimed to investigate the responsiveness of GPI-APs to cold acclimation treatment in Arabidopsis We isolated the PM, DRM, and apoplast fractions separately and, in addition, GPI-AP-enriched fractions were prepared from the PM preparation. Label-free quantitative shotgun proteomics identified a number of GPI-APs (163 proteins). Among them, some GPI-APs such as fasciclin-like arabinogalactan proteins and glycerophosphoryldiester phosphodiesterase-like proteins predominantly increased in PM- and GPI-AP-enriched fractions while the changes of GPI-APs in the DRM and apoplast fractions during cold acclimation were considerably different from those of other fractions. These proteins are thought to be associated with cell wall structure and properties. Therefore, this study demonstrated that each GPI-AP responded to cold acclimation in a different manner, suggesting that these changes during cold acclimation are involved in rearrangement of the extracellular matrix including the cell wall towards acquisition of freezing tolerance. PMID:27471282

  14. Sortases and the Art of Anchoring Proteins to the Envelopes of Gram-Positive Bacteria

    PubMed Central

    Marraffini, Luciano A.; DeDent, Andrea C.; Schneewind, Olaf

    2006-01-01

    The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their environment. Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provides bacterial envelopes with species- and strain-specific properties that are ultimately responsible for bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. Surface proteins typically carry two topogenic sequences, i.e., N-terminal signal peptides and C-terminal sorting signals. Sortases catalyze a transpeptidation reaction by first cleaving a surface protein substrate at the cell wall sorting signal. The resulting acyl enzyme intermediates between sortases and their substrates are then resolved by the nucleophilic attack of amino groups, typically provided by the cell wall cross bridges of peptidoglycan precursors. The surface protein linked to peptidoglycan is then incorporated into the envelope and displayed on the microbial surface. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis. PMID:16524923

  15. Expression and clinical significance of A‐kinase anchor protein 4 in lung adenocarcinoma tissue

    PubMed Central

    Guo, Kang; Yu, Xiao‐Yun; Yu, Zhuang; Xu, Ping

    2015-01-01

    Abstract Background The A‐kinase anchor proteins (AKAP) are a growing family of scaffolding proteins involved in the occurrence, proliferation, and metastasis of tumors by controlling intracellular signals. In this study, the expression and significance of AKAP4 were analyzed in patients with lung adenocarcinoma and adjacent non‐cancerous tissues. Methods Using reverse transcriptase‐polymerase chain reaction and Western blot, AKAP4 messenger ribonucleic acid (mRNA) and protein expression levels were measured in 108 cases of lung adenocarcinoma and adjacent non‐cancerous tissues. Results AKAP4 mRNA and protein were expressed in lung adenocarcinoma tissues, but not in adjacent non‐cancerous tissues. The expression of AKAP4 mRNA and protein was closely associated with lymphatic metastasis (P < 0.05), but had no relationship with stage, differentiation degree, gender, age or smoking (P > 0.05). AKAP4 expression had an adverse effect on the overall survival rate (P < 0.05). Conclusion The expression of AKAP4 was high in lung adenocarcinoma tissue, which may be closely related to the lymphatic metastasis of lung adenocarcinoma. AKAP4 may be a novel lung adenocarcinoma molecule marker and a predictor of poor prognosis. PMID:27148411

  16. Generation of Glycosylphosphatidylinositol Anchor Protein-Deficient Blood Cells From Human Induced Pluripotent Stem Cells

    PubMed Central

    Yuan, Xuan; Braunstein, Evan M.; Ye, Zhaohui; Liu, Cyndi F.; Chen, Guibin; Zou, Jizhong; Cheng, Linzhao

    2013-01-01

    PIG-A is an X-linked gene required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; thus, PIG-A mutant cells have a deficiency or absence of all GPI-anchored proteins (GPI-APs). Acquired mutations in hematopoietic stem cells result in the disease paroxysmal nocturnal hemoglobinuria, and hypomorphic germline PIG-A mutations lead to severe developmental abnormalities, seizures, and early death. Human induced pluripotent stem cells (iPSCs) can differentiate into cell types derived from all three germ layers, providing a novel developmental system for modeling human diseases. Using PIG-A gene targeting and an inducible PIG-A expression system, we have established, for the first time, a conditional PIG-A knockout model in human iPSCs that allows for the production of GPI-AP-deficient blood cells. PIG-A-null iPSCs were unable to generate hematopoietic cells or any cells expressing the CD34 marker and were defective in generating mesodermal cells expressing KDR/VEGFR2 (kinase insert domain receptor) and CD56 markers. In addition, PIG-A-null iPSCs had a block in embryonic development prior to mesoderm differentiation that appears to be due to defective signaling through bone morphogenetic protein 4. However, early inducible PIG-A transgene expression allowed for the generation of GPI-AP-deficient blood cells. This conditional PIG-A knockout model should be a valuable tool for studying the importance of GPI-APs in hematopoiesis and human development. PMID:24113066

  17. Abolishing Cell Wall Glycosylphosphatidylinositol-Anchored Proteins in Candida albicans Enhances Recognition by Host Dectin-1.

    PubMed

    Shen, Hui; Chen, Si Min; Liu, Wei; Zhu, Fang; He, Li Juan; Zhang, Jun Dong; Zhang, Shi Qun; Yan, Lan; Xu, Zheng; Xu, Guo Tong; An, Mao Mao; Jiang, Yuan Ying

    2015-07-01

    Fungi can shield surface pathogen-associated molecular patterns (PAMPs) for evading host immune attack. The most common and opportunistic human pathogen, Candida albicans, can shield β-(1 3)-glucan on the cell wall, one of the major PAMPs, to avoid host phagocyte Dectin-1 recognition. The way to interfere in the shielding process for more effective antifungal defense is not well established. In this study, we found that deletion of the C. albicans GPI7 gene, which was responsible for adding ethanolaminephosphate to the second mannose in glycosylphosphatidylinositol (GPI) biosynthesis, could block the attachment of most GPI-anchored cell wall proteins (GPI-CWPs) to the cell wall and subsequently unmask the concealed β-(1,3)-glucan. Neutrophils could kill the uncloaked gpi7 mutant more efficiently with an augmented respiratory burst. The gpi7 mutant also stimulated Dectin-1-dependent immune responses of macrophages, including activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways and secretion of specific cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and IL-12p40. Furthermore, the gpi7 null mutant could induce an enhanced inflammatory response through promoting significant recruitment of neutrophils and monocytes and could stimulate stronger Th1 and Th17 cell responses to fungal infections in vivo. These in vivo phenotypes also were Dectin-1 dependent. Thus, we assume that GPI-CWPs are involved in the immune mechanism of C. albicans escaping from host recognition by Dectin-1. Our studies also indicate that the blockage of GPI anchor synthesis is a strategy to inhibit C. albicans evading host recognition. PMID:25895969

  18. The surface-anchored NanA protein promotes pneumococcal brain endothelial cell invasion.

    PubMed

    Uchiyama, Satoshi; Carlin, Aaron F; Khosravi, Arya; Weiman, Shannon; Banerjee, Anirban; Quach, Darin; Hightower, George; Mitchell, Tim J; Doran, Kelly S; Nizet, Victor

    2009-08-31

    In humans, Streptococcus pneumoniae (SPN) is the leading cause of bacterial meningitis, a disease with high attributable mortality and frequent permanent neurological sequelae. The molecular mechanisms underlying the central nervous system tropism of SPN are incompletely understood, but include a primary interaction of the pathogen with the blood-brain barrier (BBB) endothelium. All SPN strains possess a gene encoding the surface-anchored sialidase (neuraminidase) NanA, which cleaves sialic acid on host cells and proteins. Here, we use an isogenic SPN NanA-deficient mutant and heterologous expression of the protein to show that NanA is both necessary and sufficient to promote SPN adherence to and invasion of human brain microvascular endothelial cells (hBMECs). NanA-mediated hBMEC invasion depends only partially on sialidase activity, whereas the N-terminal lectinlike domain of the protein plays a critical role. NanA promotes SPN-BBB interaction in a murine infection model, identifying the protein as proximal mediator of CNS entry by the pathogen. PMID:19687228

  19. Distinct Pathways Mediate the Sorting of Tail-Anchored Proteins to the Plastid Outer Envelope

    PubMed Central

    Dhanoa, Preetinder K.; Richardson, Lynn G. L.; Smith, Matthew D.; Gidda, Satinder K.; Henderson, Matthew P. A.; Andrews, David W.; Mullen, Robert T.

    2010-01-01

    Background Tail-anchored (TA) proteins are a distinct class of membrane proteins that are sorted post-translationally to various organelles and function in a number of important cellular processes, including redox reactions, vesicular trafficking and protein translocation. While the molecular targeting signals and pathways responsible for sorting TA proteins to their correct intracellular destinations in yeasts and mammals have begun to be characterized, relatively little is known about TA protein biogenesis in plant cells, especially for those sorted to the plastid outer envelope. Methodology/Principal Findings Here we investigated the biogenesis of three plastid TA proteins, including the 33-kDa and 34-kDa GTPases of the translocon at the outer envelope of chloroplasts (Toc33 and Toc34) and a novel 9-kDa protein of unknown function that we define here as an outer envelope TA protein (OEP9). Using a combination of in vivo and in vitro assays we show that OEP9 utilizes a different sorting pathway than that used by Toc33 and Toc34. For instance, while all three TA proteins interact with the cytosolic OEP chaperone/receptor, AKR2A, the plastid targeting information within OEP9 is distinct from that within Toc33 and Toc34. Toc33 and Toc34 also appear to differ from OEP9 in that their insertion is dependent on themselves and the unique lipid composition of the plastid outer envelope. By contrast, the insertion of OEP9 into the plastid outer envelope occurs in a proteinaceous-dependent, but Toc33/34-independent manner and membrane lipids appear to serve primarily to facilitate normal thermodynamic integration of this TA protein. Conclusions/Significance Collectively, the results provide evidence in support of at least two sorting pathways for plastid TA outer envelope proteins and shed light on not only the complex diversity of pathways involved in the targeting and insertion of proteins into plastids, but also the molecular mechanisms that underlie the delivery of TA

  20. mRNA encoding Sec61β, a tail-anchored protein, is localized on the endoplasmic reticulum

    PubMed Central

    Cui, Xianying A.; Zhang, Hui; Ilan, Lena; Liu, Ai Xin; Kharchuk, Iryna; Palazzo, Alexander F.

    2015-01-01

    ABSTRACT Although one pathway for the post-translational targeting of tail-anchored proteins to the endoplasmic reticulum (ER) has been well defined, it is unclear whether additional pathways exist. Here, we provide evidence that a subset of mRNAs encoding tail-anchored proteins, including Sec61β and nesprin-2, is partially localized to the surface of the ER in mammalian cells. In particular, Sec61b mRNA can be targeted to, and later maintained on, the ER using both translation-dependent and -independent mechanisms. Our data suggests that this process is independent of p180 (also known as RRBP1), a known mRNA receptor on the ER, and the transmembrane domain recognition complex (TRC) pathway components, TRC40 (also known as ASNA1) and BAT3 (also known as BAG6). In addition, our data indicates that Sec61b mRNA might access translocon-bound ribosomes. Our results show that certain tail-anchored proteins are likely to be synthesized directly on the ER, and this facilitates their membrane insertion. Thus, it is clear that mammalian cells utilize multiple mechanisms to ensure efficient targeting of tail-anchored proteins to the surface of the ER. PMID:26272916

  1. PscI is a type III secretion needle anchoring protein with in vitro polymerization capacities.

    PubMed

    Monlezun, Laura; Liebl, David; Fenel, Daphna; Grandjean, Teddy; Berry, Alice; Schoehn, Guy; Dessein, Rodrigue; Faudry, Eric; Attree, Ina

    2015-04-01

    The export of bacterial toxins across the bacterial envelope requires the assembly of complex, membrane-embedded protein architectures. Pseudomonas aeruginosa employs type III secretion (T3S) injectisome to translocate exotoxins directly into the cytoplasm of a target eukaryotic cell. This multi-protein channel crosses two bacterial membranes and extends further as a needle through which the proteins travel. We show in this work that PscI, proposed to form the T3S system (T3SS) inner rod, possesses intrinsic properties to polymerize into flexible and regularly twisted fibrils and activates IL-1β production in mouse bone marrow macrophages in vitro. We also found that point mutations within C-terminal amphipathic helix of PscI alter needle assembly in vitro and T3SS function in cell infection assays, suggesting that this region is essential for an efficient needle assembly. The overexpression of PscF partially compensates for the absence of the inner rod in PscI-deficient mutant by forming a secretion-proficient injectisome. All together, we propose that the polymerized PscI in P. aeruginosa optimizes the injectisome function by anchoring the needle within the envelope-embedded complex of the T3S secretome and - contrary to its counterpart in Salmonella - is not involved in substrate switching. PMID:25614137

  2. A biochemical analysis of the constraints of tail-anchored protein biogenesis

    PubMed Central

    Leznicki, Pawel; Warwicker, Jim; High, Stephen

    2011-01-01

    TA (tail-anchored) proteins utilize distinct biosynthetic pathways, including TRC40 (transmembrane domain recognition complex of 40 kDa)-mediated, chaperone-dependent and/or unassisted routes to the ER (endoplasmic reticulum) membrane. We have addressed the flexibility of cytosolic components participating in these pathways, and explored the thermodynamic constraints of their membrane insertion, by exploiting recombinant forms of Sec61β and Cytb5 (cytochrome b5) bearing covalent modifications within their TA region. In both cases, efficient membrane insertion relied on cytosolic factors capable of accommodating a surprising range of covalent modifications to the TA region. For Sec61β, we found that both SGTA (small glutamine-rich tetratricopeptide repeat-containing protein α) and TRC40 can bind this substrate with a singly PEGylated TA region. However, by introducing two PEG [poly(ethylene glycol)] moieties, TRC40 binding can be prevented, resulting in a block of subsequent membrane integration. Although TRC40 can bind Sec61β polypeptides singly PEGylated at different locations, membrane insertion is more sensitive to the precise location of PEG attachment. Modelling and experimentation indicate that this post-TRC40 effect results from an increased energetic cost of inserting different PEGylated TA regions into the lipid bilayer. We therefore propose that the membrane integration of TA proteins delivered via TRC40 is strongly dependent upon underlying thermodynamics, and speculate that their insertion is via a phospholipid-mediated process. PMID:21466504

  3. Anchoring of an immunogenic Plasmodium falciparum circumsporozoite protein on the surface of Dictyostelium discoideum.

    PubMed

    Reymond, C D; Beghdadi-Rais, C; Roggero, M; Duarte, E A; Desponds, C; Bernard, M; Groux, D; Matile, H; Bron, C; Corradin, G

    1995-05-26

    The circumsporozoite protein (CSP), a major antigen of Plasmodium falciparum, was expressed in the slime mold Dictyostelium discoideum. Fusion of the parasite protein to a leader peptide derived from Dictyostelium contact site A was essential for expression. The natural parasite surface antigen, however, was not detected at the slime mold cell surface as expected but retained intracellularly. Removal of the last 23 amino acids resulted in secretion of CSP, suggesting that the C-terminal segment of the CSP, rather than an ectoplasmic domain, was responsible for retention. Cell surface expression was obtained when the CSP C-terminal segment was replaced by the D. discoideum contact site A glycosyl phosphatidylinositol anchor signal sequence. Mice were immunized with Dictyostelium cells harboring CSP at their surface. The raised antibodies recognized two different regions of the CSP. Anti-sporozoite titers of these sera were equivalent to anti-peptide titers detected by enzyme-linked immunosorbent assay. Thus, cell surface targeting of antigens can be obtained in Dictyostelium, generating sporozoite-like cells having potentials for vaccination, diagnostic tests, or basic studies involving parasite cell surface proteins. PMID:7759554

  4. SMN affects membrane remodelling and anchoring of the protein synthesis machinery.

    PubMed

    Gabanella, Francesca; Pisani, Cinzia; Borreca, Antonella; Farioli-Vecchioli, Stefano; Ciotti, Maria Teresa; Ingegnere, Tiziano; Onori, Annalisa; Ammassari-Teule, Martine; Corbi, Nicoletta; Canu, Nadia; Monaco, Lucia; Passananti, Claudio; Di Certo, Maria Grazia

    2016-02-15

    Disconnection between membrane signalling and actin networks can have catastrophic effects depending on cell size and polarity. The survival motor neuron (SMN) protein is ubiquitously involved in assembly of spliceosomal small nuclear ribonucleoprotein particles. Other SMN functions could, however, affect cellular activities driving asymmetrical cell surface expansions. Genes able to mitigate SMN deficiency operate within pathways in which SMN can act, such as mRNA translation, actin network and endocytosis. Here, we found that SMN accumulates at membrane protrusions during the dynamic rearrangement of the actin filaments. In addition to localization data, we show that SMN interacts with caveolin-1, which mediates anchoring of translation machinery components. Importantly, SMN deficiency depletes the plasma membrane of ribosomes, and this correlates with the failure of fibroblasts to extend membrane protrusions. These findings strongly support a relationship between SMN and membrane dynamics. We propose that SMN could assembly translational platforms associated with and governed by the plasma membrane. This activity could be crucial in cells that have an exacerbated interdependence of membrane remodelling and local protein synthesis. PMID:26743087

  5. A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division

    PubMed Central

    Manor, Uri; Bartholomew, Sadie; Golani, Gonen; Christenson, Eric; Kozlov, Michael; Higgs, Henry; Spudich, James; Lippincott-Schwartz, Jennifer

    2015-01-01

    Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division. DOI: http://dx.doi.org/10.7554/eLife.08828.001 PMID:26305500

  6. The naturally competent strain Streptococcus thermophilus LMD-9 as a new tool to anchor heterologous proteins on the cell surface

    PubMed Central

    2014-01-01

    Background From fundamental studies to industrial processes, synthesis of heterologous protein by micro-organisms is widely employed. The secretion of soluble heterologous proteins in the extracellular medium facilitates their recovery, while their attachment to the cell surface permits the use of the recombinant host cells as protein or peptide supports. One of the key points to carry out heterologous expression is to choose the appropriate host. We propose to enlarge the panel of heterologous secretion hosts by using Streptococcus thermophilus LMD-9. This lactic acid bacterium has a generally recognised as safe status, is widely used in the manufacture of yogurts, fermented milks and cheeses, and is easy to transform by natural competence. This study demonstrates the feasibility of secretion of a heterologous protein anchored to the cell surface by S. thermophilus. For this, we used the cell envelope proteinase (CEP) PrtH of Lactobacillus helveticus CNRZ32 CIRM-BIA 103. Results Using S. thermophilus LMD-9 as the background host, three recombinant strains were constructed: i) a negative control corresponding to S. thermophilus PrtS- mutant where the prtS gene encoding its CEP was partially deleted; ii) a PrtH+ mutant expressing the L. helveticus PrtH pro-protein with its own motif (S-layer type) of cell-wall attachment and iii) a PrtH+WANS mutant expressing PrtH pro-protein with the LPXTG anchoring motif from PrtS. The PrtH + and PrtH + WANS genes expression levels were measured by RT-qPCR in the corresponding mutants and compared to that of prtS gene in the strain LMD-9. The expression levels of both fused prtH CEPs genes, regardless of the anchoring motif, reached up-to more than 76% of the wild-type prtS expression level. CEPs were sought and identified on the cell surface of LMD-9 wild-type strain, PrtH+ and PrtH+WANS mutants using shaving technique followed by peptide identification with tandem mass spectrometry, demonstrating that the heterologous secretion

  7. A Novel CXCL10-Based GPI-Anchored Fusion Protein as Adjuvant in NK-Based Tumor Therapy

    PubMed Central

    Muenchmeier, Niklas; Boecker, Sophia; Bankel, Lorenz; Hinz, Laura; Rieth, Nicole; Lapa, Constantin; Mendler, Anna N.; Noessner, Elfriede

    2013-01-01

    Background Cellular therapy is a promising therapeutic strategy for malignant diseases. The efficacy of this therapy can be limited by poor infiltration of the tumor by immune effector cells. In particular, NK cell infiltration is often reduced relative to T cells. A novel class of fusion proteins was designed to enhance the recruitment of specific leukocyte subsets based on their expression of a given chemokine receptor. The proteins are composed of an N-terminal chemokine head, the mucin domain taken from the membrane-anchored chemokine CX3CL1, and a C-terminal glycosylphosphatidylinositol (GPI) membrane anchor replacing the normal transmembrane domain allowing integration of the proteins into cell membranes when injected into a solid tumor. The mucin domain in conjunction with the chemokine head acts to specifically recruit leukocytes expressing the corresponding chemokine receptor. Methodology/Principal Findings A fusion protein comprising a CXCL10 chemokine head (CXCL10-mucin-GPI) was used for proof of concept for this approach and expressed constitutively in Chinese Hamster Ovary cells. FPLC was used to purify proteins. The recombinant proteins efficiently integrated into cell membranes in a process dependent upon the GPI anchor and were able to activate the CXCR3 receptor on lymphocytes. Endothelial cells incubated with CXCL10-mucin-GPI efficiently recruited NK cells in vitro under conditions of physiologic flow, which was shown to be dependent on the presence of the mucin domain. Experiments conducted in vivo using established tumors in mice suggested a positive effect of CXCL10-mucin-GPI on the recruitment of NK cells. Conclusions The results suggest enhanced recruitment of NK cells by CXCL10-mucin-GPI. This class of fusion proteins represents a novel adjuvant in cellular immunotherapy. The underlying concept of a chemokine head fused to the mucin domain and a GPI anchor signal sequence may be expanded into a broader family of reagents that will allow

  8. Rapid and serum-insensitive endocytotic delivery of proteins using biotinylated polymers attached via multivalent hydrophobic anchors.

    PubMed

    Tobinaga, Kyohei; Li, Cuicui; Takeo, Masafumi; Matsuda, Masayoshi; Nagai, Hiroko; Niidome, Takuro; Yamamoto, Tatsuhiro; Kishimura, Akihiro; Mori, Takeshi; Katayama, Yoshiki

    2014-03-10

    We have designed biotinylated polymers as synthetic receptors that have multiple alkyl groups for endocytotic delivery of target proteins. The polymers were stably attached to a cell surface via multivalent anchoring. The presented biotin was bound to streptavidin (SA) on the cell surface, and, via an endocytotic pathway, the cell rapidly internalized the biotinylated polymer/SA complex. The cell's uptake of the complex was not inhibited by the presence of 10% fetal bovine serum, and its efficacy for the uptake of SA was the highest when compared with commercial reagents and single-anchored-type synthetic receptors. The synthetic receptor-mediated endocytosis can be used generally for other kind of protein by using SA as an adaptor molecule between a target protein and the cell-surface presented biotin. PMID:24389131

  9. Msp1/ATAD1 maintains mitochondrial function by facilitating the degradation of mislocalized tail-anchored proteins

    PubMed Central

    Chen, Yu-Chan; Umanah, George K E; Dephoure, Noah; Andrabi, Shaida A; Gygi, Steven P; Dawson, Ted M; Dawson, Valina L; Rutter, Jared

    2014-01-01

    The majority of ER-targeted tail-anchored (TA) proteins are inserted into membranes by the Guided Entry of Tail-anchored protein (GET) system. Disruption of this system causes a subset of TA proteins to mislocalize to mitochondria. We show that the AAA+ ATPase Msp1 limits the accumulation of mislocalized TA proteins on mitochondria. Deletion of MSP1 causes the Pex15 and Gos1 TA proteins to accumulate on mitochondria when the GET system is impaired. Likely as a result of failing to extract mislocalized TA proteins, yeast with combined mutation of the MSP1 gene and the GET system exhibit strong synergistic growth defects and severe mitochondrial damage, including loss of mitochondrial DNA and protein and aberrant mitochondrial morphology. Like yeast Msp1, human ATAD1 limits the mitochondrial mislocalization of PEX26 and GOS28, orthologs of Pex15 and Gos1, respectively. GOS28 protein level is also increased in ATAD1−/− mouse tissues. Therefore, we propose that yeast Msp1 and mammalian ATAD1 are conserved members of the mitochondrial protein quality control system that might promote the extraction and degradation of mislocalized TA proteins to maintain mitochondrial integrity. PMID:24843043

  10. Binding Preferences for GPIHBP1, a GPI-Anchored Protein of Capillary Endothelial Cells

    PubMed Central

    Gin, Peter; Beigneux, Anne P.; Voss, Constance; Davies, Brandon S. J.; Beckstead, Jennifer A.; Ryan, Robert O.; Bensadoun, Andre; Fong, Loren G.; Young, Stephen G.

    2010-01-01

    Objective GPIHBP1, a glycosylphosphatidylinositol-anchored Ly6 protein of capillary endothelial cells, binds lipoprotein lipase (LPL) avidly, but its ability to bind related lipase family members has never been evaluated. We sought to define the ability of GPIHBP1 to bind other lipase family members as well as other apolipoproteins and lipoproteins. Methods and Results As judged by cell-based and cell-free binding assays, LPL binds to GPIHBP1 but other members of the lipase family do not. We also examined the binding of apoAV–phospholipid disks to GPIHBP1. ApoAV binds avidly to GPIHBP1-transfected cells; this binding requires GPIHBP1’s amino-terminal acidic domain and is independent of its cysteine-rich Ly6 domain (the latter domain is essential for LPL binding). GPIHBP1-transfected cells did not bind HDL. Chylomicrons binds avidly to GPIHBP1-transfected CHO cells, but this binding is dependent on GPIHBP1’s ability to bind LPL within the cell culture medium. Conclusions GPIHBP1 binds LPL but does not bind other lipase family members. GPIHBP1 binds apoAV but did not bind apoAI or HDL. The ability of GPIHBP1-transfected CHO cells to bind chylomicrons is mediated by LPL; chylomicron binding does not occur unless GPIHBP1 first captures LPL from the cell culture medium. PMID:20966398

  11. A novel import route for an N-anchor mitochondrial outer membrane protein aided by the TIM23 complex.

    PubMed

    Song, Jiyao; Tamura, Yasushi; Yoshihisa, Tohru; Endo, Toshiya

    2014-06-01

    The membrane topology of Om45 in the yeast mitochondrial outer membrane (OM) is under debate. Here, we confirm that Om45 is anchored to the OM from the intermembrane space (IMS) by its N-terminal hydrophobic segment. We show that import of Om45 requires the presequence receptors, Tom20 and Tom22, and the import channel of Tom40. Unlike any of the known OM proteins, Om45 import requires the TIM23 complex in the inner membrane, a translocator for presequence-containing proteins, and the membrane potential (ΔΨ). Therefore, Om45 is anchored to the OM via the IMS by a novel import pathway involving the TIM23 complex. PMID:24781694

  12. Effects of Detergents on the Redistribution of Gangliosides and GPI-anchored Proteins in Brain Tissue Sections

    PubMed Central

    Heffer-Lauc, Marija; Viljetiæ, Barbara; Vajn, Katarina; Schnaar, Ronald L.; Lauc, Gordan

    2008-01-01

    SUMMARY Gangliosides and glycosylphosphatidylinositol (GPI)-anchored proteins contain lipid tails that tether them to the outer side of the cell membrane. This mode of association with the cell membrane enables them to take part in the organization of lipid rafts, but it also permits gangliosides and GPI-anchored proteins to be actively released from one cell and inserted into the membrane of another cell. Recently, we reported that under conditions of lipid raft isolation, Triton X-100 causes significant redistribution of both gangliosides and GPI-anchored proteins. Aiming to find a less disruptive detergent, we evaluated the effects of CHAPS, Saponin, deoxycholic acid, Trappsol, Tween 20, Triton X-100, Brij 96V, Brij 98, and SDS on brain tissue sections. At room temperature, all detergents (1% concentration) extracted significant amounts of both gangliosides and Thy-1. At 4C, the extraction was weaker, but Triton X-100, CHAPS, and deoxycholic acid caused significant redistribution of GD1a and Thy-1 from gray matter into the white matter. Both redistribution and extraction were significantly augmented when sections were incubated with detergents in the presence of primary antibodies. Of the nine tested detergents, none is the ideal choice. However, Brij 96V appears to be able to sufficiently reveal myelin epitopes while causing the least amount of artifacts. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. PMID:17409378

  13. Tail-anchored Protein Insertion in Mammals: FUNCTION AND RECIPROCAL INTERACTIONS OF THE TWO SUBUNITS OF THE TRC40 RECEPTOR.

    PubMed

    Colombo, Sara Francesca; Cardani, Silvia; Maroli, Annalisa; Vitiello, Adriana; Soffientini, Paolo; Crespi, Arianna; Bram, Richard F; Benfante, Roberta; Borgese, Nica

    2016-07-15

    The GET (guided entry of tail-anchored proteins)/TRC (transmembrane recognition complex) pathway for tail-anchored protein targeting to the endoplasmic reticulum (ER) has been characterized in detail in yeast and is thought to function similarly in mammals, where the orthologue of the central ATPase, Get3, is known as TRC40 or Asna1. Get3/TRC40 function requires an ER receptor, which in yeast consists of the Get1/Get2 heterotetramer and in mammals of the WRB protein (tryptophan-rich basic protein), homologous to yeast Get1, in combination with CAML (calcium-modulating cyclophilin ligand), which is not homologous to Get2. To better characterize the mammalian receptor, we investigated the role of endogenous WRB and CAML in tail-anchored protein insertion as well as their association, concentration, and stoichiometry in rat liver microsomes and cultured cells. Functional proteoliposomes, reconstituted from a microsomal detergent extract, lost their activity when made with an extract depleted of TRC40-associated proteins or of CAML itself, whereas in vitro synthesized CAML and WRB together were sufficient to confer insertion competence to liposomes. CAML was found to be in ∼5-fold excess over WRB, and alteration of this ratio did not inhibit insertion. Depletion of each subunit affected the levels of the other one; in the case of CAML silencing, this effect was attributable to destabilization of the WRB transcript and not of WRB protein itself. These results reveal unanticipated complexity in the mutual regulation of the TRC40 receptor subunits and raise the question as to the role of the excess CAML in the mammalian ER. PMID:27226539

  14. Molecular insights into DNA binding and anchoring by the Bacillus subtilis sporulation kinetochore-like RacA protein.

    PubMed

    Schumacher, Maria A; Lee, Jeehyun; Zeng, Wenjie

    2016-06-20

    During Bacillus subtilis sporulation, segregating sister chromosomes are anchored to cell poles and the chromosome is remodeled into an elongated structure called the axial filament. Data indicate that a developmentally regulated protein called RacA is involved in these functions. To gain insight into how RacA performs these diverse processes we performed a battery of structural and biochemical analyses. These studies show that RacA contains an N-terminal winged-helix-turn-helix module connected by a disordered region to a predicted coiled-coil domain. Structures capture RacA binding the DNA using distinct protein-protein interfaces and employing adjustable DNA docking modes. This unique DNA binding mechanism indicates how RacA can both specifically recognize its GC-rich centromere and also non-specifically bind the DNA. Adjacent RacA molecules within the protein-DNA structure interact leading to DNA compaction, suggesting a mechanism for axial filament formation. We also show that the RacA C-domain coiled coil directly contacts the coiled coil region of the polar protein DivIVA, which anchors RacA and hence the chromosome to the pole. Thus, our combined data reveal unique DNA binding properties by RacA and provide insight into the DNA remodeling and polar anchorage functions of the protein. PMID:27085804

  15. Fatal Transmissible Amyloid Encephalopathy: A New Type of Prion Disease Associated with Lack of Prion Protein Membrane Anchoring

    PubMed Central

    Chesebro, Bruce; Race, Brent; Meade-White, Kimberly; LaCasse, Rachel; Race, Richard; Klingeborn, Mikael; Striebel, James; Dorward, David; McGovern, Gillian; Jeffrey, Martin

    2010-01-01

    Prion diseases are fatal neurodegenerative diseases of humans and animals characterized by gray matter spongiosis and accumulation of aggregated, misfolded, protease-resistant prion protein (PrPres). PrPres can be deposited in brain in an amyloid-form and/or non-amyloid form, and is derived from host-encoded protease-sensitive PrP (PrPsen), a protein normally anchored to the plasma membrane by glycosylphosphatidylinositol (GPI). Previously, using heterozygous transgenic mice expressing only anchorless PrP, we found that PrP anchoring to the cell membrane was required for typical clinical scrapie. However, in the present experiments, using homozygous transgenic mice expressing two-fold more anchorless PrP, scrapie infection induced a new fatal disease with unique clinical signs and altered neuropathology, compared to non-transgenic mice expressing only anchored PrP. Brain tissue of transgenic mice had high amounts of infectivity, and histopathology showed dense amyloid PrPres plaque deposits without gray matter spongiosis. In contrast, infected non-transgenic mice had diffuse non-amyloid PrPres deposits with significant gray matter spongiosis. Brain graft studies suggested that anchored PrPsen expression was required for gray matter spongiosis during prion infection. Furthermore, electron and light microscopic studies in infected transgenic mice demonstrated several pathogenic processes not seen in typical prion disease, including cerebral amyloid angiopathy and ultrastructural alterations in perivascular neuropil. These findings were similar to certain human familial prion diseases as well as to non-prion human neurodegenerative diseases, such as Alzheimer's disease. PMID:20221436

  16. Structural and genetic analyses reveal the protein SepF as a new membrane anchor for the Z ring

    PubMed Central

    Duman, Ramona; Ishikawa, Shu; Celik, Ilkay; Strahl, Henrik; Ogasawara, Naotake; Troc, Paulina; Löwe, Jan; Hamoen, Leendert W.

    2013-01-01

    A key step in bacterial cell division is the polymerization of the tubulin homolog FtsZ at midcell. FtsZ polymers are anchored to the cell membrane by FtsA and are required for the assembly of all other cell division proteins. In Gram-positive and cyanobacteria, FtsZ filaments are aligned by the protein SepF, which in vitro polymerizes into large rings that bundle FtsZ filaments. Here we describe the crystal structure of the only globular domain of SepF, located within the C-terminal region. Two-hybrid data revealed that this domain comprises the FtsZ binding site, and EM analyses showed that it is sufficient for ring formation, which is explained by the filaments in the crystals of SepF. Site-directed mutagenesis, gel filtration, and analytical ultracentrifugation indicated that dimers form the basic units of SepF filaments. High-resolution structured illumination microscopy suggested that SepF is membrane associated, and it turned out that purified SepF not only binds to lipid membranes, but also recruits FtsZ. Further genetic and biochemical analyses showed that an amphipathic helix at the N terminus functions as the membrane-binding domain, making SepF a unique membrane anchor for the FtsZ ring. This clarifies why Bacillus subtilis grows without FtsA or the putative membrane anchor EzrA and why bacteria lacking FtsA contain SepF homologs. Both FtsA and SepF use an amphipathic helix for membrane binding. These helices prefer positively curved membranes due to relaxed lipid density; therefore this type of membrane anchor may assist in keeping the Z ring positioned at the strongly curved leading edge of the developing septum. PMID:24218584

  17. The 4.1 Protein Coracle Mediates Subunit-Selective Anchoring of Drosophila Glutamate Receptors to the Postsynaptic Actin Cytoskeleton

    PubMed Central

    Chen, Kaiyun; Merino, Carlos; Sigrist, Stephan J.; Featherstone, David E.

    2005-01-01

    Glutamatergic Drosophila neuromuscular junctions contain two spatially, biophysically, and pharmacologically distinct subtypes of postsynaptic glutamate receptor (GluR). These receptor subtypes appear to be molecularly identical except that A receptors contain the subunit GluRIIA (but not GluRIIB), and B receptors contain the subunit GluRIIB (but not GluRIIA). A- and B-type receptors are coexpressed in the same cells, in which they form homotypic clusters. During development, A- and B-type receptors can be differentially regulated. The mechanisms that allow differential segregation and regulation of A- and B-type receptors are unknown. Presumably, A-and B-type receptors are differentially anchored to the membrane cytoskeleton, but essentially nothing is known about how Drosophila glutamate receptors are localized or anchored. We identified coracle, a homolog of mammalian brain 4.1 proteins, in yeast two-hybrid and genetic screens for proteins that interact with and localize Drosophila glutamate receptors. Coracle interacts with the C terminus of GluRIIA but not GluRIIB. To test whether coracle is required for glutamate receptor localization, we immunocytochemically and electrophysiologically examined receptors in coracle mutants. In coracle mutants, synaptic A-type receptors are lost, but there is no detectable change in B-type receptor function or localization. Pharmacological disruption of postsynaptic actin phenocopies the coracle mutants, suggesting that A-type receptors are anchored to the actin cytoskeleton via coracle, whereas B-type receptors are anchored at the synapse by another (yet unknown) mechanism. PMID:16014728

  18. Design and characterization of a modular membrane protein anchor to functionalize the moss Physcomitrella patens with extracellular catalytic and/or binding activities.

    PubMed

    Morath, Volker; Truong, Dong-Jiunn Jeffery; Albrecht, Florian; Polte, Ingmar; Ciccone, Rosario Adriano; Funke, Louise Friederike; Reichart, Leonie; Wolf, Christopher Guy; Brunner, Andreas-David; Fischer, Katrin; Schneider, Philipp Constantin; Brüggenthies, Johanna Barbara; Fröhlich, Fabian; Wiedemann, Gertrud; Reski, Ralf; Skerra, Arne

    2014-12-19

    Heterologous enzymes and binding proteins were secreted by the moss Physcomitrella patens or anchored extracellularly on its cell membrane in order to functionalize the apoplast as a biochemical reaction compartment. This modular membrane anchoring system utilizes the signal peptide and the transmembrane segment of the somatic embryogenesis receptor-like kinase (SERK), which were identified in a comprehensive bioinformatic analysis of the P. patens genome. By fusing the soluble enzyme NanoLuc luciferase to the signal peptide, its secretion capability was confirmed in vivo. The membrane localization of hybrid proteins comprising the SERK signal peptide, NanoLuc or other functional modules, the SERK transmembrane anchor, and a C-terminal GFP reporter was demonstrated using fluorescence microscopy as well as site-specific proteolytic release of the extracellular enzyme domain. Our membrane anchoring system enables the expression of various functional proteins in the apoplast of P. patens, empowering this photoautotrophic organism for biotechnological applications. PMID:25524107

  19. Anchoring tick salivary anti-complement proteins IRAC I and IRAC II to membrane increases their immunogenicity.

    PubMed

    Gillet, Laurent; Schroeder, Hélène; Mast, Jan; Thirion, Muriel; Renauld, Jean-Christophe; Dewals, Benjamin; Vanderplasschen, Alain

    2009-01-01

    Tick salivary proteins are promising targets for the development of anti-tick vaccines. Recently, we described two paralogous anti-complement proteins, called Ixodes ricinus anti-complement (IRAC) proteins I and II, that are co-expressed in tick I. ricinus salivary glands. However, our previous attempts to immunize rabbits against IRAC via infection with recombinant Bovine herpesvirus 4 (BoHV-4) vectors invariably failed although both recombinants expressed high levels of functional IRAC proteins in vitro. As IRAC are soluble monovalent antigens, one of the possible explanations is that monovalent ligation of the B-cell receptor induces receptor activation but fails to promote antigen presentation, a phenomenon that is thought to induce a state of B-cell tolerance. In the present study, we tried to increase IRAC immunogenicity by expressing them as oligovalent antigens. To this end, IRAC were fused to membrane anchors and BoHV-4 vectors expressing these recombinant forms were produced. The immunization potentials of recombinant viruses expressing either secreted or transmembrane IRAC proteins were then compared. While the former did not induce a detectable immune response against IRAC, the latter led to high titres of anti-IRAC antibodies that only marginally affected tick blood feeding. All together, the data presented in this study demonstrate that the immunogenicity of a soluble antigen can be greatly improved by anchoring it in membrane. PMID:19531344

  20. Growth factor-induced activation of a kinase activity which causes regulatory phosphorylation of p42/microtubule-associated protein kinase.

    PubMed Central

    L'Allemain, G; Her, J H; Wu, J; Sturgill, T W; Weber, M J

    1992-01-01

    p42/microtubule-associated protein kinase (p42mapk) is activated by tyrosine and threonine phosphorylation, and its regulatory phosphorylation is likely to be important in signalling pathways involved in growth control, secretion, and differentiation. Here we show that treatment of quiescent 3T3 cells with diverse agonists results in the appearance of an activity capable of causing the in vitro phosphorylation of p42mapk on the regulatory tyrosine and to a lesser extent on the regulatory threonine, resulting in enzymatic activation of the p42mapk. This p42mapk-activating activity is capable of phosphorylating a kinase-defective p42mapk mutant, thus confirming its activity as a kinase. Images PMID:1314951

  1. A C-terminal Membrane Anchor Affects the Interactions of Prion Proteins with Lipid Membranes*

    PubMed Central

    Chu, Nam K.; Shabbir, Waheed; Bove-Fenderson, Erin; Araman, Can; Lemmens-Gruber, Rosa; Harris, David A.; Becker, Christian F. W.

    2014-01-01

    Membrane attachment via a C-terminal glycosylphosphatidylinositol anchor is critical for conversion of PrPC into pathogenic PrPSc. Therefore the effects of the anchor on PrP structure and function need to be deciphered. Three PrP variants, including full-length PrP (residues 23–231, FL_PrP), N-terminally truncated PrP (residues 90–231, T_PrP), and PrP missing its central hydrophobic region (Δ105–125, ΔCR_PrP), were equipped with a C-terminal membrane anchor via a semisynthesis strategy. Analyses of the interactions of lipidated PrPs with phospholipid membranes demonstrated that C-terminal membrane attachment induces a different binding mode of PrP to membranes, distinct from that of non-lipidated PrPs, and influences the biochemical and conformational properties of PrPs. Additionally, fluorescence-based assays indicated pore formation by lipidated ΔCR_PrP, a variant that is known to be highly neurotoxic in transgenic mice. This finding was supported by using patch clamp electrophysiological measurements of cultured cells. These results provide new evidence for the role of the membrane anchor in PrP-lipid interactions, highlighting the importance of the N-terminal and the central hydrophobic domain in these interactions. PMID:25217642

  2. A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins

    PubMed Central

    Ghamrawi, Sarah; Gastebois, Amandine; Zykwinska, Agata; Vandeputte, Patrick; Marot, Agnès; Mabilleau, Guillaume; Cuenot, Stéphane; Bouchara, Jean-Philippe

    2015-01-01

    Scedosporium boydii is a pathogenic filamentous fungus that causes a wide range of human infections, notably respiratory infections in patients with cystic fibrosis. The development of new therapeutic strategies targeting S. boydii necessitates a better understanding of the physiology of this fungus and the identification of new molecular targets. In this work, we studied the conidium-to-germ tube transition using a variety of techniques including scanning and transmission electron microscopy, atomic force microscopy, two-phase partitioning, microelectrophoresis and cationized ferritin labeling, chemical force spectroscopy, lectin labeling, and nanoLC-MS/MS for cell wall GPI-anchored protein analysis. We demonstrated that the cell wall undergoes structural changes with germination accompanied with a lower hydrophobicity, electrostatic charge and binding capacity to cationized ferritin. Changes during germination also included a higher accessibility of some cell wall polysaccharides to lectins and less CH3/CH3 interactions (hydrophobic adhesion forces mainly due to glycoproteins). We also extracted and identified 20 GPI-anchored proteins from the cell wall of S. boydii, among which one was detected only in the conidial wall extract and 12 only in the mycelial wall extract. The identified sequences belonged to protein families involved in virulence in other fungi like Gelp/Gasp, Crhp, Bglp/Bgtp families and a superoxide dismutase. These results highlighted the cell wall remodeling during germination in S. boydii with the identification of a substantial number of cell wall GPI-anchored conidial or hyphal specific proteins, which provides a basis to investigate the role of these molecules in the host-pathogen interaction and fungal virulence. PMID:26038837

  3. Temperature-induced alteration of inositolphosphorylceramides in the putative glycosylated lipid precursors of Tetrahymena mimbres glycosylphosphatidylinositol-anchored proteins.

    PubMed Central

    Hung, C Y; Ko, Y G; Thompson, G A

    1995-01-01

    Tetrahymena species contain relatively prominent glycosylphosphatidylinositol (GPI)-anchored proteins as well as their putative precursor phosphatidylinositol (PI) glycans. We have characterized the lipid components of the two principal T. mimbres PI glycans. Following their purification by preparative TLC, the PI glycans were hydrolysed in methanolic HCl or NaOH, and resulting lipids were analysed by chromatography and mass spectrometry. The two PI glycans contained nearly identical lipid moieties having long-chain bases with N-linked fatty acids. The predominant long-chain base, 3-O-methylsphinganine, was first assumed to be O-methylated as an artifact of hydrolysis, but subsequently, on the basis of control experiments, it was shown to be naturally occurring. PI glycans from cells grown at 28 degrees C contained primarily palmitic acid (79%) and some stearic acid (11%), whereas the principal PI glycan from 38 degrees C-grown T. mimbres contained 65% stearic acid. In 15 degrees C-grown cells stearic acid accounted for only 2% of ceramide-bound fatty acids and was almost totally replaced by palmitic acid (95%). The distributions of fatty acids bound to T. mimbres GPI-anchored proteins [Ko, Hung and Thompson (1995) Biochem. J. 307, 115-121] were similar but not identical to those of the PI glycans described here. Temperature-induced specification of the lipid components of mature T. mimbres GPI-anchored proteins appears to be established both at the level of PI-glycan synthesis and the level of PI-glycan utilization for protein attachment. PMID:7717964

  4. A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins.

    PubMed

    Ghamrawi, Sarah; Gastebois, Amandine; Zykwinska, Agata; Vandeputte, Patrick; Marot, Agnès; Mabilleau, Guillaume; Cuenot, Stéphane; Bouchara, Jean-Philippe

    2015-01-01

    Scedosporium boydii is a pathogenic filamentous fungus that causes a wide range of human infections, notably respiratory infections in patients with cystic fibrosis. The development of new therapeutic strategies targeting S. boydii necessitates a better understanding of the physiology of this fungus and the identification of new molecular targets. In this work, we studied the conidium-to-germ tube transition using a variety of techniques including scanning and transmission electron microscopy, atomic force microscopy, two-phase partitioning, microelectrophoresis and cationized ferritin labeling, chemical force spectroscopy, lectin labeling, and nanoLC-MS/MS for cell wall GPI-anchored protein analysis. We demonstrated that the cell wall undergoes structural changes with germination accompanied with a lower hydrophobicity, electrostatic charge and binding capacity to cationized ferritin. Changes during germination also included a higher accessibility of some cell wall polysaccharides to lectins and less CH3/CH3 interactions (hydrophobic adhesion forces mainly due to glycoproteins). We also extracted and identified 20 GPI-anchored proteins from the cell wall of S. boydii, among which one was detected only in the conidial wall extract and 12 only in the mycelial wall extract. The identified sequences belonged to protein families involved in virulence in other fungi like Gelp/Gasp, Crhp, Bglp/Bgtp families and a superoxide dismutase. These results highlighted the cell wall remodeling during germination in S. boydii with the identification of a substantial number of cell wall GPI-anchored conidial or hyphal specific proteins, which provides a basis to investigate the role of these molecules in the host-pathogen interaction and fungal virulence. PMID:26038837

  5. Modification-specific proteomics of plasma membrane proteins: identification and characterization of glycosylphosphatidylinositol-anchored proteins released upon phospholipase D treatment.

    PubMed

    Elortza, Felix; Mohammed, Shabaz; Bunkenborg, Jakob; Foster, Leonard J; Nühse, Thomas S; Brodbeck, Urs; Peck, Scott C; Jensen, Ole N

    2006-04-01

    Plasma membrane proteins are displayed through diverse mechanisms, including anchoring in the extracellular leaflet via glycosylphosphatidylinositol (GPI) molecules. GPI-anchored membrane proteins (GPI-APs) are a functionally and structurally diverse protein family, and their importance is well-recognized as they are candidate cell surface biomarker molecules with potential diagnostic and therapeutic applications in molecular medicine. GPI-APs have also attracted interest in plant biotechnology because of their role in root development and cell remodeling. Using a shave-and-conquer concept, we demonstrate that phospholipase D (PLD) treatment of human and plant plasma membrane fractions leads to the release of GPI-anchored proteins that were identified and characterized by capillary liquid chromatography and tandem mass spectrometry. In contrast to phospholipase C, the PLD enzyme is not affected by structural heterogeneity of the GPI moiety, making PLD a generally useful reagent for proteomic investigations of GPI-anchored proteins in a variety of cells, tissues, and organisms. A total of 11 human GPI-APs and 35 Arabidopsis thaliana GPI-APs were identified, representing a significant addition to the number of experimentally detected GPI-APs in both species. Computational GPI-AP sequence analysis tools were investigated for the characterization of the identified GPI-APs, and these demonstrated that there is some discrepancy in their efficiency in classification of GPI-APs and the exact assignment of omega-sites. This study highlights the efficiency of an integrative proteomics approach that combines experimental and computational methods to provide the selectivity, specificity, and sensitivity required for characterization of post-translationally modified membrane proteins. PMID:16602701

  6. Dynamin-related Protein 1 Oligomerization in Solution Impairs Functional Interactions with Membrane-anchored Mitochondrial Fission Factor.

    PubMed

    Clinton, Ryan W; Francy, Christopher A; Ramachandran, Rajesh; Qi, Xin; Mears, Jason A

    2016-01-01

    Mitochondrial fission is a crucial cellular process mediated by the mechanoenzymatic GTPase, dynamin-related protein 1 (Drp1). During mitochondrial division, Drp1 is recruited from the cytosol to the outer mitochondrial membrane by one, or several, integral membrane proteins. One such Drp1 partner protein, mitochondrial fission factor (Mff), is essential for mitochondrial division, but its mechanism of action remains unexplored. Previous studies have been limited by a weak interaction between Drp1 and Mff in vitro. Through refined in vitro reconstitution approaches and multiple independent assays, we show that removal of the regulatory variable domain (VD) in Drp1 enhances formation of a functional Drp1-Mff copolymer. This protein assembly exhibits greatly stimulated cooperative GTPase activity in solution. Moreover, when Mff was anchored to a lipid template, to mimic a more physiologic environment, significant stimulation of GTPase activity was observed with both WT and ΔVD Drp1. Contrary to recent findings, we show that premature Drp1 self-assembly in solution impairs functional interactions with membrane-anchored Mff. Instead, dimeric Drp1 species are selectively recruited by Mff to initiate assembly of a functional fission complex. Correspondingly, we also found that the coiled-coil motif in Mff is not essential for Drp1 interactions, but rather serves to augment cooperative self-assembly of Drp1 proximal to the membrane. Taken together, our findings provide a mechanism wherein the multimeric states of both Mff and Drp1 regulate their collaborative interaction. PMID:26578514

  7. Molecular insights into DNA binding and anchoring by the Bacillus subtilis sporulation kinetochore-like RacA protein

    PubMed Central

    Schumacher, Maria A.; Lee, Jeehyun; Zeng, Wenjie

    2016-01-01

    During Bacillus subtilis sporulation, segregating sister chromosomes are anchored to cell poles and the chromosome is remodeled into an elongated structure called the axial filament. Data indicate that a developmentally regulated protein called RacA is involved in these functions. To gain insight into how RacA performs these diverse processes we performed a battery of structural and biochemical analyses. These studies show that RacA contains an N-terminal winged-helix-turn-helix module connected by a disordered region to a predicted coiled-coil domain. Structures capture RacA binding the DNA using distinct protein–protein interfaces and employing adjustable DNA docking modes. This unique DNA binding mechanism indicates how RacA can both specifically recognize its GC-rich centromere and also non-specifically bind the DNA. Adjacent RacA molecules within the protein–DNA structure interact leading to DNA compaction, suggesting a mechanism for axial filament formation. We also show that the RacA C-domain coiled coil directly contacts the coiled coil region of the polar protein DivIVA, which anchors RacA and hence the chromosome to the pole. Thus, our combined data reveal unique DNA binding properties by RacA and provide insight into the DNA remodeling and polar anchorage functions of the protein. PMID:27085804

  8. C lostridium difficile surface proteins are anchored to the cell wall using CWB2 motifs that recognise the anionic polymer PSII

    PubMed Central

    Willing, Stephanie E.; Candela, Thomas; Shaw, Helen Alexandra; Seager, Zoe; Mesnage, Stéphane; Fagan, Robert P.

    2015-01-01

    Summary Gram‐positive surface proteins can be covalently or non‐covalently anchored to the cell wall and can impart important properties on the bacterium in respect of cell envelope organisation and interaction with the environment. We describe here a mechanism of protein anchoring involving tandem CWB2 motifs found in a large number of cell wall proteins in the Firmicutes. In the Clostridium difficile cell wall protein family, we show the three tandem repeats of the CWB2 motif are essential for correct anchoring to the cell wall. CWB2 repeats are non‐identical and cannot substitute for each other, as shown by the secretion into the culture supernatant of proteins containing variations in the patterns of repeats. A conserved Ile Leu Leu sequence within the CWB2 repeats is essential for correct anchoring, although a preceding proline residue is dispensable. We propose a likely genetic locus encoding synthesis of the anionic polymer PSII and, using RNA knock‐down of key genes, reveal subtle effects on cell wall composition. We show that the anionic polymer PSII binds two cell wall proteins, SlpA and Cwp2, and these interactions require the CWB2 repeats, defining a new mechanism of protein anchoring in Gram‐positive bacteria. PMID:25649385

  9. Creating Order from Chaos: Cellular Regulation by Kinase Anchoring

    PubMed Central

    Scott, John D.; Dessauer, Carmen W.; Tasken, Kjetil

    2012-01-01

    Second messenger responses rely on where and when the enzymes that propagate these signals become active. Spatial and temporal organization of certain signaling enzymes is controlled in part by A-kinase anchoring proteins (AKAPs). This family of regulatory proteins was originally classified on the basis of their ability to compartmentalize the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (also known as protein kinase A, or PKA). However, it is now recognized that AKAPs position G protein–coupled receptors, adenylyl cyclases, G proteins, and their effector proteins in relation to protein kinases and signal termination enzymes such as phosphodiesterases and protein phosphatases. This arrangement offers a simple and efficient means to limit the scope, duration, and directional flow of information to sites deep within the cell. This review focuses on the pros and cons of reagents that define the biological role of kinase anchoring inside cells and discusses recent advances in our understanding of anchored second messenger signaling in the cardiovascular and immune systems. PMID:23043438

  10. Mto2p, a novel fission yeast protein required for cytoplasmic microtubule organization and anchoring of the cytokinetic actin ring.

    PubMed

    Venkatram, Srinivas; Jennings, Jennifer L; Link, Andrew; Gould, Kathleen L

    2005-06-01

    Microtubules regulate diverse cellular processes, including chromosome segregation, nuclear positioning, and cytokinesis. In many organisms, microtubule nucleation requires gamma-tubulin and associated proteins present at specific microtubule organizing centers (MTOCs). In fission yeast, interphase cytoplasmic microtubules originate from poorly characterized interphase MTOCs and spindle pole body (SPB), and during late anaphase from the equatorial MTOC (EMTOC). It has been previously shown that Mto1p (Mbo1p/Mod20p) function is important for the organization/nucleation of all cytoplasmic microtubules. Here, we show that Mto2p, a novel protein, interacts with Mto1p and is important for establishing a normal interphase cytoplasmic microtubule array. In addition, mto2Delta cells fail to establish a stable EMTOC and localize gamma-tubulin complex members to this medial structure. As predicted from these functions, Mto2p localizes to microtubules, the SPB, and the EMTOC in an Mto1p-dependent manner. mto2Delta cells fail to anchor the cytokinetic actin ring in the medial region of the cell and under conditions that mildly perturb actin structures, these rings unravel in mto2Delta cells. Our results suggest that the Mto2p and the EMTOC are critical for anchoring the cytokinetic actin ring to the medial region of the cell and for proper coordination of mitosis with cytokinesis. PMID:15800064

  11. Regulated Shift from Helical to Polar Localization of Listeria monocytogenesCell Wall-Anchored Proteins ▿ †

    PubMed Central

    Bruck, Serawit; Personnic, Nicolas; Prevost, Marie-Christine; Cossart, Pascale; Bierne, Hélène

    2011-01-01

    Many virulence factors of Gram-positive bacterial pathogens are covalently anchored to the peptidoglycan (PG) by sortase enzymes. However, for rod-shaped bacteria little is known about the spatiotemporal organization of these surface proteins in the cell wall. Here we report the three-dimensional (3D) localization of the PG-bound virulence factors InlA, InlH, InlJ, and SvpA in the envelope of Listeria monocytogenesunder different growth conditions. We found that all PG-anchored proteins are positioned along the lateral cell wall in nonoverlapping helices. However, these surface proteins can also become localized at the pole and asymmetrically distributed when specific regulatory pathways are activated. InlA and InlJ are enriched at poles when expressed at high levels in exponential-phase bacteria. InlA and InlH, which are σBdependent, specifically relocalize to the septal cell wall and subsequently to the new pole in cells entering stationary phase. The accumulation of InlA and InlH in the septal region also occurs when oxidative stress impairs bacterial growth. In contrast, the iron-dependent protein SvpA is present at the old pole and is excluded from the septum and new pole of bacteria grown under low-iron conditions. We conclude that L. monocytogenesrapidly reorganizes the spatial localization of its PG proteins in response to changes in environmental conditions such as nutrient deprivation or other stresses. This dynamic control would distribute virulence factors at specific sites during the infectious process. PMID:21725001

  12. The Crystal Structures of Yeast Get3 Suggest a Mechanism for Tail-Anchored Protein Membrane Insertion

    SciTech Connect

    Hu, Junbin; Li, Jingzhi; Qian, Xinguo; Denic, Vlad; Sha, Bingdong

    2010-08-16

    Tail-anchored (TA) proteins represent a unique class of membrane proteins that contain a single C-terminal transmembrane helix. The post-translational insertion of the yeast TA proteins into the ER membrane requires the Golgi ER trafficking (GET) complex which contains Get1, Get2 and Get3. Get3 is an ATPase that recognizes and binds the C-terminal transmembrane domain (TMD) of the TA proteins. We have determined the crystal structures of Get3 from two yeast species, S. cerevisiae and D. hansenii, respectively. These high resolution crystal structures show that Get3 contains a nucleotide-binding domain and a 'finger' domain for binding the TA protein TMD. A large hydrophobic groove on the finger domain of S. cerevisiae Get3 structure might represent the binding site for TMD of TA proteins. A hydrophobic helix from a symmetry-related Get3 molecule sits in the TMD-binding groove and mimics the TA binding scenario. Interestingly, the crystal structures of the Get3 dimers from S. cerevisiae and D. hansenii exhibit distinct conformations. The S. cerevisiae Get3 dimer structure does not contain nucleotides and maintains an 'open' conformation, while the D. hansenii Get3 dimer structure binds ADP and stays in a 'closed' conformation. We propose that the conformational changes to switch the Get3 between the open and closed conformations may facilitate the membrane insertions for TA proteins.

  13. Unique domain anchoring of Src to synaptic NMDA receptors via the mitochondrial protein NADH dehydrogenase subunit 2.

    PubMed

    Gingrich, Jeffrey R; Pelkey, Kenneth A; Fam, Sami R; Huang, Yueqiao; Petralia, Ronald S; Wenthold, Robert J; Salter, Michael W

    2004-04-20

    Src is the prototypic protein tyrosine kinase and is critical for controlling diverse cellular functions. Regions in Src define structural and functional domains conserved in many cell signaling proteins. Src also contains a region of low sequence conservation termed the unique domain, the function of which has until now remained enigmatic. Here, we show that the unique domain of Src is a protein-protein interaction region and we identify NADH dehydrogenase subunit 2 (ND2) as a Src unique domain-interacting protein. ND2 is a subunit of complex I in mitochondria, but we find that ND2 interacts with Src outside this organelle at excitatory synapses in the brain. ND2 acts as an adapter protein anchoring Src to the N-methyl-d-aspartate (NMDA) receptor complex, and is crucial for Src regulation of synaptic NMDA receptor activity. By showing an extramitochondrial action for a protein encoded in the mitochondrial genome, we identify a previously unsuspected means by which mitochondria regulate cellular function, suggesting a new paradigm that may be of general relevance for control of Src signaling. PMID:15069201

  14. New insights into the targeting of a subset of tail-anchored proteins to the outer mitochondrial membrane

    PubMed Central

    Marty, Naomi J.; Teresinski, Howard J.; Hwang, Yeen Ting; Clendening, Eric A.; Gidda, Satinder K.; Sliwinska, Elwira; Zhang, Daiyuan; Miernyk, Ján A.; Brito, Glauber C.; Andrews, David W.; Dyer, John M.; Mullen, Robert T.

    2014-01-01

    Tail-anchored (TA) proteins are a unique class of functionally diverse membrane proteins defined by their single C-terminal membrane-spanning domain and their ability to insert post-translationally into specific organelles with an Ncytoplasm-Corganelle interior orientation. The molecular mechanisms by which TA proteins are sorted to the proper organelles are not well-understood. Herein we present results indicating that a dibasic targeting motif (i.e., -R-R/K/H-X{X≠E}) identified previously in the C terminus of the mitochondrial isoform of the TA protein cytochrome b5, also exists in many other A. thaliana outer mitochondrial membrane (OMM)-TA proteins. This motif is conspicuously absent, however, in all but one of the TA protein subunits of the translocon at the outer membrane of mitochondria (TOM), suggesting that these two groups of proteins utilize distinct biogenetic pathways. Consistent with this premise, we show that the TA sequences of the dibasic-containing proteins are both necessary and sufficient for targeting to mitochondria, and are interchangeable, while the TA regions of TOM proteins lacking a dibasic motif are necessary, but not sufficient for localization, and cannot be functionally exchanged. We also present results from a comprehensive mutational analysis of the dibasic motif and surrounding sequences that not only greatly expands the functional definition and context-dependent properties of this targeting signal, but also led to the identification of other novel putative OMM-TA proteins. Collectively, these results provide important insight to the complexity of the targeting pathways involved in the biogenesis of OMM-TA proteins and help define a consensus targeting motif that is utilized by at least a subset of these proteins. PMID:25237314

  15. Involvement of GPI-anchored lipid transfer proteins in the development of seed coats and pollen in Arabidopsis thaliana.

    PubMed

    Edstam, Monika M; Edqvist, Johan

    2014-09-01

    The non-specific lipid transfer proteins (nsLTPs) constitute a large protein family specific for plants. Proteins from the family are found in all land plants but have not been identified in green algae. Their in vivo functions are still disputed although evidence is accumulating for a role of these proteins in cuticle development. In a previous study, we performed a co-expression analysis of glycosylphosphatidylinositol (GPI)-anchored nsLTPs (LTPGs), which suggested that these proteins are also involved in the accumulation of suberin and sporopollenin. Here, we follow up the previous co-expression study by characterizing the phenotypes of Arabidopsis thaliana lines with insertions in LTPG genes. The observed phenotypes include an inability to limit tetrazolium salt uptake in seeds, development of hair-like structures on seeds, altered pollen morphologies and decreased levels of ω-hydroxy fatty acids in seed coats. The observed phenotypes give further support for a role in suberin and sporopollenin biosynthesis or deposition in A. thaliana. PMID:24460633

  16. A role for protein kinase CK2 in cell proliferation: evidence using a kinase-inactive mutant of CK2 catalytic subunit alpha.

    PubMed

    Lebrin, F; Chambaz, E M; Bianchini, L

    2001-04-12

    Protein kinase CK2 is an ubiquitous and pleiotropic Ser/Thr protein kinase composed of two catalytic (alpha and/or alpha') and two regulatory (beta) subunits generally combined to form alpha(2)beta(2), alphaalpha'beta(2), or alpha'(2)beta(2) heterotetramers. To gain more insight into the role of CK2 in the control of proliferation in mammalian cells, overexpression of isolated CK2 subunits alpha, alpha', or beta was carried out in two fibroblast cell lines: NIH3T3 and CCL39. To interfere with CK2 cellular functions, cells were also transfected with a kinase-inactive mutant of CK2alpha catalytic subunit: CK2alpha-K68A. In NIH3T3 cells, overexpression of either wild-type subunit (alpha, alpha' or beta) had no effect on cell proliferation. In contrast, overexpression of the CK2alpha kinase-deficient mutant induced a marked inhibition of cell proliferation. This resulted from a defect in G1/S progression as demonstrated in transient transfection experiments in both NIH3T3 and CCL39 cells using BrdU incorporation measurements and in CCL39 clones stably overexpressing the CK2alpha-K68A mutant by growth curve analysis. We demonstrated that the kinase-negative mutant has the capacity to integrate the endogenous CK2 subunit pool both as an isolated kinase-inactive alpha subunit and as associated to the beta subunit in a kinase-inactive tetramer. Finally we showed that expression of the kinase-inactive mutant interferes with phosphorylation of an endogenous CK2 substrate; we speculate that optimal phosphorylation of target proteins by CK2 is required to achieve optimal cell cycle progression. PMID:11360185

  17. Intermolecular Interaction between Anchoring Subunits Specify Subcellular Targeting and Function of RGS Proteins in Retina ON-Bipolar Neurons.

    PubMed

    Sarria, Ignacio; Orlandi, Cesare; McCall, Maureen A; Gregg, Ronald G; Martemyanov, Kirill A

    2016-03-01

    In vertebrate retina, light responses generated by the rod photoreceptors are transmitted to the second-order neurons, the ON-bipolar cells (ON-BC), and this communication is indispensible for vision in dim light. In ON-BCs, synaptic transmission is initiated by the metabotropic glutamate receptor, mGluR6, that signals via the G-protein Go to control opening of the effector ion channel, TRPM1. A key role in this process belongs to the GTPase Activating Protein (GAP) complex that catalyzes Go inactivation upon light-induced suppression of glutamate release in rod photoreceptors, thereby driving ON-BC depolarization to changes in synaptic input. The GAP complex has a striking molecular complexity. It contains two Regulator of G-protein Signaling (RGS) proteins RGS7 and RGS11 that directly act on Go and two adaptor subunits: RGS Anchor Protein (R9AP) and the orphan receptor, GPR179. Here we examined the organizational principles of the GAP complex in ON-BCs. Biochemical experiments revealed that RGS7 binds to a conserved site in GPR179 and that RGS11 in vivo forms a complex only with R9AP. R9AP and GPR179 are further integrated via direct protein-protein interactions involving their cytoplasmic domains. Elimination of GPR179 prevents postsynaptic accumulation of R9AP. Furthermore, concurrent knock-out of both R9AP and RGS7 does not reconfigure the GAP complex and completely abolishes synaptic transmission, resulting in a novel mouse model of night blindness. Based on these results, we propose a model of hierarchical assembly and function of the GAP complex that supports ON-BCs visual signaling. PMID:26961947

  18. Anchoring of Protein Kinase A by ERM (Ezrin-Radixin-Moesin) Proteins Is Required for Proper Netrin Signaling through DCC (Deleted in Colorectal Cancer)*

    PubMed Central

    Deming, Paula B.; Campbell, Shirley L.; Stone, Jamie B.; Rivard, Robert L.; Mercier, Alison L.; Howe, Alan K.

    2015-01-01

    Netrin-1, acting through its principal receptor DCC (deleted in colorectal cancer), serves as an axon guidance cue during neural development and also contributes to vascular morphogenesis, epithelial migration, and the pathogenesis of some tumors. Several lines of evidence suggest that netrin-DCC signaling can regulate and be regulated by the cAMP-dependent protein kinase, PKA, although the molecular details of this relationship are poorly understood. Specificity in PKA signaling is often achieved through differential subcellular localization of the enzyme by interaction with protein kinase A anchoring proteins (AKAPs). Here, we show that AKAP function is required for DCC-mediated activation of PKA and phosphorylation of cytoskeletal regulatory proteins of the Mena/VASP (vasodilator-stimulated phosphoprotein) family. Moreover, we show that DCC and PKA physically interact and that this association is mediated by the ezrin-radixin-moesin (ERM) family of plasma membrane-actin cytoskeleton cross-linking proteins. Silencing of ERM protein expression inhibits DCC-PKA interaction, DCC-mediated PKA activation, and phosphorylation of Mena/VASP proteins as well as growth cone morphology and neurite outgrowth. Finally, although expression of wild-type radixin partially rescued growth cone morphology and tropism toward netrin in ERM-knockdown cells, expression of an AKAP-deficient mutant of radixin did not fully rescue growth cone morphology and switched netrin tropism from attraction to repulsion. These data support a model in which ERM-mediated anchoring of PKA activity to DCC is required for proper netrin/DCC-mediated signaling. PMID:25575591

  19. Structure and dynamics of the conserved protein GPI anchor core inserted into detergent micelles.

    PubMed

    Chevalier, Franck; Lopez-Prados, Javier; Groves, Patrick; Perez, Serge; Martín-Lomas, Manuel; Nieto, Pedro M

    2006-10-01

    A suitable approach which combines nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations have been used to study the structure and the dynamics of the glycosylphosphatidylinositol (GPI) anchor Manalphal-2Manalpha1-6Manalphal -4GlcNalpha1-6myo-inositol-1-OPO(3)-sn-1,2-dimyristoylglycerol (1) incorporated into dodecylphosphatidylcholine (DPC) micelles. The results have been compared to those previously obtained for the products obtainable from (1) after phospholipase cleavage, in aqueous solution. Relaxation and diffusion NMR experiments were used to establish the formation of stable aggregates and the insertion of (1) into the micelles. MD calculations were performed including explicit water, sodium and chloride ions and using the Particle Mesh Ewald approach for the evaluation of the electrostatic energy term. The MD predicted three dimensional structure and dynamics were substantiated by nuclear overhauser effect (NOE) measurements and relaxation data. The pseudopentasaccharide structure, which was not affected by incorporation of (1) into the micelle, showed a complex dynamic behaviour with a faster relative motion at the terminal mannopyranose unit and decreased mobility close to the micelle. This motion may be better described as an oscillation relative to the membrane rather than a folding event. PMID:16774909

  20. Creation of a Cellooligosaccharide-Assimilating Escherichia coli Strain by Displaying Active Beta-Glucosidase on the Cell Surface via a Novel Anchor Protein

    PubMed Central

    Tanaka, Tsutomu; Kawabata, Hitomi; Ogino, Chiaki; Kondo, Akihiko

    2011-01-01

    We demonstrated direct assimilation of cellooligosaccharide using Escherichia coli displaying beta-glucosidase (BGL). BGL from Thermobifida fusca YX (Tfu0937) was displayed on the E. coli cell surface using a novel anchor protein named Blc. This strain was grown successfully on 0.2% cellobiose, and the optical density at 600 nm (OD600) was 1.05 after 20 h. PMID:21742905

  1. Insight into Early-Stage Unfolding of GPI-Anchored Human Prion Protein.

    PubMed

    Wu, Emilia L; Qi, Yifei; Park, Soohyung; Mallajosyula, Sairam S; MacKerell, Alexander D; Klauda, Jeffery B; Im, Wonpil

    2015-11-17

    Prion diseases are fatal neurodegenerative disorders, which are characterized by the accumulation of misfolded prion protein (PrPSc) converted from a normal host cellular prion protein (PrPC). Experimental studies suggest that PrPC is enriched with α-helical structure, whereas PrPSc contains a high proportion of β-sheet. In this study, we report the impact of N-glycosylation and the membrane on the secondary structure stability utilizing extensive microsecond molecular dynamics simulations. Our results reveal that the HB (residues 173 to 194) C-terminal fragment undergoes conformational changes and helix unfolding in the absence of membrane environments because of the competition between protein backbone intramolecular and protein-water intermolecular hydrogen bonds as well as its intrinsic instability originated from the amino acid sequence. This initiation of the unfolding process of PrPC leads to a subsequent increase in the length of the HB-HC loop (residues 195 to 199) that may trigger larger rigid body motions or further unfolding around this region. Continuous interactions between prion protein and the membrane not only constrain the protein conformation but also decrease the solvent accessibility of the backbone atoms, thereby stabilizing the secondary structure, which is enhanced by N-glycosylation via additional interactions between the N-glycans and the membrane surface. PMID:26588568

  2. Anchoring of both PKA-RIIα and 14-3-3θ regulates retinoic acid induced 16 mediated phosphorylation of heat shock protein 70

    PubMed Central

    Tang, Hai-Lin; Zhu, Shi-Ying; Zhao, Lan-Juan; Ren, Hao; Zhao, Ping; Qi, Zhong-Tian; Wang, Wen

    2015-01-01

    Our previous study reported that retinoic acid induced 16 (RAI16) could enhance tumorigenesis in hepatocellular carcinoma (HCC). However, the cellular functions of RAI16 are still unclear. In this study, by immunoprecipitation and tandem (MS/MS) mass spectrometry analysis, we identified that RAI16 interacted with the type II regulatory subunit of PKA (PKA-RIIα), acting as a novel protein kinase A anchoring protein (AKAP). In addition, RAI16 also interacted with heat shock protein 70 (HSP70) and 14-3-3θ. Further studies indicated that RAI16 mediated PKA phosphorylation of HSP70 at serine 486, resulting in anti-apoptosis events. RAI16 was also phosphorylated by the anchored PKA at serine 325, which promoted the recruitment of 14-3-3θ, which, in turn, inhibited RAI16 mediated PKA phosphorylation of HSP70. These findings offer mechanism insight into RAI16 mediated anti-apoptosis signaling in HCC. PMID:25900241

  3. Membrane-anchored ubiquitin ligase complex is required for the turnover of lysosomal membrane proteins

    PubMed Central

    Li, Ming; Koshi, Tatsuhiro

    2015-01-01

    Cells must regulate the abundance and activity of numerous nutrient transporters in different organelle membranes to achieve nutrient homeostasis. As the recycling center and major storage organelle, lysosomes are essential for maintaining nutrient homeostasis. However, very little is known about mechanisms that govern the regulation of its membrane proteins. In this study, we demonstrated that changes of Zn2+ levels trigger the downregulation of vacuolar Zn2+ transporters. Low Zn2+ levels cause the degradation of the influx transporter Cot1, whereas high Zn2+ levels trigger the degradation of the efflux channel Zrt3. The degradation process depends on the vacuole membrane recycling and degradation pathway. Unexpectedly, we identified a RING domain–containing E3 ligase Tul1 and its interacting proteins in the Dsc complex that are important for the ubiquitination of Cot1 and partial ubiquitination of Zrt3. Our study demonstrated that the Dsc complex can function at the vacuole to regulate the composition and lifetime of vacuolar membrane proteins. PMID:26527740

  4. FlaF is a β-sandwich protein that anchors the archaellum in the archaeal cell envelope by binding the S-layer protein

    SciTech Connect

    Banerjee, Ankan; Tsai, Chi -Lin; Chaudhury, Paushali; Tripp, Patrick; Arvai, Andrew  S.; Ishida, Justin  P.; Tainer, John  A.; Albers, Sonja -Verena

    2015-05-01

    Archaea employ the archaellum, a type IV pilus-like nanomachine, for swimming motility. In the crenarchaeon Sulfolobus acidocaldarius, the archaellum consists of seven proteins: FlaB/X/G/F/H/I/J. FlaF is conserved and essential for archaellum assembly but no FlaF structures exist. Here, we truncated the FlaF N terminus and solved 1.5-Å and 1.65-Å resolution crystal structures of this monotopic membrane protein. Structures revealed an N-terminal α-helix and an eight-strand β-sandwich, immunoglobulin-like fold with striking similarity to S-layer proteins. Crystal structures, X-ray scattering, and mutational analyses suggest dimer assembly is needed for in vivo function. The sole cell envelope component of S. acidocaldarius is a paracrystalline S-layer, and FlaF specifically bound to S-layer protein, suggesting that its interaction domain is located in the pseudoperiplasm with its N-terminal helix in the membrane. From these data, FlaF may act as the previously unknown archaellum stator protein that anchors the rotating archaellum to the archaeal cell envelope.

  5. FlaF is a β-sandwich protein that anchors the archaellum in the archaeal cell envelope by binding the S-layer protein

    DOE PAGESBeta

    Banerjee, Ankan; Tsai, Chi -Lin; Chaudhury, Paushali; Tripp, Patrick; Arvai, Andrew  S.; Ishida, Justin  P.; Tainer, John  A.; Albers, Sonja -Verena

    2015-05-01

    Archaea employ the archaellum, a type IV pilus-like nanomachine, for swimming motility. In the crenarchaeon Sulfolobus acidocaldarius, the archaellum consists of seven proteins: FlaB/X/G/F/H/I/J. FlaF is conserved and essential for archaellum assembly but no FlaF structures exist. Here, we truncated the FlaF N terminus and solved 1.5-Å and 1.65-Å resolution crystal structures of this monotopic membrane protein. Structures revealed an N-terminal α-helix and an eight-strand β-sandwich, immunoglobulin-like fold with striking similarity to S-layer proteins. Crystal structures, X-ray scattering, and mutational analyses suggest dimer assembly is needed for in vivo function. The sole cell envelope component of S. acidocaldarius is amore » paracrystalline S-layer, and FlaF specifically bound to S-layer protein, suggesting that its interaction domain is located in the pseudoperiplasm with its N-terminal helix in the membrane. From these data, FlaF may act as the previously unknown archaellum stator protein that anchors the rotating archaellum to the archaeal cell envelope.« less

  6. FlaF Is a β-Sandwich Protein that Anchors the Archaellum in the Archaeal Cell Envelope by Binding the S-Layer Protein

    PubMed Central

    Banerjee, Ankan; Tsai, Chi-Lin; Chaudhury, Paushali; Tripp, Patrick; Arvai, Andrew S.; Ishida, Justin P.; Tainer, John A.; Albers, Sonja-Verena

    2015-01-01

    Summary Archaea employ the archaellum, a type IV pilus-like nanomachine, for swimming motility. In the crenarchaeon Sulfolobus acidocaldarius, the archaellum consists of seven proteins: FlaB/X/G/F/H/I/J. FlaF is conserved and essential for archaellum assembly but no FlaF structures exist. Here, we truncated the FlaF N terminus and solved 1.5-Å and 1.65-Å resolution crystal structures of this monotopic membrane protein. Structures revealed an N-terminal α-helix and an eight-strand β-sandwich, immunoglobulin-like fold with striking similarity to S-layer proteins. Crystal structures, X-ray scattering, and mutational analyses suggest dimer assembly is needed for in vivo function. The sole cell envelope component of S. acidocaldarius is a paracrystalline S-layer, and FlaF specifically bound to S-layer protein, suggesting that its interaction domain is located in the pseudoperiplasm with its N-terminal helix in the membrane. From these data, FlaF may act as the previously unknown archaellum stator protein that anchors the rotating archaellum to the archaeal cell envelope. PMID:25865246

  7. Glycosylphosphatidylinositol-anchored proteins as chaperones and co-receptors for FERONIA receptor kinase signaling in Arabidopsis

    PubMed Central

    Li, Chao; Yeh, Fang-Ling; Cheung, Alice Y; Duan, Qiaohong; Kita, Daniel; Liu, Ming-Che; Maman, Jacob; Luu, Emily J; Wu, Brendan W; Gates, Laura; Jalal, Methun; Kwong, Amy; Carpenter, Hunter; Wu, Hen-Ming

    2015-01-01

    The Arabidopsis receptor kinase FERONIA (FER) is a multifunctional regulator for plant growth and reproduction. Here we report that the female gametophyte-expressed glycosylphosphatidylinositol-anchored protein (GPI-AP) LORELEI and the seedling-expressed LRE-like GPI-AP1 (LLG1) bind to the extracellular juxtamembrane region of FER and show that this interaction is pivotal for FER function. LLG1 interacts with FER in the endoplasmic reticulum and on the cell surface, and loss of LLG1 function induces cytoplasmic retention of FER, consistent with transport of FER from the endoplasmic reticulum to the plasma membrane in a complex with LLG1. We further demonstrate that LLG1 is a component of the FER-regulated RHO GTPase signaling complex and that fer and llg1 mutants display indistinguishable growth, developmental and signaling phenotypes, analogous to how lre and fer share similar reproductive defects. Together our results support LLG1/LRE acting as a chaperone and co-receptor for FER and elucidate a mechanism by which GPI-APs enable the signaling capacity of a cell surface receptor. DOI: http://dx.doi.org/10.7554/eLife.06587.001 PMID:26052747

  8. PbsP, a cell wall-anchored protein that binds plasminogen to promote hematogenous dissemination of group B Streptococcus.

    PubMed

    Buscetta, Marco; Firon, Arnaud; Pietrocola, Giampiero; Biondo, Carmelo; Mancuso, Giuseppe; Midiri, Angelina; Romeo, Letizia; Galbo, Roberta; Venza, Mario; Venza, Isabella; Kaminski, Pierre-Alexandre; Gominet, Myriam; Teti, Giuseppe; Speziale, Pietro; Trieu-Cuot, Patrick; Beninati, Concetta

    2016-07-01

    Streptococcus agalactiae (Group B Streptococcus or GBS) is a leading cause of invasive infections in neonates whose virulence is dependent on its ability to interact with cells and host components. We here characterized a surface protein with a critical function in GBS pathophysiology. This adhesin, designated PbsP, possesses two Streptococcal Surface Repeat domains, a methionine and lysine-rich region, and a LPXTG cell wall-anchoring motif. PbsP mediates plasminogen (Plg) binding both in vitro and in vivo and we showed that cell surface-bound Plg can be activated into plasmin by tissue plasminogen activator to increase the bacterial extracellular proteolytic activity. Absence of PbsP results in a decreased bacterial transmigration across brain endothelial cells and impaired virulence in a murine model of infection. PbsP is conserved among the main GBS lineages and is a major plasminogen adhesin in non-CC17 GBS strains. Importantly, immunization of mice with recombinant PbsP confers protective immunity. Our results indicate that GBS have evolved different strategies to recruit Plg which indicates that the ability to acquire cell surface proteolytic activity is essential for the invasiveness of this bacterium. PMID:26888569

  9. Impaired FcϵRI stability, signaling, and effector functions in murine mast cells lacking glycosylphosphatidylinositol-anchored proteins

    PubMed Central

    Wu, Ping; Eastham-Anderson, Jeffrey; Kinoshita, Taroh; Brown, Eric J.

    2011-01-01

    A key event and potential therapeutic target in allergic and asthmatic diseases is signaling by the IgE receptor FcϵRI, which depends on its interactions with Src family kinases (SFK). Here we tested the hypothesis that glycosylphosphatidylinositiol-anchored proteins (GPI-AP) are involved in FcϵRI signaling, based on previous observations that GPI-AP colocalize with and mediate activation of SFK. We generated mice with a hematopoietic cell-specific GPI-AP deficiency by targeted disruption of the GPI biosynthesis gene PigA. In these mice, IgE-mediated passive cutaneous anaphylaxis was largely abolished. PigA-deficient mast cells cultured from these mice showed impaired degranulation in response to stimulation with IgE and antigen in vitro, despite normal IgE binding and antigen-induced FcϵRI aggregation. On stimulation of these cells with IgE and antigen, coprecipitation of the FcϵRI α-chain with the γ-chain and β-chain was markedly reduced. As a result, IgE/antigen–induced FcϵRI-Lyn association and γ-chain tyrosine phosphorylation were both impaired in PigA-deficient cells. These data provide genetic evidence for an unanticipated key role of GPI-AP in FcϵRI interchain interactions and early FcϵRI signaling events, necessary for antigen-induced mast cell degranulation. PMID:21865342

  10. Glycosylphosphatidylinositol-anchored proteins as chaperones and co-receptors for FERONIA receptor kinase signaling in Arabidopsis.

    PubMed

    Li, Chao; Yeh, Fang-Ling; Cheung, Alice Y; Duan, Qiaohong; Kita, Daniel; Liu, Ming-Che; Maman, Jacob; Luu, Emily J; Wu, Brendan W; Gates, Laura; Jalal, Methun; Kwong, Amy; Carpenter, Hunter; Wu, Hen-Ming

    2015-01-01

    The Arabidopsis receptor kinase FERONIA (FER) is a multifunctional regulator for plant growth and reproduction. Here we report that the female gametophyte-expressed glycosylphosphatidylinositol-anchored protein (GPI-AP) LORELEI and the seedling-expressed LRE-like GPI-AP1 (LLG1) bind to the extracellular juxtamembrane region of FER and show that this interaction is pivotal for FER function. LLG1 interacts with FER in the endoplasmic reticulum and on the cell surface, and loss of LLG1 function induces cytoplasmic retention of FER, consistent with transport of FER from the endoplasmic reticulum to the plasma membrane in a complex with LLG1. We further demonstrate that LLG1 is a component of the FER-regulated RHO GTPase signaling complex and that fer and llg1 mutants display indistinguishable growth, developmental and signaling phenotypes, analogous to how lre and fer share similar reproductive defects. Together our results support LLG1/LRE acting as a chaperone and co-receptor for FER and elucidate a mechanism by which GPI-APs enable the signaling capacity of a cell surface receptor. PMID:26052747

  11. Display of active enzymes on the cell surface of Escherichia coli using PgsA anchor protein and their application to bioconversion.

    PubMed

    Narita, Junya; Okano, Kenji; Tateno, Toshihiro; Tanino, Takanori; Sewaki, Tomomitsu; Sung, Moon-Hee; Fukuda, Hideki; Kondo, Akihiko

    2006-05-01

    We have developed a novel Escherichia coli cell surface display system by employing PgsA as an anchoring motif. In our display system, C-terminal fusion to PgsA anchor protein from Bacillus subtilis was used. The enzymes selected for display were alpha-amylase (AmyA) from Streptococcus bovis 148 and lipase B (CALB) from Candida antarctica. The molecular mass values of AmyA and CALB are approximately 77 and 34 kDa, respectively. The enzymes were displayed on the surface as a fusion protein with a FLAG peptide tag at the C terminus. Both the PgsA-AmyA-FLAG and PgsA-CALB-FLAG fusion proteins were shown to be displayed by immunofluorescence labeling using anti-FLAG antibody. The displayed enzymes were active forms, and AmyA and CALB activities reached 990 U/g (dry cell weight) and 4.6 U/g (dry cell weight), respectively. AmyA-displaying E. coli cells grew utilizing cornstarch as the sole carbon source, while CALB-displaying E. coli cells catalyzed enantioselective transesterification, indicating that they are effective whole-cell biocatalysts. Since a target enzyme with a size of 77 kDa and an industrially useful lipase have been successfully displayed on the cell surface of E. coli for the first time, PgsA protein is probably a useful anchoring motif to display various enzymes. PMID:16133338

  12. Membrane Anchoring of Aminoacyl-tRNA Synthetases by Convergent Acquisition of a Novel Protein Domain*

    PubMed Central

    Olmedo-Verd, Elvira; Santamaría-Gómez, Javier; Ochoa de Alda, Jesús A. G.; Ribas de Pouplana, Lluis; Luque, Ignacio

    2011-01-01

    Four distinct aminoacyl-tRNA synthetases (aaRSs) found in some cyanobacterial species contain a novel protein domain that bears two putative transmembrane helices. This CAAD domain is present in glutamyl-, isoleucyl-, leucyl-, and valyl-tRNA synthetases, the latter of which has probably recruited the domain more than once during evolution. Deleting the CAAD domain from the valyl-tRNA synthetase of Anabaena sp. PCC 7120 did not significantly modify the catalytic properties of this enzyme, suggesting that it does not participate in its canonical tRNA-charging function. Multiple lines of evidence suggest that the function of the CAAD domain is structural, mediating the membrane anchorage of the enzyme, although membrane localization of aaRSs has not previously been described in any living organism. Synthetases containing the CAAD domain were localized in the intracytoplasmic thylakoid membranes of cyanobacteria and were largely absent from the plasma membrane. The CAAD domain was necessary and apparently sufficient for protein targeting to membranes. Moreover, localization of aaRSs in thylakoids was important under nitrogen limiting conditions. In Anabaena, a multicellular filamentous cyanobacterium often used as a model for prokaryotic cell differentiation, valyl-tRNA synthetase underwent subcellular relocation at the cell poles during heterocyst differentiation, a process also dependent on the CAAD domain. PMID:21965654

  13. Dolichol phosphate mannose synthase is required in vivo for glycosyl phosphatidylinositol membrane anchoring, O mannosylation, and N glycosylation of protein in Saccharomyces cerevisiae.

    PubMed Central

    Orlean, P

    1990-01-01

    Glycosyl phosphatidylinositol (GPI) anchoring, N glycosylation, and O mannosylation of protein occur in the rough endoplasmic reticulum and involve transfer of precursor structures that contain mannose. Direct genetic evidence is presented that dolichol phosphate mannose (Dol-P-Man) synthase, which transfers mannose from GDPMan to the polyisoprenoid dolichol phosphate, is required in vivo for all three biosynthetic pathways leading to these covalent modifications of protein in yeast cells. Temperature-sensitive yeast mutants were isolated after in vitro mutagenesis of the yeast DPM1 gene. At the nonpermissive temperature of 37 degrees C, the dpm1 mutants were blocked in [2-3H]myo-inositol incorporation into protein and accumulated a lipid that could be radiolabeled with both [2-3H]myo-inositol and [2-3H]glucosamine and met existing criteria for an intermediate in GPI anchor biosynthesis. The likeliest explanation for these results is that Dol-P-Man donates the mannose residues needed for completion of the GPI anchor precursor lipid before it can be transferred to protein. Dol-P-Man synthase is also required in vivo for N glycosylation of protein, because (i) dpm1 cells were unable to make the full-length precursor Dol-PP-GlcNAc2Man9Glc3 and instead accumulated the intermediate Dol-PP-GlcNAc2Man5 in their pool of lipid-linked precursor oligosaccharides and (ii) truncated, endoglycosidase H-resistant oligosaccharides were transferred to the N-glycosylated protein invertase after a shift to 37 degrees C. Dol-P-Man synthase is also required in vivo for O mannosylation of protein, because chitinase, normally a 150-kDa O-mannosylated protein, showed a molecular size of 60 kDa, the size predicted for the unglycosylated protein, after shift of the dpm1 mutant to the nonpermissive temperature. Images PMID:2146492

  14. Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

    SciTech Connect

    Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.; E-mail: andy.blakely@vanderbilt.edu

    2005-08-05

    The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH{sub 2}-terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking.

  15. AKAP-Anchored PKA Maintains Neuronal L-type Calcium Channel Activity and NFAT Transcriptional Signaling

    PubMed Central

    Murphy, Jonathan G.; Sanderson, Jennifer L.; Gorski, Jessica A.; Scott, John D.; Catterall, William A.; Sather, William A.; Dell’Acqua, Mark L.

    2014-01-01

    Summary In neurons, Ca2+ influx through L-type voltage-gated Ca2+ channels (LTCC) couples electrical activity to changes in transcription. LTCC activity is elevated by the cAMP-dependent protein kinase (PKA) and depressed by the Ca2+-dependent phosphatase calcineurin (CaN), with both enzymes localized to the channel by A-kinase anchoring protein (AKAP) 79/150. AKAP79/150 anchoring of CaN also promotes LTCC activation of transcription through dephosphorylation of the nuclear factor of activated T-cells (NFAT). We report here that genetic disruption of PKA anchoring to AKAP79/150 also interferes with LTCC activation of CaN-NFAT signaling in neurons. Disruption of AKAP-PKA anchoring promoted redistribution of the kinase out of dendritic spines, profound decreases in LTCC phosphorylation and Ca2+ influx, and impaired NFAT movement to the nucleus and activation of transcription. Our findings support a model wherein basal activity of AKAP79/150-anchored PKA opposes CaN to preserve LTCC phosphorylation, thereby sustaining LTCC activation of CaN-NFAT signaling to the neuronal nucleus. PMID:24835999

  16. Beyond anchoring: the expanding role of the hendra virus fusion protein transmembrane domain in protein folding, stability, and function.

    PubMed

    Smith, Everett Clinton; Culler, Megan R; Hellman, Lance M; Fried, Michael G; Creamer, Trevor P; Dutch, Rebecca Ellis

    2012-03-01

    While work with viral fusion proteins has demonstrated that the transmembrane domain (TMD) can affect protein folding, stability, and membrane fusion promotion, the mechanism(s) remains poorly understood. TMDs could play a role in fusion promotion through direct TMD-TMD interactions, and we have recently shown that isolated TMDs from three paramyxovirus fusion (F) proteins interact as trimers using sedimentation equilibrium (SE) analysis (E. C. Smith, et al., submitted for publication). Immediately N-terminal to the TMD is heptad repeat B (HRB), which plays critical roles in fusion. Interestingly, addition of HRB decreased the stability of the trimeric TMD-TMD interactions. This result, combined with previous findings that HRB forms a trimeric coiled coil in the prefusion form of the whole protein though HRB peptides fail to stably associate in isolation, suggests that the trimeric TMD-TMD interactions work in concert with elements in the F ectodomain head to stabilize a weak HRB interaction. Thus, changes in TMD-TMD interactions could be important in regulating F triggering and refolding. Alanine insertions between the TMD and HRB demonstrated that spacing between these two regions is important for protein stability while not affecting TMD-TMD interactions. Additional mutagenesis of the C-terminal end of the TMD suggests that β-branched residues within the TMD play a role in membrane fusion, potentially through modulation of TMD-TMD interactions. Our results support a model whereby the C-terminal end of the Hendra virus F TMD is an important regulator of TMD-TMD interactions and show that these interactions help hold HRB in place prior to the triggering of membrane fusion. PMID:22238302

  17. Epstein-Barr Viral BNLF2a Protein Hijacks the Tail-anchored Protein Insertion Machinery to Block Antigen Processing by the Transport Complex TAP*

    PubMed Central

    Wycisk, Agnes I.; Lin, Jiacheng; Loch, Sandra; Hobohm, Kathleen; Funke, Jessica; Wieneke, Ralph; Koch, Joachim; Skach, William R.; Mayerhofer, Peter U.; Tampé, Robert

    2011-01-01

    Virus-infected cells are eliminated by cytotoxic T lymphocytes, which recognize viral epitopes displayed on major histocompatibility complex class I molecules at the cell surface. Herpesviruses have evolved sophisticated strategies to escape this immune surveillance. During the lytic phase of EBV infection, the viral factor BNLF2a interferes with antigen processing by preventing peptide loading of major histocompatibility complex class I molecules. Here we reveal details of the inhibition mechanism of this EBV protein. We demonstrate that BNLF2a acts as a tail-anchored protein, exploiting the mammalian Asna-1/WRB (Get3/Get1) machinery for posttranslational insertion into the endoplasmic reticulum membrane, where it subsequently blocks antigen translocation by the transporter associated with antigen processing (TAP). BNLF2a binds directly to the core TAP complex arresting the ATP-binding cassette transporter in a transport-incompetent conformation. The inhibition mechanism of EBV BNLF2a is distinct and mutually exclusive of other viral TAP inhibitors. PMID:21984826

  18. Subcellular Partitioning of Protein Tyrosine Phosphatase 1B to the Endoplasmic Reticulum and Mitochondria Depends Sensitively on the Composition of Its Tail Anchor

    PubMed Central

    Fueller, Julia; Egorov, Mikhail V.; Walther, Kirstin A.; Sabet, Ola; Mallah, Jana; Grabenbauer, Markus; Kinkhabwala, Ali

    2015-01-01

    The canonical protein tyrosine phosphatase PTP1B is an important regulator of diverse cellular signaling networks. PTP1B has long been thought to exert its influence solely from its perch on the endoplasmic reticulum (ER); however, an additional subpopulation of PTP1B has recently been detected in mitochondria extracted from rat brain tissue. Here, we show that PTP1B’s mitochondrial localization is general (observed across diverse mammalian cell lines) and sensitively dependent on the transmembrane domain length, C-terminal charge and hydropathy of its short (≤35 amino acid) tail anchor. Our electron microscopy of specific DAB precipitation revealed that PTP1B localizes via its tail anchor to the outer mitochondrial membrane (OMM), with fluorescence lifetime imaging microscopy establishing that this OMM pool contributes to the previously reported cytoplasmic interaction of PTP1B with endocytosed epidermal growth factor receptor. We additionally examined the mechanism of PTP1B’s insertion into the ER membrane through heterologous expression of PTP1B’s tail anchor in wild-type yeast and yeast mutants of major conserved ER insertion pathways: In none of these yeast strains was ER targeting significantly impeded, providing in vivo support for the hypothesis of spontaneous membrane insertion (as previously demonstrated in vitro). Further functional elucidation of the newly recognized mitochondrial pool of PTP1B will likely be important for understanding its complex roles in cellular responses to external stimuli, cell proliferation and diseased states. PMID:26431424

  19. Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells.

    PubMed

    Zhang, Bo; Zuo, Fanglei; Yu, Rui; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

    2015-01-01

    Adhesion to host cells is considered important for Lactobacillus plantarum as well as other lactic acid bacteria (LAB) to persist in human gut and thus exert probiotic effects. Here, we sequenced the genome of Lt. plantarum strain NL42 originating from a traditional Chinese dairy product, performed comparative genomic analysis and characterized a novel adhesion factor. The genome of NL42 was highly divergent from its closest neighbors, especially in six large genomic regions. NL42 harbors a total of 42 genes encoding adhesion-associated proteins; among them, cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif. Expression of cwaA in Lactococcus lactis significantly increased its autoaggregation and hydrophobicity, and conferred the new ability to adhere to human colonic epithelial HT-29 cells by targeting cellular surface proteins, and not carbohydrate moieties, for CwaA adhesion. In addition, the recombinant Lc. lactis inhibited adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells, mainly by exclusion. We conclude that CwaA is a novel adhesion factor in Lt. plantarum and a potential candidate for improving the adhesion ability of probiotics or other bacteria of interest. PMID:26370773

  20. Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells

    PubMed Central

    Zhang, Bo; Zuo, Fanglei; Yu, Rui; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

    2015-01-01

    Adhesion to host cells is considered important for Lactobacillus plantarum as well as other lactic acid bacteria (LAB) to persist in human gut and thus exert probiotic effects. Here, we sequenced the genome of Lt. plantarum strain NL42 originating from a traditional Chinese dairy product, performed comparative genomic analysis and characterized a novel adhesion factor. The genome of NL42 was highly divergent from its closest neighbors, especially in six large genomic regions. NL42 harbors a total of 42 genes encoding adhesion-associated proteins; among them, cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif. Expression of cwaA in Lactococcus lactis significantly increased its autoaggregation and hydrophobicity, and conferred the new ability to adhere to human colonic epithelial HT-29 cells by targeting cellular surface proteins, and not carbohydrate moieties, for CwaA adhesion. In addition, the recombinant Lc. lactis inhibited adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells, mainly by exclusion. We conclude that CwaA is a novel adhesion factor in Lt. plantarum and a potential candidate for improving the adhesion ability of probiotics or other bacteria of interest. PMID:26370773

  1. Backbone conformational flexibility of the lipid modified membrane anchor of the human N-Ras protein investigated by solid-state NMR and molecular dynamics simulation.

    PubMed

    Vogel, Alexander; Reuther, Guido; Roark, Matthew B; Tan, Kui-Thong; Waldmann, Herbert; Feller, Scott E; Huster, Daniel

    2010-02-01

    The lipid modified human N-Ras protein, implicated in human cancer development, is of particular interest due to its membrane anchor that determines the activity and subcellular location of the protein. Previous solid-state NMR investigations indicated that this membrane anchor is highly dynamic, which may be indicative of backbone conformational flexibility. This article aims to address if a dynamic exchange between three structural models exist that had been determined previously. We applied a combination of solid-state nuclear magnetic resonance (NMR) methods and replica exchange molecular dynamics (MD) simulations using a Ras peptide that represents the terminal seven amino acids of the human N-Ras protein. Analysis of correlations between the conformations of individual amino acids revealed that Cys 181 and Met 182 undergo collective conformational exchange. Two major structures constituting about 60% of all conformations could be identified. The two conformations found in the simulation are in rapid exchange, which gives rise to low backbone order parameters and nuclear spin relaxation as measured by experimental NMR methods. These parameters were also determined from two 300 ns conventional MD simulations, providing very good agreement with the experimental data. PMID:19819220

  2. Ultrasonic/Sonic Anchor

    NASA Technical Reports Server (NTRS)

    Bar-Cohen, Yoseph; Sherrit, Stewart

    2009-01-01

    The ultrasonic/sonic anchor (U/S anchor) is an anchoring device that drills a hole for itself in rock, concrete, or other similar material. The U/S anchor is a recent addition to a series of related devices, the first of which were reported in "Ultrasonic/Sonic Drill/Corers With Integrated Sensors"

  3. Coexpression patterns indicate that GPI-anchored non-specific lipid transfer proteins are involved in accumulation of cuticular wax, suberin and sporopollenin.

    PubMed

    Edstam, Monika M; Blomqvist, Kristina; Eklöf, Anna; Wennergren, Uno; Edqvist, Johan

    2013-12-01

    The non-specific lipid transfer proteins (nsLTP) are unique to land plants. The nsLTPs are characterized by a compact structure with a central hydrophobic cavity and can be classified to different types based on sequence similarity, intron position or spacing between the cysteine residues. The type G nsLTPs (LTPGs) have a GPI-anchor in the C-terminal region which attaches the protein to the exterior side of the plasma membrane. The function of these proteins, which are encoded by large gene families, has not been systematically investigated so far. In this study we have explored microarray data to investigate the expression pattern of the LTPGs in Arabidopsis and rice. We identified that the LTPG genes in each plant can be arranged in three expression modules with significant coexpression within the modules. According to expression patterns and module sizes, the Arabidopsis module AtI is functionally equivalent to the rice module OsI, AtII corresponds to OsII and AtIII is functionally comparable to OsIII. Starting from modules AtI, AtII and AtIII we generated extended networks with Arabidopsis genes coexpressed with the modules. Gene ontology analyses of the obtained networks suggest roles for LTPGs in the synthesis or deposition of cuticular waxes, suberin and sporopollenin. The AtI-module is primarily involved with cuticular wax, the AtII-module with suberin and the AtIII-module with sporopollenin. Further transcript analysis revealed that several transcript forms exist for several of the LTPG genes in both Arabidopsis and rice. The data suggests that the GPI-anchor attachment and localization of LTPGs may be controlled to some extent by alternative splicing. PMID:23893219

  4. Structure of the Sgt2/Get5 complex provides insights into GET-mediated targeting of tail-anchored membrane proteins

    PubMed Central

    Simon, Aline C.; Simpson, Peter J.; Goldstone, Rachael M.; Krysztofinska, Ewelina M.; Murray, James W.; High, Stephen; Isaacson, Rivka L.

    2013-01-01

    Small, glutamine-rich, tetratricopeptide repeat protein 2 (Sgt2) is the first known port of call for many newly synthesized tail-anchored (TA) proteins released from the ribosome and destined for the GET (Guided Entry of TA proteins) pathway. This leads them to the residential membrane of the endoplasmic reticulum via an alternative to the cotranslational, signal recognition particle-dependent mechanism that their topology denies them. In yeast, the first stage of the GET pathway involves Sgt2 passing TA proteins on to the Get4/Get5 complex through a direct interaction between the N-terminal (NT) domain of Sgt2 and the ubiquitin-like (UBL) domain of Get5. Here we characterize this interaction at a molecular level by solving both a solution structure of Sgt2_NT, which adopts a unique helical fold, and a crystal structure of the Get5_UBL. Furthermore, using reciprocal chemical shift perturbation data and experimental restraints, we solve a structure of the Sgt2_NT/Get5_UBL complex, validate it via site-directed mutagenesis, and empirically determine its stoichiometry using relaxation experiments and isothermal titration calorimetry. Taken together, these data provide detailed structural information about the interaction between two key players in the coordinated delivery of TA protein substrates into the GET pathway. PMID:23297211

  5. Bradyrhizobium japonicum TlpA, a novel membrane-anchored thioredoxin-like protein involved in the biogenesis of cytochrome aa3 and development of symbiosis.

    PubMed Central

    Loferer, H; Bott, M; Hennecke, H

    1993-01-01

    We report the discovery of a bacterial gene, tlpA, that codes for a hitherto unknown type of thioredoxin-like protein. The gene was found in the course of studying a Tn5 insertion mutant of the soybean root nodule symbiont Bradyrhizobium japonicum. The TlpA protein shared up to 31% amino acid sequence identity with various eukaryotic and prokaryotic thioredoxins and protein disulfide isomerases, and possessed a characteristic active-site sequence, Trp-Cys-Val-Pro-Cys. In contrast to all members of the thioredoxin family known to date, TlpA was shown to be anchored to the cytoplasmic membrane by means of an N-terminal transmembrane domain, while the active site-containing part of the protein faced the periplasm. The tlpA mutant had a pleiotropic phenotype in that it was defective in the development of a nitrogen fixing endosymbiosis and exhibited a strongly decreased oxidase activity, as compared with the wild-type. Holocytochrome aa3 was spectroscopically undetectable in the mutant, whereas the apoprotein of subunit one (CoxA) of this oxidase was still synthesized and incorporated into the cytoplasmic membrane. Since cytochrome aa3 is not a prerequisite for the development of symbiosis, the results suggest that TlpA is involved in at least two independent cellular processes, one of which is an essential periplasmic step in the maturation of cytochrome aa3. Images PMID:8253065

  6. TssK Is a Trimeric Cytoplasmic Protein Interacting with Components of Both Phage-like and Membrane Anchoring Complexes of the Type VI Secretion System*

    PubMed Central

    Zoued, Abdelrahim; Durand, Eric; Bebeacua, Cecilia; Brunet, Yannick R.; Douzi, Badreddine; Cambillau, Christian; Cascales, Eric; Journet, Laure

    2013-01-01

    The Type VI secretion system (T6SS) is a macromolecular machine that mediates bacteria-host or bacteria-bacteria interactions. The T6SS core apparatus assembles from 13 proteins that form two sub-assemblies: a phage-like complex and a trans-envelope complex. The Hcp, VgrG, TssE, and TssB/C subunits are structurally and functionally related to components of the tail of contractile bacteriophages. This phage-like structure is thought to be anchored to the membrane by a trans-envelope complex composed of the TssJ, TssL, and TssM proteins. However, how the two sub-complexes are connected remains unknown. Here we identify TssK, a protein that establishes contacts with the two T6SS sub-complexes through direct interactions with TssL, Hcp, and TssC. TssK is a cytoplasmic protein assembling trimers that display a three-armed shape, as revealed by TEM and SAXS analyses. Fluorescence microscopy experiments further demonstrate the requirement of TssK for sheath assembly. Our results suggest a central role for TssK by linking both complexes during T6SS assembly. PMID:23921384

  7. Glycosylphosphatidylinositol Anchor Modification Machinery Deficiency Is Responsible for the Formation of Pro-Prion Protein (PrP) in BxPC-3 Protein and Increases Cancer Cell Motility.

    PubMed

    Yang, Liheng; Gao, Zhenxing; Hu, Lipeng; Wu, Guiru; Yang, Xiaowen; Zhang, Lihua; Zhu, Ying; Wong, Boon-Seng; Xin, Wei; Sy, Man-Sun; Li, Chaoyang

    2016-02-19

    The normal cellular prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein. However, in pancreatic ductal adenocarcinoma cell lines, such as BxPC-3, PrP exists as a pro-PrP retaining its glycosylphosphatidylinositol (GPI) peptide signaling sequence. Here, we report the identification of another pancreatic ductal adenocarcinoma cell line, AsPC-1, which expresses a mature GPI-anchored PrP. Comparison of the 24 genes involved in the GPI anchor modification pathway between AsPC-1 and BxPC-3 revealed 15 of the 24 genes, including PGAP1 and PIG-F, were down-regulated in the latter cells. We also identified six missense mutations in DPM2, PIG-C, PIG-N, and PIG-P alongside eight silent mutations. When BxPC-3 cells were fused with Chinese hamster ovary (CHO) cells, which lack endogenous PrP, pro-PrP was successfully converted into mature GPI-anchored PrP. Expression of the individual gene, such as PGAP1, PIG-F, or PIG-C, into BxPC-3 cells does not result in phosphoinositide-specific phospholipase C sensitivity of PrP. However, when PIG-F but not PIG-P is expressed in PGAP1-expressing BxPC-3 cells, PrP on the surface of the cells becomes phosphoinositide-specific phospholipase C-sensitive. Thus, low expression of PIG-F and PGAP1 is the major factor contributing to the accumulation of pro-PrP. More importantly, BxPC-3 cells expressing GPI-anchored PrP migrate much slower than BxPC-3 cells bearing pro-PrP. In addition, GPI-anchored PrP-bearing AsPC-1 cells also migrate slower than pro-PrP bearing BxPC-3 cells, although both cells express filamin A. "Knocking out" PRNP in BxPC-3 cell drastically reduces its migration. Collectively, these results show that multiple gene irregularity in BxPC-3 cells is responsible for the formation of pro-PrP, and binding of pro-PrP to filamin A contributes to enhanced tumor cell motility. PMID:26683373

  8. Transcriptional activation of the anchoring protein SAP97 by heat shock factor (HSF)-1 stabilizes Kv1.5 channels in HL-1 cells

    PubMed Central

    Ting, YK; Morikawa, K; Kurata, Y; Li, P; Bahrudin, U; Mizuta, E; Kato, M; Miake, J; Yamamoto, Y; Yoshida, A; Murata, M; Inoue, T; Nakai, A; Shiota, G; Higaki, K; Nanba, E; Ninomiya, H; Shirayoshi, Y; Hisatome, I

    2011-01-01

    BACKGROUND AND PURPOSE The expression of voltage-dependent K+ channels (Kv) 1.5 is regulated by members of the heat shock protein (Hsp) family. We examined whether the heat shock transcription factor 1 (HSF-1) and its inducer geranylgeranylacetone (GGA) could affect the expression of Kv1.5 channels and its anchoring protein, synapse associated protein 97 (SAP97). EXPERIMENTAL APPROACH Transfected mouse atrial cardiomyocytes (HL-1 cells) and COS7 cells were subjected to luciferase reporter gene assay and whole-cell patch clamp. Protein and mRNA extracts were subjected to Western blot and quantitative real-time polymerase chain reaction. KEY RESULTS Heat shock of HL-1 cells induced expression of Hsp70, HSF-1, SAP97 and Kv1.5 proteins. These effects were reproduced by wild-type HSF-1. Both heat shock and expression of HSF-1, but not the R71G mutant, increased the SAP97 mRNA level. Small interfering RNA (siRNA) against SAP97 abolished HSF-1-induced increase of Kv1.5 and SAP97 proteins. A luciferase reporter gene assay revealed that the SAP97 promoter region (from −919 to −740) that contains heat shock elements (HSEs) was required for this induction. Suppression of SIRT1 function either by nicotinamide or siRNA decreased the level of SAP97 mRNA. SIRT1 activation by resveratrol had opposing effects. A treatment of the cells with GGA increased the level of SAP97 mRNA, Kv1.5 proteins and IKur current, which could be modified with either resveratrol or nicotinamide. CONCLUSIONS AND IMPLICATIONS HSF-1 induced transcription of SAP97 through SIRT1-dependent interaction with HSEs; the increase in SAP97 resulted in stabilization of Kv1.5 channels. These effects were mimicked by GGA. PMID:21232033

  9. A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis

    SciTech Connect

    Rosiere, T.K.; Marrs, J.A.; Bouck, G.B. )

    1990-04-01

    The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD binding domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39.

  10. GhLTPG1, a cotton GPI-anchored lipid transfer protein, regulates the transport of phosphatidylinositol monophosphates and cotton fiber elongation

    PubMed Central

    Deng, Ting; Yao, Hongyan; Wang, Jin; Wang, Jun; Xue, Hongwei; Zuo, Kaijing

    2016-01-01

    The cotton fibers are seed trichomes that elongate from the ovule epidermis. Polar lipids are required for the quick enlargement of cell membrane and fiber cell growth, however, how lipids are transported from the ovules into the developing fibers remains less known. Here, we reported the functional characterization of GhLTPG1, a GPI-anchored lipid transport protein, during cotton fiber elongation. GhLTPG1 was abundantly expressed in elongating cotton fibers and outer integument of the ovules, and GhLTPG1 protein was located on cell membrane. Biochemical analysis showed that GhLTPG1 specifically bound to phosphatidylinositol mono-phosphates (PtdIns3P, PtdIns4P and PtdIns5P) in vitro and transported PtdInsPs from the synthesis places to the plasma membranes in vivo. Expression of GhLTPG1 in Arabidopsis caused an increased number of trichomes, and fibers in GhLTPG1-knockdown cotton plants exhibited significantly reduced length, decreased polar lipid content, and repression of fiber elongation-related genes expression. These results suggested that GhLTPG1 protein regulates the cotton fiber elongation through mediating the transport of phosphatidylinositol monophosphates. PMID:27311358

  11. GhLTPG1, a cotton GPI-anchored lipid transfer protein, regulates the transport of phosphatidylinositol monophosphates and cotton fiber elongation.

    PubMed

    Deng, Ting; Yao, Hongyan; Wang, Jin; Wang, Jun; Xue, Hongwei; Zuo, Kaijing

    2016-01-01

    The cotton fibers are seed trichomes that elongate from the ovule epidermis. Polar lipids are required for the quick enlargement of cell membrane and fiber cell growth, however, how lipids are transported from the ovules into the developing fibers remains less known. Here, we reported the functional characterization of GhLTPG1, a GPI-anchored lipid transport protein, during cotton fiber elongation. GhLTPG1 was abundantly expressed in elongating cotton fibers and outer integument of the ovules, and GhLTPG1 protein was located on cell membrane. Biochemical analysis showed that GhLTPG1 specifically bound to phosphatidylinositol mono-phosphates (PtdIns3P, PtdIns4P and PtdIns5P) in vitro and transported PtdInsPs from the synthesis places to the plasma membranes in vivo. Expression of GhLTPG1 in Arabidopsis caused an increased number of trichomes, and fibers in GhLTPG1-knockdown cotton plants exhibited significantly reduced length, decreased polar lipid content, and repression of fiber elongation-related genes expression. These results suggested that GhLTPG1 protein regulates the cotton fiber elongation through mediating the transport of phosphatidylinositol monophosphates. PMID:27311358

  12. Roles of the protruding loop of factor B essential for the localization of lipoproteins (LolB) in the anchoring of bacterial triacylated proteins to the outer membrane.

    PubMed

    Hayashi, Yumi; Tsurumizu, Ryoji; Tsukahara, Jun; Takeda, Kazuki; Narita, Shin-ichiro; Mori, Makiko; Miki, Kunio; Tokuda, Hajime

    2014-04-11

    The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes. LolB is itself a lipoprotein anchored to outer membranes, although the membrane anchor is functionally dispensable. LolB then localizes lipoproteins to outer membranes through largely unknown mechanisms. The crystal structure of LolB is similar to that of LolA, and it possesses a hydrophobic cavity that accommodates acyl chains of lipoproteins. To elucidate the molecular function of LolB, a periplasmic version of LolB, mLolB, was mutagenized at various conserved residues. Despite the lack of acyl chains, most defective mutants were insoluble. However, a derivative with glutamate in place of leucine 68 was soluble and unable to localize lipoproteins to outer membranes. This leucine is present in a loop protruding from mLolB into an aqueous environment, and no analogous loop is present in LolA. Thus, leucine 68 was replaced with other residues. Replacement by acidic, but not hydrophobic, residues generated for the first time mLolB derivatives that can accept but cannot localize lipoproteins to outer membranes. Moreover, deletion of the leucine with neighboring residues impaired the lipoprotein receptor activity. Based on these observations, the roles of the protruding loop of LolB in the last step of lipoprotein sorting are discussed. PMID:24569999

  13. The nuclear pore complex protein ALADIN is anchored via NDC1 but not via POM121 and GP210 in the nuclear envelope

    SciTech Connect

    Kind, Barbara; Koehler, Katrin; Lorenz, Mike; Huebner, Angela

    2009-12-11

    The nuclear pore complex (NPC) consists of {approx}30 different proteins and provides the only sites for macromolecular transport between cytoplasm and nucleus. ALADIN was discovered as a new member of the NPC. Mutations in ALADIN are known to cause triple A syndrome, a rare autosomal recessive disorder characterized by adrenal insufficiency, alacrima, and achalasia. The function and exact location of the nucleoporin ALADIN within the NPC multiprotein complex is still unclear. Using a siRNA-based approach we downregulated the three known membrane integrated nucleoporins NDC1, GP210, and POM121 in stably expressing GFP-ALADIN HeLa cells. We identified NDC1 but not GP210 and POM121 as the main anchor of ALADIN within the NPC. Solely the depletion of NDC1 caused mislocalization of ALADIN. Vice versa, the depletion of ALADIN led also to disappearance of NDC1 at the NPC. However, the downregulation of two further membrane-integral nucleoporins GP210 and POM121 had no effect on ALADIN localization. Furthermore, we could show a direct association of NDC1 and ALADIN in NPCs by fluorescence resonance energy transfer (FRET) measurements. Based on our findings we conclude that ALADIN is anchored in the nuclear envelope via NDC1 and that this interaction gets lost, if ALADIN is mutated. The loss of integration of ALADIN in the NPC is a main pathogenetic aspect for the development of the triple A syndrome and suggests that the interaction between ALADIN and NDC1 may be involved in the pathogenesis of the disease.

  14. Roles of the Protruding Loop of Factor B Essential for the Localization of Lipoproteins (LolB) in the Anchoring of Bacterial Triacylated Proteins to the Outer Membrane*

    PubMed Central

    Hayashi, Yumi; Tsurumizu, Ryoji; Tsukahara, Jun; Takeda, Kazuki; Narita, Shin-ichiro; Mori, Makiko; Miki, Kunio; Tokuda, Hajime

    2014-01-01

    The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes. LolB is itself a lipoprotein anchored to outer membranes, although the membrane anchor is functionally dispensable. LolB then localizes lipoproteins to outer membranes through largely unknown mechanisms. The crystal structure of LolB is similar to that of LolA, and it possesses a hydrophobic cavity that accommodates acyl chains of lipoproteins. To elucidate the molecular function of LolB, a periplasmic version of LolB, mLolB, was mutagenized at various conserved residues. Despite the lack of acyl chains, most defective mutants were insoluble. However, a derivative with glutamate in place of leucine 68 was soluble and unable to localize lipoproteins to outer membranes. This leucine is present in a loop protruding from mLolB into an aqueous environment, and no analogous loop is present in LolA. Thus, leucine 68 was replaced with other residues. Replacement by acidic, but not hydrophobic, residues generated for the first time mLolB derivatives that can accept but cannot localize lipoproteins to outer membranes. Moreover, deletion of the leucine with neighboring residues impaired the lipoprotein receptor activity. Based on these observations, the roles of the protruding loop of LolB in the last step of lipoprotein sorting are discussed. PMID:24569999

  15. Anchoring skeletal muscle development and disease: the role of ankyrin repeat domain containing proteins in muscle physiology.

    PubMed

    Tee, Jin-Ming; Peppelenbosch, Maikel P

    2010-08-01

    The ankyrin repeat is a protein module with high affinity for other ankyrin repeats based on strong Van der Waals forces. The resulting dimerization is unusually resistant to both mechanical forces and alkanization, making this module exceedingly useful for meeting the extraordinary demands of muscle physiology. Many aspects of muscle function are controlled by the superfamily ankyrin repeat domain containing proteins, including structural fixation of the contractile apparatus to the muscle membrane by ankyrins, the archetypical member of the family. Additionally, other ankyrin repeat domain containing proteins critically control the various differentiation steps during muscle development, with Notch and developmental stage-specific expression of the members of the Ankyrin repeat and SOCS box (ASB) containing family of proteins controlling compartment size and guiding the various steps of muscle specification. Also, adaptive responses in fully formed muscle require ankyrin repeat containing proteins, with Myotrophin/V-1 ankyrin repeat containing proteins controlling the induction of hypertrophic responses following excessive mechanical load, and muscle ankyrin repeat proteins (MARPs) acting as protective mechanisms of last resort following extreme demands on muscle tissue. Knowledge on mechanisms governing the ordered expression of the various members of superfamily of ankyrin repeat domain containing proteins may prove exceedingly useful for developing novel rational therapy for cardiac disease and muscle dystrophies. PMID:20515317

  16. Tromp1, a putative rare outer membrane protein, is anchored by an uncleaved signal sequence to the Treponema pallidum cytoplasmic membrane.

    PubMed Central

    Akins, D R; Robinson, E; Shevchenko, D; Elkins, C; Cox, D L; Radolf, J D

    1997-01-01

    Treponema pallidum rare outer membrane protein 1 (Tromp1) has extensive sequence homology with substrate-binding proteins of ATP-binding cassette transporters. Because such proteins typically are periplasmic or cytoplasmic membrane associated, experiments were conducted to clarify Tromp1's physicochemical properties and cellular location in T. pallidum. Comparison of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities of (i) native Tromp1 and Tromp1 synthesized by coupled in vitro transcription-translation and (ii) native Tromp1 and recombinant Tromp1 lacking the N-terminal signal sequence revealed that the native protein is not processed. Other studies demonstrated that recombinant Tromp1 lacks three basic porin-like properties: (i) the ability to form aqueous channels in liposomes which permit the influx of small hydrophilic solutes, (ii) an extensive beta-sheet secondary structure, and (iii) amphiphilicity. Subsurface localization of native Tromp1 was demonstrated by immunofluorescence analysis of treponemes encapsulated in gel microdroplets, while opsonization assays failed to detect surface-exposed Tromp1. Incubation of motile treponemes with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazarine, a photoactivatable, lipophilic probe, also did not result in the detection of Tromp1 within the outer membranes of intact treponemes but, instead, resulted in the labeling of a basic 30.5-kDa presumptive outer membrane protein. Finally, analysis of fractionated treponemes revealed that native Tromp1 is associated predominantly with cell cylinders. These findings comprise a body of evidence that Tromp1 actually is anchored by an uncleaved signal sequence to the periplasmic face of the T. pallidum cytoplasmic membrane, where it likely subserves a transport-related function. PMID:9260949

  17. Display of alpha-amylase on the surface of Lactobacillus casei cells by use of the PgsA anchor protein, and production of lactic acid from starch.

    PubMed

    Narita, Junya; Okano, Kenji; Kitao, Tomoe; Ishida, Saori; Sewaki, Tomomitsu; Sung, Moon-Hee; Fukuda, Hideki; Kondo, Akihiko

    2006-01-01

    We developed a new cell surface engineering system based on the PgsA anchor protein from Bacillus subtilis. In this system, the N terminus of the target protein was fused to the PgsA protein and the resulting fusion protein was expressed on the cell surface. Using this new system, we constructed a novel starch-degrading strain of Lactobacillus casei by genetically displaying alpha-amylase from the Streptococcus bovis strain 148 with a FLAG peptide tag (AmyAF). Localization of the PgsA-AmyA-FLAG fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. The lactic acid bacteria which displayed AmyAF showed significantly elevated hydrolytic activity toward soluble starch. By fermentation using AmyAF-displaying L. casei cells, 50 g/liter of soluble starch was reduced to 13.7 g/liter, and 21.8 g/liter of lactic acid was produced within about 24 h. The yield in terms of grams of lactic acid produced per gram of carbohydrate utilized was 0.60 g per g of carbohydrate consumed at 24 h. Since AmyA was immobilized on the cells, cells were recovered after fermentation and used repeatedly. During repeated utilization of cells, the lactic acid yield was improved to 0.81 g per g of carbohydrate consumed at 72 h. These results indicate that efficient simultaneous saccharification and fermentation from soluble starch to lactic acid were carried out by recombinant L. casei cells with cell surface display of AmyA. PMID:16391053

  18. Display of α-Amylase on the Surface of Lactobacillus casei Cells by Use of the PgsA Anchor Protein, and Production of Lactic Acid from Starch

    PubMed Central

    Narita, Junya; Okano, Kenji; Kitao, Tomoe; Ishida, Saori; Sewaki, Tomomitsu; Sung, Moon-Hee; Fukuda, Hideki; Kondo, Akihiko

    2006-01-01

    We developed a new cell surface engineering system based on the PgsA anchor protein from Bacillus subtilis. In this system, the N terminus of the target protein was fused to the PgsA protein and the resulting fusion protein was expressed on the cell surface. Using this new system, we constructed a novel starch-degrading strain of Lactobacillus casei by genetically displaying α-amylase from the Streptococcus bovis strain 148 with a FLAG peptide tag (AmyAF). Localization of the PgsA-AmyA-FLAG fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. The lactic acid bacteria which displayed AmyAF showed significantly elevated hydrolytic activity toward soluble starch. By fermentation using AmyAF-displaying L. casei cells, 50 g/liter of soluble starch was reduced to 13.7 g/liter, and 21.8 g/liter of lactic acid was produced within about 24 h. The yield in terms of grams of lactic acid produced per gram of carbohydrate utilized was 0.60 g per g of carbohydrate consumed at 24 h. Since AmyA was immobilized on the cells, cells were recovered after fermentation and used repeatedly. During repeated utilization of cells, the lactic acid yield was improved to 0.81 g per g of carbohydrate consumed at 72 h. These results indicate that efficient simultaneous saccharification and fermentation from soluble starch to lactic acid were carried out by recombinant L. casei cells with cell surface display of AmyA. PMID:16391053

  19. A Plasma Membrane-Anchored Fluorescent Protein Fusion Illuminates Sieve Element Plasma Membranes in Arabidopsis and Tobacco1[W][OA

    PubMed Central

    Thompson, Matthew V.; Wolniak, Stephen M.

    2008-01-01

    Rapid acquisition of quantitative anatomical data from the sieve tubes of angiosperm phloem has been confounded by their small size, their distance from organ surfaces, and the time-consuming nature of traditional methods, such as transmission electron microscopy. To improve access to these cells, for which good anatomical data are critical, a monomeric yellow fluorescent protein (mCitrine) was N-terminally fused to a small (approximately 6 kD) membrane protein (AtRCI2A) and stably expressed in Arabidopsis thaliana (Columbia-0 ecotype) and Nicotiana tabacum (‘Samsun’) under the control of a companion cell-specific promoter (AtSUC2p). The construct, called by its abbreviation SUmCR, yielded stable sieve element (SE) plasma membrane fluorescence labeling, even after plastic (methacrylate) embedding. In conjunction with wide-field fluorescence measurements of sieve pore number and position using aniline blue-stained callose, mCitrine-labeled material was used to calculate rough estimates of sieve tube-specific conductivity for both species. The SUmCR construct also revealed a hitherto unknown expression domain of the AtSUC2 Suc-H+ symporter in the epidermis of the cell division zone of developing root tips. The success of this construct in targeting plasma membrane-anchored fluorescent proteins to SEs could be attributable to the small size of AtRCI2A or to the presence of other signals innate to AtRCI2A that permit the protein to be trafficked to SEs. The construct provides a hitherto unique entrée into companion cell-to-SE protein targeting, as well as a new tool for studying whole-plant phloem anatomy and architecture. PMID:18223149

  20. The conserved lysine of the catalytic domain of protein kinases is actively involved in the phosphotransfer reaction and not required for anchoring ATP.

    PubMed Central

    Carrera, A C; Alexandrov, K; Roberts, T M

    1993-01-01

    The study of the various protein kinases reveals that, despite their considerably diversity, they have evolved from a common origin. Eleven conserved subdomains have been described that encompass the catalytic core of these enzymes. One of these conserved regions, subdomain II, contains an invariant lysine residue present in all known protein kinase catalytic domains. Two facts have suggested that this conserved lysine of subdomain II is essential for binding ATP: (i) several investigators have demonstrated that this residue is physically proximal to the ATP molecule, and (ii) conservative substitutions at this site render the kinase inactive. However, these results are also consistent with a functional role of the conserved lysine of subdomain II in orienting or facilitating the transfer of phosphate. To study in more detail the role of subdomain II, we have generated mutants of the protein-tyrosine kinase pp56lck that have single amino acid substitutions within the area surrounding the conserved residue Lys-273 in subdomain II. When compared with wild-type pp56lck, these mutants displayed profound reductions in their phosphotransfer efficiencies and small differences in their affinities for ATP. Further, the substitution of arginine for Lys-273 resulted in a mutant protein unable to transfer the gamma-phosphate of ATP but able to bind 8-azido-ATP with an efficiency similar to that of wild-type pp56lck. These results suggest that the region including Lys-273 of subdomain II is involved in the enzymatic process of phosphate transfer, rather than in anchoring ATP. Images PMID:8421674

  1. Mass spectrometric identification of glycosylphosphatidylinositol-anchored peptides.

    PubMed

    Masuishi, Yusuke; Nomura, Ayako; Okayama, Akiko; Kimura, Yayoi; Arakawa, Noriaki; Hirano, Hisashi

    2013-10-01

    Glycosylphosphatidylinositol (GPI) anchoring is a post-translational modification widely observed among eukaryotic membrane proteins. GPI anchors are attached to proteins via the carboxy-terminus in the outer leaflet of the cell membrane, where GPI-anchored proteins (GPI-APs) perform important functions as coreceptors and enzymes. Precursors of GPI-APs (Pre-GPI-APs) contain a C-terminal hydrophobic sequence that is involved in cleavage of the signal sequence from the protein and addition of the GPI anchor by the transamidase complex. In order to confirm that a given protein contains a GPI anchor, it is essential to identify the C-terminal peptide containing the GPI-anchor modification site (ω-site). Previously, efficient identification of GPI-anchored C-terminal peptides by mass spectrometry has been difficult, in part because of complex structure of the GPI-anchor moiety. We developed a method to experimentally identify GPI-APs and their ω-sites. In this method, a part of GPI-anchor moieties are removed from GPI-anchored peptides using phosphatidylinositol-specific phospholipase C (PI-PLC) and aqueous hydrogen fluoride (HF), and peptide sequence is then determined by mass spectrometry. Using this method, we successfully identified 10 GPI-APs and 12 ω-sites in the cultured ovarian adenocarcinoma cells, demonstrating that this method is useful for identifying efficiently GPI-APs. PMID:24001144

  2. Complement Factor H-Related 5-Hybrid Proteins Anchor Properdin and Activate Complement at Self-Surfaces.

    PubMed

    Chen, Qian; Manzke, Melanie; Hartmann, Andrea; Büttner, Maike; Amann, Kerstin; Pauly, Diana; Wiesener, Michael; Skerka, Christine; Zipfel, Peter F

    2016-05-01

    C3 glomerulopathy (C3G) is a severe kidney disease for which no specific therapy exists. The causes of C3G are heterogeneous, and defective complement regulation is often linked to C3G pathogenesis. Copy number variations in the complement factor H-related (CFHR) gene cluster on chromosome 1q32 and CFHR5 mutant proteins associate with this disease. Here, we identified CFHR5 as a pattern recognition protein that binds to damaged human endothelial cell surfaces and to properdin, the human complement activator. We found the two N-terminal short consensus repeat domains of CFHR5 contact properdin and mediate dimer formation. These properdin-binding segments are duplicated in two mutant CFHR5 proteins, CFHR2-CFHR5Hyb from German patients with C3G and CFHR5Dup from Cypriot patients with C3G. Each of these mutated proteins assembled into large multimeric complexes and, compared to CFHR5, bound damaged human cell surfaces and properdin with greater intensity and exacerbated local complement activation. This enhanced surface binding and properdin recruitment was further evidenced in the mesangia of a transplanted and explanted kidney from a German patient with a CFHR2-CFHR5Hyb protein. Enhanced properdin staining correlated with local complement activation with C3b and C5b-9 deposition on the mesangial cell surface in vitro This gain of function in complement activation for two disease-associated CFHR5 mutants describes a new disease mechanism of C3G, which is relevant for defining appropriate treatment options for this disorder. PMID:26432903

  3. A role for the CaM Kinase II related anchoring protein (αkap) in maintaining the stability of nicotinic Acetylcholine Receptors

    PubMed Central

    Mouslim, Chakib; Aittaleb, Mohamed; Hume, Richard I.; Akaaboune, Mohammed

    2012-01-01

    αkap, a muscle specific anchoring protein encoded within the Camk2a gene is thought to play a role in targeting multiple calcium/calmodulin kinase II isoforms to specific subcellular locations. Here we demonstrate a novel function of αkap in stabilizing nicotinic acetylcholine receptors (AChR). Knockdown of αkap expression with shRNA significantly enhanced the degradation of AChR α-subunits (AChRα), leading to fewer and smaller AChR clusters on the surface of differentiated C2C12 myotubes. Mutagenesis and biochemical studies in HEK293T cells revealed that αkap promoted AChRα stability by a ubiquitin-dependent mechanism. In the absence of αkap, AChRα was heavily ubiquitinated and the number of AChRα was increased by proteasome inhibitors. However, in the presence of αkap, AChRα was less ubiquitinated and proteasome inhibitors had almost no effect on AChRα accumulation. The major sites of AChRα ubiquitination reside within the large intracellular loop and mutations of critical lysine residues in this loop to arginine increased AChRα stability in the absence of αkap. These results provide an unexpected mechanism by which αkap controls receptor trafficking onto the surface of muscle cells, and thus the maintenance of postsynaptic receptor density and synaptic function. PMID:22496563

  4. Anchors for Education Reforms

    ERIC Educational Resources Information Center

    Alok, Kumar

    2012-01-01

    Education reforms, considering their significance, deserve better methods than mere "trial and error." This article conceptualizes a network of six anchors for education reforms: education policy, education system, curriculum, pedagogy, assessment, and teacher education. It establishes the futility to reform anchors in isolation and anticipates…

  5. Identification and Characterization of a Novel Issatchenkia orientalis GPI-Anchored Protein, IoGas1, Required for Resistance to Low pH and Salt Stress.

    PubMed

    Matsushika, Akinori; Negi, Kanako; Suzuki, Toshihiro; Goshima, Tetsuya; Hoshino, Tamotsu

    2016-01-01

    The use of yeasts tolerant to acid (low pH) and salt stress is of industrial importance for several bioproduction processes. To identify new candidate genes having potential roles in low-pH tolerance, we screened an expression genomic DNA library of a multiple-stress-tolerant yeast, Issatchenkia orientalis (Pichia kudriavzevii), for clones that allowed Saccharomyces cerevisiae cells to grow under highly acidic conditions (pH 2.0). A genomic DNA clone containing two putative open reading frames was obtained, of which the putative protein-coding gene comprising 1629 bp was retransformed into the host. This transformant grew significantly at pH 2.0, and at pH 2.5 in the presence of 7.5% Na2SO4. The predicted amino acid sequence of this new gene, named I. orientalis GAS1 (IoGAS1), was 60% identical to the S. cerevisiae Gas1 protein, a glycosylphosphatidylinositol-anchored protein essential for maintaining cell wall integrity, and 58-59% identical to Candida albicans Phr1 and Phr2, pH-responsive proteins implicated in cell wall assembly and virulence. Northern hybridization analyses indicated that, as for the C. albicans homologs, IoGAS1 expression was pH-dependent, with expression increasing with decreasing pH (from 4.0 to 2.0) of the medium. These results suggest that IoGAS1 represents a novel pH-regulated system required for the adaptation of I. orientalis to environments of diverse pH. Heterologous expression of IoGAS1 complemented the growth and morphological defects of a S. cerevisiae gas1Δ mutant, demonstrating that IoGAS1 and the corresponding S. cerevisiae gene play similar roles in cell wall biosynthesis. Site-directed mutagenesis experiments revealed that two conserved glutamate residues (E161 and E262) in the IoGas1 protein play a crucial role in yeast morphogenesis and tolerance to low pH and salt stress. Furthermore, overexpression of IoGAS1 in S. cerevisiae remarkably improved the ethanol fermentation ability at pH 2.5, and at pH 2.0 in the presence of

  6. Salmonella Effectors SseF and SseG Interact with Mammalian Protein ACBD3 (GCP60) To Anchor Salmonella-Containing Vacuoles at the Golgi Network

    PubMed Central

    Yu, Xiu-Jun; Liu, Mei

    2016-01-01

    ABSTRACT Following infection of mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) replicates within membrane-bound compartments known as Salmonella-containing vacuoles (SCVs). The Salmonella pathogenicity island 2 type III secretion system (SPI-2 T3SS) translocates approximately 30 different effectors across the vacuolar membrane. SseF and SseG are two such effectors that are required for SCVs to localize close to the Golgi network in infected epithelial cells. In a yeast two-hybrid assay, SseG and an N-terminal variant of SseF interacted directly with mammalian ACBD3, a multifunctional cytosolic Golgi network-associated protein. Knockdown of ACBD3 by small interfering RNA (siRNA) reduced epithelial cell Golgi network association of wild-type bacteria, phenocopying the effect of null mutations of sseG or sseF. Binding of SseF to ACBD3 in infected cells required the presence of SseG. A single-amino-acid mutant of SseG and a double-amino-acid mutant of SseF were obtained that did not interact with ACBD3 in Saccharomyces cerevisiae. When either of these was produced together with the corresponding wild-type effector by Salmonella in infected cells, they enabled SCV-Golgi network association and interacted with ACBD3. However, these properties were lost and bacteria displayed an intracellular replication defect when cells were infected with Salmonella carrying both mutant genes. Knockdown of ACBD3 resulted in a replication defect of wild-type bacteria but did not further attenuate the growth defect of a ΔsseFG mutant strain. We propose a model in which interaction between SseF and SseG enables both proteins to bind ACBD3, thereby anchoring SCVs at the Golgi network and facilitating bacterial replication. PMID:27406559

  7. The SrkA Kinase Is Part of the SakA Mitogen-Activated Protein Kinase Interactome and Regulates Stress Responses and Development in Aspergillus nidulans

    PubMed Central

    Jaimes-Arroyo, Rafael; Lara-Rojas, Fernando; Bayram, Özgür; Valerius, Oliver; Braus, Gerhard H.

    2015-01-01

    Fungi and many other eukaryotes use specialized mitogen-activated protein kinases (MAPK) of the Hog1/p38 family to transduce environmental stress signals. In Aspergillus nidulans, the MAPK SakA and the transcription factor AtfA are components of a central multiple stress-signaling pathway that also regulates development. Here we characterize SrkA, a putative MAPK-activated protein kinase, as a novel component of this pathway. ΔsrkA and ΔsakA mutants share a derepressed sexual development phenotype. However, ΔsrkA mutants are not sensitive to oxidative stress, and in fact, srkA inactivation partially suppresses the sensitivity of ΔsakA mutant conidia to H2O2, tert-butyl-hydroperoxide (t-BOOH), and menadione. In the absence of stress, SrkA shows physical interaction with nonphosphorylated SakA in the cytosol. We show that H2O2 induces a drastic change in mitochondrial morphology consistent with a fission process and the relocalization of SrkA to nuclei and mitochondria, depending on the presence of SakA. SakA-SrkA nuclear interaction is also observed during normal asexual development in dormant spores. Using SakA and SrkA S-tag pulldown and purification studies coupled to mass spectrometry, we found that SakA interacts with SrkA, the stress MAPK MpkC, the PPT1-type phosphatase AN6892, and other proteins involved in cell cycle regulation, DNA damage response, mRNA stability and protein synthesis, mitochondrial function, and other stress-related responses. We propose that oxidative stress induces DNA damage and mitochondrial fission and that SakA and SrkA mediate cell cycle arrest and regulate mitochondrial function during stress. Our results provide new insights into the mechanisms by which SakA and SrkA regulate the remodelling of cell physiology during oxidative stress and development. PMID:25820520

  8. Therapeutic strategies for anchored kinases and phosphatases: exploiting short linear motifs and intrinsic disorder

    PubMed Central

    Nygren, Patrick J.; Scott, John D.

    2015-01-01

    Phosphorylation events that occur in response to the second messenger cAMP are controlled spatially and temporally by protein kinase A (PKA) interacting with A-kinase anchoring proteins (AKAPs). Recent advances in understanding the structural basis for this interaction have reinforced the hypothesis that AKAPs create spatially constrained signaling microdomains. This has led to the realization that the PKA/AKAP interface is a potential drug target for modulating a plethora of cell-signaling events. Pharmacological disruption of kinase–AKAP interactions has previously been explored for disease treatment and remains an interesting area of research. However, disrupting or enhancing the association of phosphatases with AKAPs is a therapeutic concept of equal promise, particularly since they oppose the actions of many anchored kinases. Accordingly, numerous AKAPs bind phosphatases such as protein phosphatase 1 (PP1), calcineurin (PP2B), and PP2A. These multimodal signaling hubs are equally able to control the addition of phosphate groups onto target substrates, as well as the removal of these phosphate groups. In this review, we describe recent advances in structural analysis of kinase and phosphatase interactions with AKAPs, and suggest future possibilities for targeting these interactions for therapeutic benefit. PMID:26283967

  9. cPLA2α and EHD1 interact and regulate the vesiculation of cholesterol-rich, GPI-anchored, protein-containing endosomes

    PubMed Central

    Cai, Bishuang; Caplan, Steve; Naslavsky, Naava

    2012-01-01

    The lipid modifier phospholipase A2 catalyzes the hydrolysis of phospholipids to inverted-cone–shaped lysophospholipids that contribute to membrane curvature and/or tubulation. Conflicting findings exist regarding the function of cytosolic phospholipase A2 (cPLA2) and its role in membrane regulation at the Golgi and early endosomes. However, no studies addressed the role of cPLA2 in the regulation of cholesterol-rich membranes that contain glycosylphosphatidylinositol-anchored proteins (GPI-APs). Our studies support a role for cPLA2α in the vesiculation of GPI-AP–containing membranes, using endogenous CD59 as a model for GPI-APs. On cPLA2α depletion, CD59-containing endosomes became hypertubular. Moreover, accumulation of lysophospholipids induced by a lysophospholipid acyltransferase inhibitor extensively vesiculated CD59-containing endosomes. However, overexpression of cPLA2α did not increase the endosomal vesiculation, implying a requirement for additional factors. Indeed, depletion of the “pinchase” EHD1, a C-terminal Eps15 homology domain (EHD) ATPase, also induced hypertubulation of CD59-containing endosomes. Furthermore, EHD1 and cPLA2α demonstrated in situ proximity (<40 nm) and interacted in vivo. The results presented here provide evidence that the lipid modifier cPLA2α and EHD1 are involved in the vesiculation of CD59-containing endosomes. We speculate that cPLA2α induces membrane curvature and allows EHD1, possibly in the context of a complex, to sever the curved membranes into vesicles. PMID:22456504

  10. Bivalent Ligation of the Collagen-binding Modules of Fibronectin by SFS, a Non-anchored Bacterial Protein of Streptococcus equi*

    PubMed Central

    Ma, Wenjiang; Ma, Hanqing; Fogerty, Frances J.; Mosher, Deane F.

    2015-01-01

    SFS is a non-anchored protein of Streptococcus equi subspecies equi that causes upper respiratory infection in horses. SFS has been shown to bind to fibronectin (FN) and block interaction of FN with type I collagen. We have characterized interactions of a recombinant 60-mer polypeptide, R1R2, with FN. R1R2 contains two copies of collagen-like 19-residue repeats. Experiments utilizing various FN fragments and epitope-mapped anti-FN monoclonal antibodies located the binding site to 8-9FNI modules of the gelatin-binding domain. Fluorescence polarization and competitive enzyme-linked assays demonstrated that R1R2 binds preferentially to compact dimeric FN rather than monomeric constructs containing 8-9FNI or a large dimeric FN construct that is constitutively in an extended conformation. In contrast to bacterial peptides that bind 2–5FNI in addition to 8-9FNI, R1R2 did not cause conformational extension of FN as assessed by a conformationally sensitive antibody. Equilibrium and stopped-flow binding assays and size exclusion chromatography were compatible with a two-step binding reaction in which each of the repeats of R1R2 interacts with one of the subunits of dimeric FN, resulting in a stable complex with a slow koff. In addition to not binding to type I collagen, the R1R2·FN complex incorporated less efficiently into extracellular matrix than free FN. Thus, R1R2 binds to FN utilizing features of compact soluble FN and in doing so interferes with the organization of the extracellular matrix. A similar bivalent binding strategy may underlie the collagen-FN interaction. PMID:25525266

  11. Anchor Trial Launch

    Cancer.gov

    NCI has launched a multicenter phase III clinical trial called the ANCHOR Study -- Anal Cancer HSIL (High-grade Squamous Intraepithelial Lesion) Outcomes Research Study -- to determine if treatment of HSIL in HIV-infected individuals can prevent anal canc

  12. A Kinase Inhibitor Screen Reveals Protein Kinase C-dependent Endocytic Recycling of ErbB2 in Breast Cancer Cells*

    PubMed Central

    Bailey, Tameka A.; Luan, Haitao; Tom, Eric; Bielecki, Timothy Alan; Mohapatra, Bhopal; Ahmad, Gulzar; George, Manju; Kelly, David L.; Natarajan, Amarnath; Raja, Srikumar M.; Band, Vimla; Band, Hamid

    2014-01-01

    ErbB2 overexpression drives oncogenesis in 20–30% cases of breast cancer. Oncogenic potential of ErbB2 is linked to inefficient endocytic traffic into lysosomes and preferential recycling. However, regulation of ErbB2 recycling is incompletely understood. We used a high-content immunofluorescence imaging-based kinase inhibitor screen on SKBR-3 breast cancer cells to identify kinases whose inhibition alters the clearance of cell surface ErbB2 induced by Hsp90 inhibitor 17-AAG. Less ErbB2 clearance was observed with broad-spectrum PKC inhibitor Ro 31-8220. A similar effect was observed with Go 6976, a selective inhibitor of classical Ca2+-dependent PKCs (α, β1, βII, and γ). PKC activation by PMA promoted surface ErbB2 clearance but without degradation, and ErbB2 was observed to move into a juxtanuclear compartment where it colocalized with PKC-α and PKC-δ together with the endocytic recycling regulator Arf6. PKC-α knockdown impaired the juxtanuclear localization of ErbB2. ErbB2 transit to the recycling compartment was also impaired upon PKC-δ knockdown. PMA-induced Erk phosphorylation was reduced by ErbB2 inhibitor lapatinib, as well as by knockdown of PKC-δ but not that of PKC-α. Our results suggest that activation of PKC-α and -δ mediates a novel positive feedback loop by promoting ErbB2 entry into the endocytic recycling compartment, consistent with reported positive roles for these PKCs in ErbB2-mediated tumorigenesis. As the endocytic recycling compartment/pericentrion has emerged as a PKC-dependent signaling hub for G-protein-coupled receptors, our findings raise the possibility that oncogenesis by ErbB2 involves previously unexplored PKC-dependent endosomal signaling. PMID:25225290

  13. Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling.

    PubMed

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

    2016-07-12

    Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein-protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein-protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions. PMID:27357676

  14. Anchoring of FRET Sensors—A Requirement for Spatiotemporal Resolution

    PubMed Central

    Ivanova, Elena V.; Figueroa, Ricardo A.; Gatsinzi, Tom; Hallberg, Einar; Iverfeldt, Kerstin

    2016-01-01

    FRET biosensors have become a routine tool for investigating mechanisms and components of cell signaling. Strategies for improving them for particular applications are continuously sought. One important aspect to consider when designing FRET probes is the dynamic distribution and propagation of signals within living cells. We have addressed this issue by directly comparing an anchored (taFS) to a non-anchored (naFS) cleavable FRET sensor. We chose a microtubule-associated protein tau as an anchor, as microtubules are abundant throughout the cytosol of cells. We show that tau-anchored FRET sensors are concentrated at the cytoskeleton and enriched in the neurite-like processes of cells, providing high intensity of the total signal. In addition, anchoring limits the diffusion of the sensor, enabling spatiotemporally resolved monitoring of subcellular variations in enzyme activity. Thus, anchoring is an important aspect to consider when designing FRET sensors for deeper understanding of cell signaling. PMID:27196902

  15. Anchoring of FRET Sensors-A Requirement for Spatiotemporal Resolution.

    PubMed

    Ivanova, Elena V; Figueroa, Ricardo A; Gatsinzi, Tom; Hallberg, Einar; Iverfeldt, Kerstin

    2016-01-01

    FRET biosensors have become a routine tool for investigating mechanisms and components of cell signaling. Strategies for improving them for particular applications are continuously sought. One important aspect to consider when designing FRET probes is the dynamic distribution and propagation of signals within living cells. We have addressed this issue by directly comparing an anchored (taFS) to a non-anchored (naFS) cleavable FRET sensor. We chose a microtubule-associated protein tau as an anchor, as microtubules are abundant throughout the cytosol of cells. We show that tau-anchored FRET sensors are concentrated at the cytoskeleton and enriched in the neurite-like processes of cells, providing high intensity of the total signal. In addition, anchoring limits the diffusion of the sensor, enabling spatiotemporally resolved monitoring of subcellular variations in enzyme activity. Thus, anchoring is an important aspect to consider when designing FRET sensors for deeper understanding of cell signaling. PMID:27196902

  16. AnchorDock: Blind and Flexible Anchor-Driven Peptide Docking.

    PubMed

    Ben-Shimon, Avraham; Niv, Masha Y

    2015-05-01

    The huge conformational space stemming from the inherent flexibility of peptides is among the main obstacles to successful and efficient computational modeling of protein-peptide interactions. Current peptide docking methods typically overcome this challenge using prior knowledge from the structure of the complex. Here we introduce AnchorDock, a peptide docking approach, which automatically targets the docking search to the most relevant parts of the conformational space. This is done by precomputing the free peptide's structure and by computationally identifying anchoring spots on the protein surface. Next, a free peptide conformation undergoes anchor-driven simulated annealing molecular dynamics simulations around the predicted anchoring spots. In the challenging task of a completely blind docking test, AnchorDock produced exceptionally good results (backbone root-mean-square deviation ≤ 2.2Å, rank ≤15) for 10 of 13 unbound cases tested. The impressive performance of AnchorDock supports a molecular recognition pathway that is driven via pre-existing local structural elements. PMID:25914054

  17. The calmodulin-like proteins AtCML4 and AtCML5 are single-pass membrane proteins targeted to the endomembrane system by an N-terminal signal anchor sequence

    PubMed Central

    Ruge, Henning; Flosdorff, Sandra; Ebersberger, Ingo; Chigri, Fatima; Vothknecht, Ute C.

    2016-01-01

    Calmodulins (CaMs) are important mediators of Ca2+ signals that are found ubiquitously in all eukaryotic organisms. Plants contain a unique family of calmodulin-like proteins (CMLs) that exhibit greater sequence variance compared to canonical CaMs. The Arabidopsis thaliana proteins AtCML4 and AtCML5 are members of CML subfamily VII and possess a CaM domain comprising the characteristic double pair of EF-hands, but they are distinguished from other members of this subfamily and from canonical CaMs by an N-terminal extension of their amino acid sequence. Transient expression of yellow fluorescent protein-tagged AtCML4 and AtCML5 under a 35S-promoter in Nicotiana benthamiana leaf cells revealed a spherical fluorescence pattern. This pattern was confirmed by transient expression in Arabidopsis protoplasts under the native promoter. Co-localization analyses with various endomembrane marker proteins suggest that AtCML4 and AtCML5 are localized to vesicular structures in the interphase between Golgi and the endosomal system. Further studies revealed AtCML5 to be a single-pass membrane protein that is targeted into the endomembrane system by an N-terminal signal anchor sequence. Self-assembly green fluorescent protein and protease protection assays support a topology with the CaM domain exposed to the cytosolic surface and not the lumen of the vesicles, indicating that AtCML5 could sense Ca2+ signals in the cytosol. Phylogenetic analysis suggests that AtCML4 and AtCML5 are closely related paralogues originating from a duplication event within the Brassicaceae family. CML4/5-like proteins seem to be universally present in eudicots but are absent in some monocots. Together these results show that CML4/5-like proteins represent a flowering plant-specific subfamily of CMLs with a potential function in vesicle transport within the plant endomembrane system. PMID:27029353

  18. The calmodulin-like proteins AtCML4 and AtCML5 are single-pass membrane proteins targeted to the endomembrane system by an N-terminal signal anchor sequence.

    PubMed

    Ruge, Henning; Flosdorff, Sandra; Ebersberger, Ingo; Chigri, Fatima; Vothknecht, Ute C

    2016-06-01

    Calmodulins (CaMs) are important mediators of Ca(2+) signals that are found ubiquitously in all eukaryotic organisms. Plants contain a unique family of calmodulin-like proteins (CMLs) that exhibit greater sequence variance compared to canonical CaMs. The Arabidopsis thaliana proteins AtCML4 and AtCML5 are members of CML subfamily VII and possess a CaM domain comprising the characteristic double pair of EF-hands, but they are distinguished from other members of this subfamily and from canonical CaMs by an N-terminal extension of their amino acid sequence. Transient expression of yellow fluorescent protein-tagged AtCML4 and AtCML5 under a 35S-promoter in Nicotiana benthamiana leaf cells revealed a spherical fluorescence pattern. This pattern was confirmed by transient expression in Arabidopsis protoplasts under the native promoter. Co-localization analyses with various endomembrane marker proteins suggest that AtCML4 and AtCML5 are localized to vesicular structures in the interphase between Golgi and the endosomal system. Further studies revealed AtCML5 to be a single-pass membrane protein that is targeted into the endomembrane system by an N-terminal signal anchor sequence. Self-assembly green fluorescent protein and protease protection assays support a topology with the CaM domain exposed to the cytosolic surface and not the lumen of the vesicles, indicating that AtCML5 could sense Ca(2+) signals in the cytosol. Phylogenetic analysis suggests that AtCML4 and AtCML5 are closely related paralogues originating from a duplication event within the Brassicaceae family. CML4/5-like proteins seem to be universally present in eudicots but are absent in some monocots. Together these results show that CML4/5-like proteins represent a flowering plant-specific subfamily of CMLs with a potential function in vesicle transport within the plant endomembrane system. PMID:27029353

  19. Anchoring the Deficit of the Anchor Deficit: Dyslexia or Attention?

    ERIC Educational Resources Information Center

    Willburger, Edith; Landerl, Karin

    2010-01-01

    In the anchoring deficit hypothesis of dyslexia ("Trends Cogn. Sci.", 2007; 11: 458-465), it is proposed that perceptual problems arise from the lack of forming a perceptual anchor for repeatedly presented stimuli. A study designed to explicitly test the specificity of the anchoring deficit for dyslexia is presented. Four groups, representing all…

  20. Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling

    PubMed Central

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

    2016-01-01

    Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein–protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein–protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions. PMID:27357676

  1. Blind-Anchor-Nut-Installation Fixture (BANIF)

    NASA Technical Reports Server (NTRS)

    Willey, Norman F., Jr.; Linker, James F.

    1994-01-01

    Blind-anchor-nut-installation fixture, BANIF, developed for replacing or installing anchor nuts in blind holes or other inaccessible places. Attachment of anchor nut to BANIF enables placement of anchor nut on blind side of component.

  2. Bellow seal and anchor

    DOEpatents

    Mansure, Arthur J.

    2001-01-01

    An annular seal is made of a collapsible bellows. The bellows can function as an anchor or a seal and is easily set into position using relative component movement. The bellows folds can be slanted and their outer sealing edges can have different profiles to meet expected conditions. The bellows is expanded for insertion to reduce its outer dimension and sets by compaction as a result of relative movement. The bellows can be straight or tapered and is settable with a minimal axial force.

  3. Lightness, brightness, and anchoring.

    PubMed

    Anderson, Barton L; Whitbread, Michael; de Silva, Chamila

    2014-01-01

    The majority of work in lightness perception has evaluated the perception of lightness using flat, matte, two-dimensional surfaces. In such contexts, the amount of light reaching the eye contains a conflated mixture of the illuminant and surface lightness. A fundamental puzzle of lightness perception is understanding how it is possible to experience achromatic surfaces as specific achromatic shades in the face of this ambiguity. It has been argued that the perception of lightness in such contexts implies that the visual system imposes an "anchoring rule" whereby a specific relative luminance (the highest) serves as a fixed point in the mapping of image luminance onto the lightness scale ("white"). We conducted a series of experiments to explicitly test this assertion in contexts where this mapping seemed most unlikely-namely, low-contrast images viewed in dim illumination. Our results provide evidence that the computational ambiguity in mapping luminance onto lightness is reflected in perceptual experience. The perception of the highest luminance in a two-dimensional Mondrian display varied monotonically with its brightness, ranging from midgray to white. Similar scaling occurred for the lowest luminance and, by implication, all other luminance values. We conclude that the conflation between brightness and lightness in two-dimensional Mondrian displays is reflected in perception and find no support for the claim that any specific relative luminance value acts as a fixed anchor point in this mapping function. PMID:25104828

  4. COBRA, an Arabidopsis Extracellular Glycosyl-Phosphatidyl Inositol-Anchored Protein, Specifically Controls Highly Anisotropic Expansion through Its Involvement in Cellulose Microfibril OrientationW⃞

    PubMed Central

    Roudier, François; Fernandez, Anita G.; Fujita, Miki; Himmelspach, Regina; Borner, Georg H.H.; Schindelman, Gary; Song, Shuang; Baskin, Tobias I.; Dupree, Paul; Wasteneys, Geoffrey O.; Benfey, Philip N.

    2005-01-01

    The orientation of cell expansion is a process at the heart of plant morphogenesis. Cellulose microfibrils are the primary anisotropic material in the cell wall and thus are likely to be the main determinant of the orientation of cell expansion. COBRA (COB) has been identified previously as a potential regulator of cellulose biogenesis. In this study, characterization of a null allele, cob-4, establishes the key role of COB in controlling anisotropic expansion in most developing organs. Quantitative polarized-light and field-emission scanning electron microscopy reveal that loss of anisotropic expansion in cob mutants is accompanied by disorganization of the orientation of cellulose microfibrils and subsequent reduction of crystalline cellulose. Analyses of the conditional cob-1 allele suggested that COB is primarily implicated in microfibril deposition during rapid elongation. Immunodetection analysis in elongating root cells revealed that, in agreement with its substitution by a glycosylphosphatidylinositol anchor, COB was polarly targeted to both the plasma membrane and the longitudinal cell walls and was distributed in a banding pattern perpendicular to the longitudinal axis via a microtubule-dependent mechanism. Our observations suggest that COB, through its involvement in cellulose microfibril orientation, is an essential factor in highly anisotropic expansion during plant morphogenesis. PMID:15849274

  5. KRE genes are required for β-1,6-glucan synthesis, maintenance of capsule architecture and cell wall protein anchoring in Cryptococcus neoformans

    PubMed Central

    Gilbert, Nicole M.; Donlin, Maureen J.; Gerik, Kimberly J.; Specht, Charles A.; Djordjevic, Julianne T.; Wilson, Christabel F.; Sorrell, Tania C.; Lodge, Jennifer K.

    2010-01-01

    Summary The polysaccharide β-1,6-glucan is a major component of the cell wall of Cryptococcus neoformans, but its function has not been investigated in this fungal pathogen. We have identified and characterized seven genes, belonging to the KRE family, which are putatively involved in β-1,6-glucan synthesis. The H99 deletion mutants kre5Δ and kre6Δskn1Δ contained less cell wall β-1,6-glucan, grew slowly with an aberrant morphology, were highly sensitive to environmental and chemical stress and were avirulent in a mouse inhalation model of infection. These two mutants displayed alterations in cell wall chitosan and the exopolysaccharide capsule, a primary cryptococcal virulence determinant. The cell wall content of the GPI-anchored phospholipase B1 (Plb1) enzyme, which is required for cryptococcal cell wall integrity and virulence, was reduced in kre5Δ and kre6Δskn1Δ. Our results indicate that KRE5, KRE6 and SKN1 are involved in β-1,6-glucan synthesis, maintenance of cell wall integrity and retention of mannoproteins and known cryptococcal virulence factors in the cell wall of C. neoformans. This study sets the stage for future investigations into the function of this abundant cell wall polymer. PMID:20384682

  6. Monitoring Lipid Anchor Organization in Cell Membranes by PIE-FCCS

    PubMed Central

    2012-01-01

    This study examines the dynamic co-localization of lipid-anchored fluorescent proteins in living cells using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) and fluorescence lifetime analysis. Specifically, we look at the pairwise co-localization of anchors from lymphocyte cell kinase (LCK: myristoyl, palmitoyl, palmitoyl), RhoA (geranylgeranyl), and K-Ras (farnesyl) proteins in different cell types. In Jurkat cells, a density-dependent increase in cross-correlation among RhoA anchors is observed, while LCK anchors exhibit a more moderate increase and broader distribution. No correlation was detected among K-Ras anchors or between any of the different anchor types studied. Fluorescence lifetime data reveal no significant Förster resonance energy transfer in any of the data. In COS 7 cells, minimal correlation was detected among LCK or RhoA anchors. Taken together, these observations suggest that some lipid anchors take part in anchor-specific co-clustering with other existing clusters of native proteins and lipids in the membrane. Importantly, these observations do not support a simple interpretation of lipid anchor-mediated organization driven by partitioning based on binary lipid phase separation. PMID:22631607

  7. SpeB modulates fibronectin-dependent internalization of Streptococcus pyogenes by efficient proteolysis of cell-wall-anchored protein F1.

    PubMed

    Nyberg, Patrik; Rasmussen, Magnus; Von Pawel-Rammingen, Ulrich; Björck, Lars

    2004-05-01

    SpeB is a cysteine proteinase and virulence determinant secreted by the important human pathogen Streptococcus pyogenes. Recent investigations have suggested a role for SpeB in streptococcal entry into human cells. However, conflicting data concerning the contribution of SpeB to internalization have been presented. Protein F1 is a cell-wall-attached fibronectin (Fn)-binding protein that is present in a majority of streptococcal isolates and is important for internalization. This study shows that protein F1 is efficiently degraded by SpeB, and that removal of protein F1 from the bacterial surface leads to reduced internalization. Whereas M1 protein and protein H, two additional surface proteins of S. pyogenes that bind human plasma proteins, are protected from proteolytic degradation by their ligands, protein F1 is readily cleaved by SpeB also when in complex with Fn. This finding, and the connection between the presence of Fn at the bacterial surface and entry into human cells, suggest that SpeB plays a role in the regulation of the internalization process. PMID:15133117

  8. Granular Simulation of NEO Anchoring

    NASA Technical Reports Server (NTRS)

    Mazhar, Hammad

    2011-01-01

    NASA is interested in designing a spacecraft capable of visiting a Near Earth Object (NEO), performing experiments, and then returning safely. Certain periods of this mission will require the spacecraft to remain stationary relative to the NEO. Such situations require an anchoring mechanism that is compact, easy to deploy and upon mission completion, easily removed. The design philosophy used in the project relies on the simulation capability of a multibody dynamics physics engine. On Earth it is difficult to create low gravity conditions and testing in low gravity environments, whether artificial or in space is costly and therefore not feasible. Through simulation, gravity can be controlled with great accuracy, making it ideally suited to analyze the problem at hand. Using Chrono::Engine [1], a simulation package capable of utilizing massively parallel GPU hardware, several validation experiments will be performed. Once there is sufficient confidence, modeling of the NEO regolith interaction will begin after which the anchor tests will be performed and analyzed. The outcome of this task is a study with an analysis of several different anchor designs, along with a recommendation on which anchor is better suited to the task of anchoring. With the anchors tested against a range of parameters relating to soil, environment and anchor penetration angles/velocities on a NEO.

  9. Regulation of lipid biosynthesis, sliding motility, and biofilm formation by a membrane-anchored nucleoid-associated protein of Mycobacterium tuberculosis.

    PubMed

    Ghosh, Soumitra; Indi, Shantinath S; Nagaraja, Valakunja

    2013-04-01

    Bacteria use a number of small basic proteins for organization and compaction of their genomes. By their interaction with DNA, these nucleoid-associated proteins (NAPs) also influence gene expression. Rv3852, a NAP of Mycobacterium tuberculosis, is conserved among the pathogenic and slow-growing species of mycobacteria. Here, we show that the protein predominantly localizes in the cell membrane and that the carboxy-terminal region with the propensity to form a transmembrane helix is necessary for its membrane localization. The protein is involved in genome organization, and its ectopic expression in Mycobacterium smegmatis resulted in altered nucleoid morphology, defects in biofilm formation, sliding motility, and change in apolar lipid profile. We demonstrate its crucial role in regulating the expression of KasA, KasB, and GroEL1 proteins, which are in turn involved in controlling the surface phenotypes in mycobacteria. PMID:23396914

  10. Regulation of Lipid Biosynthesis, Sliding Motility, and Biofilm Formation by a Membrane-Anchored Nucleoid-Associated Protein of Mycobacterium tuberculosis

    PubMed Central

    Ghosh, Soumitra; Indi, Shantinath S.

    2013-01-01

    Bacteria use a number of small basic proteins for organization and compaction of their genomes. By their interaction with DNA, these nucleoid-associated proteins (NAPs) also influence gene expression. Rv3852, a NAP of Mycobacterium tuberculosis, is conserved among the pathogenic and slow-growing species of mycobacteria. Here, we show that the protein predominantly localizes in the cell membrane and that the carboxy-terminal region with the propensity to form a transmembrane helix is necessary for its membrane localization. The protein is involved in genome organization, and its ectopic expression in Mycobacterium smegmatis resulted in altered nucleoid morphology, defects in biofilm formation, sliding motility, and change in apolar lipid profile. We demonstrate its crucial role in regulating the expression of KasA, KasB, and GroEL1 proteins, which are in turn involved in controlling the surface phenotypes in mycobacteria. PMID:23396914

  11. Bone cement improves suture anchor fixation.

    PubMed

    Giori, Nicholas J; Sohn, David H; Mirza, Faisal M; Lindsey, Derek P; Lee, Arthur T

    2006-10-01

    Suture anchor fixation failure can occur if the anchor pulls out of bone. We hypothesized that suture anchor fixation can be augmented with polymethylmethacrylate cement, and that polymethylmethacrylate can be used to improve fixation in a stripped anchor hole. Six matched cadaveric proximal humeri were used. On one side, suture anchors were placed and loaded to failure using a ramped cyclic loading protocol. The stripped anchor holes then were injected with approximately 1 cc polymethylmethacrylate, and anchors were replaced and tested again. In the contralateral humerus, polymethylmethacrylate was injected into anchor holes before anchor placement and testing. In unstripped anchors, polymethylmethacrylate increased the number of cycles to failure by 34% and failure load by 71% compared with anchors not augmented with polymethylmethacrylate. Polymethylmethacrylate haugmentation of stripped anchors increased the cycles to failure by 31% and failure load by 111% compared with unstripped uncemented anchors. No difference was found in cycles to failure or failure load between cemented stripped anchors and cemented unstripped anchors. Polymethylmethacrylate can be used to augment fixation, reducing the risk of anchor pull-out failure, regardless whether the suture anchor hole is stripped or unstripped. PMID:16702922

  12. TbGT8 is a bifunctional glycosyltransferase that elaborates N-linked glycans on a protein phosphatase AcP115 and a GPI-anchor modifying glycan in Trypanosoma brucei.

    PubMed

    Nakanishi, Masayuki; Karasudani, Moe; Shiraishi, Takahiro; Hashida, Kazunori; Hino, Mami; Ferguson, Michael A J; Nomoto, Hiroshi

    2014-06-01

    The procyclic form of Trypanosoma brucei expresses procyclin surface glycoproteins with unusual glycosylphosphatidylinositol-anchor side chain structures that contain branched N-acetyllactosamine and lacto-N-biose units. The glycosyltransferase TbGT8 is involved in the synthesis of the branched side chain through its UDP-GlcNAc: βGal β1-3N-acetylglucosaminyltransferase activity. Here, we explored the role of TbGT8 in the mammalian bloodstream form of the parasite with a tetracycline-inducible conditional null T. brucei mutant for TbGT8. Under non-permissive conditions, the mutant showed significantly reduced binding to tomato lectin, which recognizes poly-N-acetyllactosamine-containing glycans. Lectin pull-down assays revealed differences between the wild type and TbGT8 null-mutant T. brucei, notably the absence of a broad protein band with an approximate molecular weight of 110 kDa in the mutant lysate. Proteomic analysis revealed that the band contained several glycoproteins, including the acidic ecto-protein phosphatase AcP115, a stage-specific glycoprotein in the bloodstream form of T. brucei. Western blotting with an anti-AcP115 antibody revealed that AcP115 was approximately 10kDa smaller in the mutant. Enzymatic de-N-glycosylation demonstrated that the underlying protein cores were the same, suggesting that the 10-kDa difference was due to differences in N-linked glycans. Immunofluorescence microscopy revealed the colocalization of hemagglutinin epitope-tagged TbGT8 and the Golgi-associated protein GRASP. These data suggest that TbGT8 is involved in the construction of complex poly-N-acetyllactosamine-containing type N-linked and GPI-linked glycans in the Golgi of the bloodstream and procyclic parasite forms, respectively. PMID:24508870

  13. Towards zirconium phosphonate-based microarrays for probing DNA-protein interactions: critical influence of the location of the probe anchoring groups.

    PubMed

    Monot, Julien; Petit, Marc; Lane, Sarah M; Guisle, Isabelle; Léger, Jean; Tellier, Charles; Talham, Daniel R; Bujoli, Bruno

    2008-05-14

    Terminal phosphate groups on double-stranded DNA probes bind strongly to glass substrates coated with a zirconium phosphonate monolayer, and probes immobilized in this way as microarrays can be used to detect protein targets. The sensitivity of the microarray was shown to be enhanced by the use of a polyguanine segment ((G)n , n > or = 5) as a spacer between the phosphate linker and the protein interaction domain. More importantly, the presence of phosphate linkers on both ends of the dsDNA probes leads to significant enhancement of target capture. The relevant characteristics of the different probes when bound to the surface were determined, by the original use of a combination of surface characterization techniques (XPS, AFM, and Sarfus). In this context, the location of the phosphate linkers in the duplex probes was found to result in different probe surface coverage and presentation on the surface, which affect subsequent interactions with the target protein. PMID:18407629

  14. Binding kinetics of membrane-anchored receptors and ligands: Molecular dynamics simulations and theory

    NASA Astrophysics Data System (ADS)

    Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard; Weikl, Thomas R.

    2015-12-01

    The adhesion of biological membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. Central questions are how the binding kinetics of these proteins is affected by the membranes and by the membrane anchoring of the proteins. In this article, we (i) present detailed data for the binding of membrane-anchored proteins from coarse-grained molecular dynamics simulations and (ii) provide a theory that describes how the binding kinetics depends on the average separation and thermal roughness of the adhering membranes and on the anchoring, lengths, and length variations of the proteins. An important element of our theory is the tilt of bound receptor-ligand complexes and transition-state complexes relative to the membrane normals. This tilt results from an interplay of the anchoring energy and rotational entropy of the complexes and facilitates the formation of receptor-ligand bonds at membrane separations smaller than the preferred separation for binding. In our simulations, we have considered both lipid-anchored and transmembrane receptor and ligand proteins. We find that the binding equilibrium constant and binding on-rate constant of lipid-anchored proteins are considerably smaller than the binding constant and on-rate constant of rigid transmembrane proteins with identical binding domains.

  15. Molecular characterization of rice OsBIANK1, encoding a plasma membrane-anchored ankyrin repeat protein, and its inducible expression in defense responses.

    PubMed

    Zhang, Xinchun; Li, Dayong; Zhang, Huijuan; Wang, Xiaoe; Zheng, Zhong; Song, Fengming

    2010-02-01

    A rice gene, OsBIANK1, encoding a protein containing a typical ankyrin repeat domain, was cloned and identified. The OsBIANK1 protein, consisting of 329 amino acids, contains a conserved ankyrin repeat domain with two ankyrin repeats organized in tandem and was showed to be localized on cytoplasmic membrane during transient expression in onion epidermal cells. Expression of OsBIANK1 was induced by treatment with benzothiadiazole (BTH), a chemical inducer capable of inducing disease resistance response in rice. In BTH-treated rice seedlings, expression of OsBIANK1 was further induced by infection with Magnaporthe grisea, the rice blast fungus, as compared with those in water-treated seedlings. Our preliminary results confirm previous evidences that OsBIANK1 may be involved in regulation of disease resistance response in rice. PMID:19288292

  16. Anchoring International sets new water depth record

    SciTech Connect

    Noble, H.J.

    1983-07-01

    Santa Barbara Channel has a history steeped in firsts in techniques for the production of offshore oil. Landscaped drilling and production islands, production piers, and directional drilling from land rigs to production under the channel, to name a few. The latest such project was handled by Anchoring International, Inc., a pipe line anchoring company headquartered in Houston, Texas. Contracted by Healy Tibbets Construction Company, prime contractor, Anchoring was commissioned to handle a new deep water record breaking anchoring job. The job was to anchor J-tube extensions in 820 feet of water--the deepest pipe line anchoring job ever undertaken. In most shallow water pipe line anchoring jobs, anchors and anchor installation unit placement over the pipe line is handled from a crane topside with visual assist from divers. However, due to the extreme depth of this project, the installation unit with anchors had to be modified for submersible operator-assisted placement capability. Anchoring International handled the anchor design and installation equipment, and submersible operator assistance was furnished by Oceaneering, International. WASP and JIM atmospheric diving systems were used. All ocean bottom activities were monitored topside with the JERED video-equipped remote controlled vehicle. Since the weight of the anchor sets and power installation unit are minimum, the entire operation was conducted from a small boat sufficient to carry dive equipment and the anchor installation unit power supply. A small pedestal crane was used to lower and retrieve the anchor installation unit.

  17. DjlA, a Membrane-Anchored DnaJ-Like Protein, Is Required for Cytotoxicity of Clam Pathogen Vibrio tapetis to Hemocytes▿

    PubMed Central

    Lakhal, Fatma; Bury-Moné, Stéphanie; Nomane, Yanoura; Le Goïc, Nelly; Paillard, Christine; Jacq, Annick

    2008-01-01

    DjlA is an inner membrane cochaperone belonging to the DnaJ family, which has been shown to be involved in Legionella sp. pathogenesis. In this study, we explored the role of this protein in the physiology and virulence of Vibrio tapetis, the etiological agent of brown ring disease (BRD) in Manila clam (Ruditapes philippinarum). Analysis of the djlA locus in V. tapetis revealed a putative organization in an operon with a downstream gene that we designated duf924Vt, which encodes a conserved protein with an unknown function and has homologues in bacteria and eukaryotes. djlA mutants displayed a reduced growth rate and showed an important loss of cytotoxic activity against R. philippinarum hemocytes in vitro, which could be restored by extrachromosomal expression of wild-type djlAVt but not duf924Vt. These results are in keeping with the potential importance of DjlA for bacterial pathogenicity and open new perspectives for understanding the mechanism of action of this protein in the novel V. tapetis-R. philippinarum interaction model. PMID:18641167

  18. Effect of secondary anchor amino acid substitutions on the immunogenic properties of an HLA-A*0201-restricted T cell epitope derived from the Trypanosoma cruzi KMP-11 protein.

    PubMed

    Lasso, Paola; Cárdenas, Constanza; Guzmán, Fanny; Rosas, Fernando; Thomas, María Carmen; López, Manuel Carlos; González, John Mario; Cuéllar, Adriana; Campanera, Josep Maria; Luque, F Javier; Puerta, Concepción Judith

    2016-04-01

    The TcTLE peptide (TLEEFSAKL) is a CD8(+) T cell HLA-A*0201-restricted epitope derived from the Trypanosoma cruzi KMP-11 protein that is efficiently processed, presented and recognized by CD8(+) T cells from chagasic patients. Since the immunogenic properties of wild-type epitopes may be enhanced by suitable substitutions in secondary anchor residues, we have studied the effect of introducing specific mutations at position 3, 6 and 7 of the TcTLE peptide. Mutations (E3L, S6V and A7F) were chosen on the basis of in silico predictions and in vitro assays were performed to determine the TcTLE-modified peptide binding capacity to the HLA-A*0201 molecule. In addition, the functional activity of peptide-specific CD8(+) T cells in HLA-A2(+) chagasic patients was also interrogated. In contrast to bioinformatics predictions, the TcTLE-modified peptide was found to have lower binding affinity and stability than the original peptide. Nevertheless, CD8(+) T cells from chronic chagasic patients recognized the TcTLE-modified peptide producing TNF-α and INF-γ and expressing CD107a/b, though in less extension than the response triggered by the original peptide. Overall, although the amino acids at positions 3, 6 and 7 of TcTLE are critical for the peptide affinity, they have a limited effect on the immunogenic properties of the TcTLE epitope. PMID:26854383

  19. The levels of epithelial anchor proteins β-catenin and zona occludens-1 are altered by E7 of human papillomaviruses 5 and 8.

    PubMed

    Heuser, Sandra; Hufbauer, Martin; Marx, Benjamin; Tok, Ali; Majewski, Slawomir; Pfister, Herbert; Akgül, Baki

    2016-02-01

    Infection with viruses of the genus Betapapillomavirus, β-human papillomaviruses (β-HPV), is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and HPV8 in patients with the skin disease epidermodysplasia verruciformis (EV). The relocalization of the junctional bridging proteins β-catenin and zona occludens-1 (ZO-1) from the adherens and tight junctions are common processes of the epithelial-mesenchymal transition (EMT) associated with tumour invasion. Here, we report that β-catenin and ZO-1 are strongly upregulated by the E7 oncoproteins of HPV5 and HPV8 in keratinocytes grown in organotypic skin cultures. Although the membrane-tethered form of β-catenin was elevated, no signs of β-catenin activity within the canonical Wnt signalling pathway could be detected. The upregulation of β-catenin and ZO-1 could also be confirmed in the skin of HPV8 transgenic mice as well as in cutaneous squamous cell carcinomas of EV patients. These data provide the first evidence that β-catenin and ZO-1 are direct targets of E7 of the oncogenic β-HPV types 5 and 8. The ability to deregulate these epithelial junction proteins may contribute to the oncogenic potential of these viruses in human skin. PMID:26645068

  20. Anchoring a Leviathan: How the Nuclear Membrane Tethers the Genome

    PubMed Central

    Czapiewski, Rafal; Robson, Michael I.; Schirmer, Eric C.

    2016-01-01

    It is well established that the nuclear envelope has many distinct direct connections to chromatin that contribute to genome organization. The functional consequences of genome organization on gene regulation are less clear. Even less understood is how interactions of lamins and nuclear envelope transmembrane proteins (NETs) with chromatin can produce anchoring tethers that can withstand the physical forces of and on the genome. Chromosomes are the largest molecules in the cell, making megadalton protein structures like the nuclear pore complexes and ribosomes seem small by comparison. Thus to withstand strong forces from chromosome dynamics an anchoring tether is likely to be much more complex than a single protein-protein or protein-DNA interaction. Here we will briefly review known NE-genome interactions that likely contribute to spatial genome organization, postulate in the context of experimental data how these anchoring tethers contribute to gene regulation, and posit several hypotheses for the physical nature of these tethers that need to be investigated experimentally. Significantly, disruption of these anchoring tethers and the subsequent consequences for gene regulation could explain how mutations in nuclear envelope proteins cause diseases ranging from muscular dystrophy to lipodystrophy to premature aging progeroid syndromes. The two favored hypotheses for nuclear envelope protein involvement in disease are (1) weakening nuclear and cellular mechanical stability, and (2) disrupting genome organization and gene regulation. Considerable experimental support has been obtained for both. The integration of both mechanical and gene expression defects in the disruption of anchoring tethers could provide a unifying hypothesis consistent with both. PMID:27200088

  1. Bone Anchored Hearing Aid

    PubMed Central

    2002-01-01

    Executive Summary Objective The objective of this health technology policy assessment was to determine the effectiveness and cost-effectiveness of bone-anchored hearing aid (BAHA) in improving the hearing of people with conduction or mixed hearing loss. The Technology The (BAHA) is a bone conduction hearing device that includes a titanium fixture permanently implanted into the mastoid bone of the skull and an external percutaneous sound processor. The sound processor is attached to the fixture by means of a skin penetrating abutment. Because the device bypasses the middle ear and directly stimulates the cochlea, it has been recommended for individuals with conduction hearing loss or discharging middle ear infection. The titanium implant is expected to last a lifetime while the external sound processor is expected to last 5 years. The total initial device cost is approximately $5,300 and the external sound processor costs approximately $3,500. Review of BAHA by the Medical Advisory Secretariat The Medical Advisory Secretariat’s review is a descriptive synthesis of findings from 36 research articles published between January 1990 and May 2002. Summary of Findings No randomized controlled studies were found. The evidence was derived from level 4 case series with relative small sample sizes (ranging from 30-188). The majority of the studies have follow-up periods of eight years or longer. All except one study were based on monaural BAHA implant on the side with the best bone conduction threshold. Safety Level 4 evidence showed that BAHA has been be implanted safely in adults and children with success rates of 90% or higher in most studies. No mortality or life threatening morbidity has been reported. Revision rates for tissue reduction or resiting were generally under 10% for adults but have been reported to be as high as 25% in pediatric studies. Adverse skin reaction around the skin penetration site was the most common complication reported. Most of these

  2. Microgravity Drill and Anchor System

    NASA Technical Reports Server (NTRS)

    Parness, Aaron; Frost, Matthew A.; King, Jonathan P.

    2013-01-01

    This work is a method to drill into a rock surface regardless of the gravitational field or orientation. The required weight-on-bit (WOB) is supplied by a self-contained anchoring mechanism. The system includes a rotary percussive coring drill, forming a complete sampling instrument usable by robot or human. This method of in situ sample acquisition using micro - spine anchoring technology enables several NASA mission concepts not currently possible with existing technology, including sampling from consolidated rock on asteroids, providing a bolt network for astronauts visiting a near-Earth asteroid, and sampling from the ceilings or vertical walls of lava tubes and cliff faces on Mars. One of the most fundamental parameters of drilling is the WOB; essentially, the load applied to the bit that allows it to cut, creating a reaction force normal to the surface. In every drilling application, there is a minimum WOB that must be maintained for the system to function properly. In microgravity (asteroids and comets), even a small WOB could not be supported conventionally by the weight of the robot or astronaut. An anchoring mechanism would be needed to resist the reactions, or the robot or astronaut would push themselves off the surface and into space. The ability of the system to anchor itself to a surface creates potential applications that reach beyond use in low gravity. The use of these anchoring mechanisms as end effectors on climbing robots has the potential of vastly expanding the scope of what is considered accessible terrain. Further, because the drill is supported by its own anchor rather than by a robotic arm, the workspace is not constrained by the reach of such an arm. Yet, if the drill is on a robotic arm, it has the benefit of not reflecting the forces of drilling back to the arm s joints. Combining the drill with the anchoring feet will create a highly mobile, highly stable, and highly reliable system. The drilling system s anchor uses hundreds of

  3. Permanent ground anchors: Nicholson design criteria

    NASA Astrophysics Data System (ADS)

    Nicholson, P. J.; Uranowski, D. D.; Wycliffe-Jones, P. T.

    1982-09-01

    The methods used by Nicholson Construction Company in the design of permanent ground anchors specifically as related to retaining walls are discussed. Basic soil parameters, design concepts, drilling and grouting methods for ground anchors are discussed. Particular emphasis is placed on anchors founded in soil rather than rock formations. Also, soil properties necessary for the proper design of anchored retaining walls are detailed. The second chapter of the report is devoted to a general discussion of retaining wall and anchor design. In addition, a design example of an anchored retaining wall is presented in a step by step manner.

  4. Whole genome sequencing of phage resistant Bacillus anthracis mutants reveals an essential role for cell surface anchoring protein CsaB in phage AP50c adsorption

    PubMed Central

    2012-01-01

    Background Spontaneous Bacillus anthracis mutants resistant to infection by phage AP50c (AP50R) exhibit a mucoid colony phenotype and secrete an extracellular matrix. Methods Here we utilized a Roche/454-based whole genome sequencing approach to identify mutations that are candidates for conferring AP50c phage resistance, followed by genetic deletion and complementation studies to validate the whole genome sequence data and demonstrate that the implicated gene is necessary for AP50c phage infection. Results Using whole genome sequence data, we mapped the relevant mutations in six AP50R strains to csaB. Eleven additional spontaneous mutants, isolated in two different genetic backgrounds, were screened by PCR followed by Sanger sequencing of the csaB gene. In each spontaneous mutant, we found either a non-synonymous substitution, a nonsense mutation, or a frame-shift mutation caused by single nucleotide polymorphisms or a 5 base pair insertion in csaB. All together, 5 and 12 of the 17 spontaneous mutations are predicted to yield altered full length and truncated CsaB proteins respectively. As expected from these results, a targeted deletion or frame-shift mutations introduced into csaB in a different genetic background, in a strain not exposed to AP50c, resulted in a phage resistant phenotype. Also, substitution of a highly conserved histidine residue with an alanine residue (H270A) in CsaB resulted in phage resistance, suggesting that a functional CsaB is necessary for phage sensitivity. Conversely, introduction of the wild type allele of csaB in cis into the csaB deletion mutant by homologous recombination or supplying the wild type CsaB protein in trans from a plasmid restored phage sensitivity. The csaB mutants accumulated cell wall material and appeared to have a defective S-layer, whereas these phenotypes were reverted in the complemented strains. Conclusions Taken together, these data suggest an essential role for csaB in AP50c phage infection, most likely in

  5. Type II PKAs are anchored to mature insulin secretory granules in INS-1 β-cells and required for cAMP-dependent potentiation of exocytosis.

    PubMed

    Villalpando, Sabrina; Cazevieille, Chantal; Fernandez, Anne; Lamb, Ned J; Hani, El-Habib

    2016-06-01

    Specificity of the cAMP-dependent protein kinase (PKA) pathway relies on an extremely sophisticated compartmentalization mechanism of the kinase within a given cell, based on high-affinity binding of PKA tetramer pools to different A-Kinase Anchoring Proteins (AKAPs). We and others have previously shown that AKAPs-dependent PKA subcellular targeting is a requisite for optimal cAMP-dependent potentiation of insulin exocytosis. We thus hypothesized that a PKA pool may directly anchor to the secretory compartment to potentiate insulin exocytosis. Here, using immunofluorescence analyses combined to subcellular fractionations and purification of insulin secretory granules (ISGs), we identified discrete subpools of type II PKAs, RIIα and RIIβ PKAs, along with the catalytic subunit, physically associated with ISGs within pancreatic insulin-secreting β-cells. Ultrastructural analysis of native rodent β-cells confirmed in vivo the occurrence of PKA on dense-core ISGs. Isoform-selective disruption of binding of PKAs to AKAPs reinforced the requirement of type II PKA isoforms for cAMP potentiation of insulin exocytosis. This granular localization of PKA was of critical importance since siRNA-mediated depletion of either RIIα or RIIβ PKAs resulted in a significant reduction of cAMP-dependent potentiation of insulin release. The present work provides evidence for a previously unrecognized pool of type II PKAs physically anchored to the β-cell ISGs compartment and supports a non-redundant function for type II PKAs during cAMP potentiation of exocytosis. PMID:26898328

  6. Evidence for Two Distinct Binding Sites for Lipoprotein Lipase on Glycosylphosphatidylinositol-anchored High Density Lipoprotein-binding Protein 1 (GPIHBP1)*

    PubMed Central

    Reimund, Mart; Larsson, Mikael; Kovrov, Oleg; Kasvandik, Sergo; Olivecrona, Gunilla; Lookene, Aivar

    2015-01-01

    GPIHBP1 is an endothelial membrane protein that transports lipoprotein lipase (LPL) from the subendothelial space to the luminal side of the capillary endothelium. Here, we provide evidence that two regions of GPIHBP1, the acidic N-terminal domain and the central Ly6 domain, interact with LPL as two distinct binding sites. This conclusion is based on comparative binding studies performed with a peptide corresponding to the N-terminal domain of GPIHBP1, the Ly6 domain of GPIHBP1, wild type GPIHBP1, and the Ly6 domain mutant GPIHBP1 Q114P. Although LPL and the N-terminal domain formed a tight but short lived complex, characterized by fast on- and off-rates, the complex between LPL and the Ly6 domain formed more slowly and persisted for a longer time. Unlike the interaction of LPL with the Ly6 domain, the interaction of LPL with the N-terminal domain was significantly weakened by salt. The Q114P mutant bound LPL similarly to the N-terminal domain of GPIHBP1. Heparin dissociated LPL from the N-terminal domain, and partially from wild type GPIHBP1, but was unable to elute the enzyme from the Ly6 domain. When LPL was in complex with the acidic peptide corresponding to the N-terminal domain of GPIHBP1, the enzyme retained its affinity for the Ly6 domain. Furthermore, LPL that was bound to the N-terminal domain interacted with lipoproteins, whereas LPL bound to the Ly6 domain did not. In summary, our data suggest that the two domains of GPIHBP1 interact independently with LPL and that the functionality of LPL depends on its localization on GPIHBP1. PMID:25873395

  7. Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses

    SciTech Connect

    Wang, Jimin Li, Yue; Modis, Yorgo

    2014-04-15

    The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1–E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. - Highlights: • Structures of pestivirus E2 proteins impose constraints on E1, E2 membrane anchors. • Atomic models of the E1 and E2 membrane anchors were generated in silico. • A “snorkeling” arginine completes the short helical hairpin in the E2 membrane anchor. • Roles in pH sensing and E1–E2 disulfide bond formation are proposed for E1 residues. • Implications for E1 ectodomain structure and disulfide bonding pattern are discussed.

  8. Data for identification of GPI-anchored peptides and ω-sites in cancer cell lines.

    PubMed

    Masuishi, Yusuke; Kimura, Yayoi; Arakawa, Noriaki; Hirano, Hisashi

    2016-06-01

    We present data obtained using a focused proteomics approach to identify the glycosylphosphatidylinositol (GPI)-anchored peptides in 19 human cancer cell lines. GPI-anchored proteins (GPI-APs), which localize to the outer leaflet of the membrane microdomains commonly referred to as lipid rafts play important roles in diverse biological processes. Due to the complex structure of the GPI-anchor moiety, it has been difficult to identify GPI-anchored peptide sequences on the proteomic scale by database searches using tools such as MASCOT. Here we provide data from 73 ω-sites derived from 49 GPI-APs in 19 human cancer cell lines. This article contains data related to the research article entitled "Identification of glycosylphosphatidylinositol-anchored proteins and ω-sites using TiO2-based affinity purification followed by hydrogen fluoride treatment" (Masuishi et al., 2016) [1]. PMID:27141528

  9. Data for identification of GPI-anchored peptides and ω-sites in cancer cell lines

    PubMed Central

    Masuishi, Yusuke; Kimura, Yayoi; Arakawa, Noriaki; Hirano, Hisashi

    2016-01-01

    We present data obtained using a focused proteomics approach to identify the glycosylphosphatidylinositol (GPI)-anchored peptides in 19 human cancer cell lines. GPI-anchored proteins (GPI-APs), which localize to the outer leaflet of the membrane microdomains commonly referred to as lipid rafts play important roles in diverse biological processes. Due to the complex structure of the GPI-anchor moiety, it has been difficult to identify GPI-anchored peptide sequences on the proteomic scale by database searches using tools such as MASCOT. Here we provide data from 73 ω-sites derived from 49 GPI-APs in 19 human cancer cell lines. This article contains data related to the research article entitled “Identification of glycosylphosphatidylinositol-anchored proteins and ω-sites using TiO2-based affinity purification followed by hydrogen fluoride treatment” (Masuishi et al., 2016) [1]. PMID:27141528

  10. Multimerization of Glycosylphosphatidylinositol-anchored High Density Lipoprotein-binding Protein 1 (GPIHBP1) and Familial Chylomicronemia from a Serine-to-Cysteine Substitution in GPIHBP1 Ly6 Domain*

    PubMed Central

    Plengpanich, Wanee; Young, Stephen G.; Khovidhunkit, Weerapan; Bensadoun, André; Karnman, Hirankorn; Ploug, Michael; Gårdsvoll, Henrik; Leung, Calvin S.; Adeyo, Oludotun; Larsson, Mikael; Muanpetch, Suwanna; Charoen, Supannika; Fong, Loren G.; Niramitmahapanya, Sathit; Beigneux, Anne P.

    2014-01-01

    GPIHBP1, a glycosylphosphatidylinositol-anchored glycoprotein of microvascular endothelial cells, binds lipoprotein lipase (LPL) within the interstitial spaces and transports it across endothelial cells to the capillary lumen. The ability of GPIHBP1 to bind LPL depends on the Ly6 domain, a three-fingered structure containing 10 cysteines and a conserved pattern of disulfide bond formation. Here, we report a patient with severe hypertriglyceridemia who was homozygous for a GPIHBP1 point mutation that converted a serine in the GPIHBP1 Ly6 domain (Ser-107) to a cysteine. Two hypertriglyceridemic siblings were homozygous for the same mutation. All three homozygotes had very low levels of LPL in the preheparin plasma. We suspected that the extra cysteine in GPIHBP1-S107C might prevent the trafficking of the protein to the cell surface, but this was not the case. However, nearly all of the GPIHBP1-S107C on the cell surface was in the form of disulfide-linked dimers and multimers, whereas wild-type GPIHBP1 was predominantly monomeric. An insect cell GPIHBP1 expression system confirmed the propensity of GPIHBP1-S107C to form disulfide-linked dimers and to form multimers. Functional studies showed that only GPIHBP1 monomers bind LPL. In keeping with that finding, there was no binding of LPL to GPIHBP1-S107C in either cell-based or cell-free binding assays. We conclude that an extra cysteine in the GPIHBP1 Ly6 motif results in multimerization of GPIHBP1, defective LPL binding, and severe hypertriglyceridemia. PMID:24847059

  11. Multimerization of glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) and familial chylomicronemia from a serine-to-cysteine substitution in GPIHBP1 Ly6 domain.

    PubMed

    Plengpanich, Wanee; Young, Stephen G; Khovidhunkit, Weerapan; Bensadoun, André; Karnman, Hirankorn; Ploug, Michael; Gårdsvoll, Henrik; Leung, Calvin S; Adeyo, Oludotun; Larsson, Mikael; Muanpetch, Suwanna; Charoen, Supannika; Fong, Loren G; Niramitmahapanya, Sathit; Beigneux, Anne P

    2014-07-11

    GPIHBP1, a glycosylphosphatidylinositol-anchored glycoprotein of microvascular endothelial cells, binds lipoprotein lipase (LPL) within the interstitial spaces and transports it across endothelial cells to the capillary lumen. The ability of GPIHBP1 to bind LPL depends on the Ly6 domain, a three-fingered structure containing 10 cysteines and a conserved pattern of disulfide bond formation. Here, we report a patient with severe hypertriglyceridemia who was homozygous for a GPIHBP1 point mutation that converted a serine in the GPIHBP1 Ly6 domain (Ser-107) to a cysteine. Two hypertriglyceridemic siblings were homozygous for the same mutation. All three homozygotes had very low levels of LPL in the preheparin plasma. We suspected that the extra cysteine in GPIHBP1-S107C might prevent the trafficking of the protein to the cell surface, but this was not the case. However, nearly all of the GPIHBP1-S107C on the cell surface was in the form of disulfide-linked dimers and multimers, whereas wild-type GPIHBP1 was predominantly monomeric. An insect cell GPIHBP1 expression system confirmed the propensity of GPIHBP1-S107C to form disulfide-linked dimers and to form multimers. Functional studies showed that only GPIHBP1 monomers bind LPL. In keeping with that finding, there was no binding of LPL to GPIHBP1-S107C in either cell-based or cell-free binding assays. We conclude that an extra cysteine in the GPIHBP1 Ly6 motif results in multimerization of GPIHBP1, defective LPL binding, and severe hypertriglyceridemia. PMID:24847059

  12. Structure, Function and On-Off Switching of a Core Unit Contact between CheA Kinase and CheW Adaptor Protein in the Bacterial Chemosensory Array: A Disulfide Mapping and TAM-IDS Study

    PubMed Central

    Natale, Andrew M.; Duplantis, Jane L.; Piasta, Kene N.; Falke, Joseph J.

    2014-01-01

    The ultrasensitive, ultrastable bacterial chemosensory array of Escherichia coli and Salmonella typhimurium is representative of the large, conserved family of sensory arrays that control the cellular chemotaxis of motile bacteria and Archaea. The core framework of the membrane-bound array is a lattice assembled from three components: a transmembrane receptor, a cytoplasmic His kinase (CheA), and a cytoplasmic adaptor protein (CheW). Structural studies in the field have revealed the global architecture of the array and complexes between specific components, but much remains to be learned about the essential protein-protein interfaces that define array structure and transmit signals between components. This study has focused on the structure, function and on-off switching of a key contact between the kinase and adaptor proteins in the working, membrane-bound array. Specifically, the study addressed interface 1 in the putative kinase-adaptor ring where subdomain 1 of the kinase regulatory domain contacts subdomain 2 of the adaptor protein. Two independent approaches – disulfide mapping and site-directed Trp and Ala mutagenesis – were employed to (i) test the structural model of interface 1 and (ii) investigate its functional roles in both stable kinase incorporation and receptor-regulated kinase on-off switching. Studies were carried out in functional, membrane-bound arrays or in live cells. The findings reveal that crystal structures of binary and ternary complexes accurately depict the native interface in its kinase-activating on state. Furthermore, the findings indicate that at least part of the interface becomes less closely packed in its kinase-inhibiting off state. Together, the evidence shows the interface has a dual structural and signaling function that is crucial for stable kinase incorporation into the array, for kinase activation in the array on state, and likely for attractant-triggered kinase on-off switching. A model is presented that describes the

  13. Anchoring in Numeric Judgments of Visual Stimuli.

    PubMed

    Langeborg, Linda; Eriksson, Mårten

    2016-01-01

    This article investigates effects of anchoring in age estimation and estimation of quantities, two tasks which to different extents are based on visual stimuli. The results are compared to anchoring in answers to classic general knowledge questions that rely on semantic knowledge. Cognitive load was manipulated to explore possible differences between domains. Effects of source credibility, manipulated by differing instructions regarding the selection of anchor values (no information regarding anchor selection, information that the anchors are randomly generated or information that the anchors are answers from an expert) on anchoring were also investigated. Effects of anchoring were large for all types of judgments but were not affected by cognitive load or by source credibility in either one of the researched domains. A main effect of cognitive load on quantity estimations and main effects of source credibility in the two visually based domains indicate that the manipulations were efficient. Implications for theoretical explanations of anchoring are discussed. In particular, because anchoring did not interact with cognitive load, the results imply that the process behind anchoring in visual tasks is predominantly automatic and unconscious. PMID:26941684

  14. Anchoring in Numeric Judgments of Visual Stimuli

    PubMed Central

    Langeborg, Linda; Eriksson, Mårten

    2016-01-01

    This article investigates effects of anchoring in age estimation and estimation of quantities, two tasks which to different extents are based on visual stimuli. The results are compared to anchoring in answers to classic general knowledge questions that rely on semantic knowledge. Cognitive load was manipulated to explore possible differences between domains. Effects of source credibility, manipulated by differing instructions regarding the selection of anchor values (no information regarding anchor selection, information that the anchors are randomly generated or information that the anchors are answers from an expert) on anchoring were also investigated. Effects of anchoring were large for all types of judgments but were not affected by cognitive load or by source credibility in either one of the researched domains. A main effect of cognitive load on quantity estimations and main effects of source credibility in the two visually based domains indicate that the manipulations were efficient. Implications for theoretical explanations of anchoring are discussed. In particular, because anchoring did not interact with cognitive load, the results imply that the process behind anchoring in visual tasks is predominantly automatic and unconscious. PMID:26941684

  15. Anchor for Fiberglas Guy Rod

    NASA Technical Reports Server (NTRS)

    Wilson, A. H.

    1982-01-01

    Solution to problem of anchoring fiberglas guy rods to install nut with threads on outer circumference, followed by aluminum sleeve. Sleeve has opening oval at upper and round at bottom end. End of rod is split so fiberglas wedge can be inserted to form V-shaped end. Spread end of rod fits into tapered hole in sleeve and threaded aluminum coupling is put over rod and sleeve.

  16. Independent control of polar and azimuthal anchoring.

    PubMed

    Anquetil-Deck, C; Cleaver, D J; Bramble, J P; Atherton, T J

    2013-07-01

    Monte Carlo simulation, experiment, and continuum theory are used to examine the anchoring exhibited by a nematic liquid crystal at a patterned substrate comprising a periodic array of rectangles that, respectively, promote vertical and planar alignment. It is shown that the easy axis and effective anchoring energy promoted by such surfaces can be readily controlled by adjusting the design of the pattern. The calculations reveal rich behavior: for strong anchoring, as exhibited by the simulated system, for rectangle ratios ≥2 the nematic aligns in the direction of the long edge of the rectangles, the azimuthal anchoring coefficient changing with pattern shape. In weak anchoring scenarios, however, including our experimental systems, preferential anchoring is degenerate between the two rectangle diagonals. Bistability between diagonally aligned and edge-aligned arrangement is predicted for intermediate combinations of anchoring coefficient and system length scale. PMID:23944468

  17. Electrostatically anchored branched brush layers.

    PubMed

    Liu, Xiaoyan; Dedinaite, Andra; Rutland, Mark; Thormann, Esben; Visnevskij, Ceslav; Makuska, Ricardas; Claesson, Per M

    2012-11-01

    A novel type of block copolymer has been synthesized. It consists of a linear cationic block and an uncharged bottle-brush block. The nonionic bottle-brush block contains 45 units long poly(ethylene oxide) side chains. This polymer was synthesized with the intention of creating branched brush layers firmly physisorbed to negatively charged surfaces via the cationic block, mimicking the architecture (but not the chemistry) of bottle-brush molecules suggested to be present on the cartilage surface, and contributing to the efficient lubrication of synovial joints. The adsorption properties of the diblock copolymer as well as of the two blocks separately were studied on silica surfaces using quartz crystal microbalance with dissipation monitoring (QCM-D) and optical reflectometry. The adsorption kinetics data highlight that the diblock copolymers initially adsorb preferentially parallel to the surface with both the cationic block and the uncharged bottle-brush block in contact with the surface. However, as the adsorption proceeds, a structural change occurs within the layer, and the PEO bottle-brush block extends toward solution, forming a surface-anchored branched brush layer. As the adsorption plateau is reached, the diblock copolymer layer is 46-48 nm thick, and the water content in the layer is above 90 wt %. The combination of strong electrostatic anchoring and highly hydrated branched brush structures provide strong steric repulsion, low friction forces, and high load bearing capacity. The strong electrostatic anchoring also provides high stability of preadsorbed layers under different ionic strength conditions. PMID:23046176

  18. Evidence that a kinase distinct from protein kinase C and phosphatidylinositol 3-kinase mediates ligation-dependent serine/threonine phosphorylation of the T-lymphocyte co-stimulatory molecule CD28.

    PubMed Central

    Parry, R V; Olive, D; Westwick, J; Sansom, D M; Ward, S G

    1997-01-01

    The CD28 cytoplasmic tail contains several potential phosphorylation sites for the serine/threonine kinase protein kinase C (PKC) and/or proline-directed serine/threonine kinases, such as extracellular signal-regulated kinases. We demonstrate that ligation of CD28 by B7.1 results in strong serine/threonine phosphorylation of CD28. It is unlikely that ligation-stimulated phosphorylation of CD28 is mediated via activation of PKC, since it was not prevented by pre-treatment of Jurkat cells with inhibitors of PKC, and it was not mimicked by treatment with PKC activators such as PMA. Nevertheless, despite for lack of detectable effects of PMA treatment on CD28 phosphorylation, PMA did partially inhibit the association of CD28 with the putative signalling molecule phosphatidylinositol 3-kinase (PI 3-kinase) and the subsequent accumulation of PtdIns(3,4,5)P3. PI 3-kinase exhibits dual specificity as both a lipid kinase and a protein serine kinase, and site-specific mutagenesis of the Tyr173 residue in the CD28 cytoplasmic tail, which abolishes CD28 coupling to PI 3-kinase [Pages, Ragueneau, Rottapel, Truneh, Nunes, Imbert and Olive (1994) Nature (London) 369, 327-329], also prevents ligation-stimulated phosphorylation of CD28. However, the two PI 3-kinase inhibitors wortmannin and LY294002 had no effect on phosphorylation of CD28 after ligation by B7.1. This study therefore demonstrates that (1) a CD28-activated serine/threonine kinase distinct from both PKC and PI 3-kinase mediates ligation-stimulated CD28 phosphorylation, and (2) the PMA-stimulated down-regulation of the coupling of CD28 to PI 3-kinase is not due to PMA-stimulated phosphorylation of CD28. PMID:9337876

  19. Characterization of the C-terminal ER membrane anchor of PTP1B

    SciTech Connect

    Anderie, Ines Schulz, Irene; Schmid, Andreas

    2007-09-10

    The tyrosine phosphatase PTP1B is an important regulator of cell function. In living cells PTP1B activity is restricted to the vicinity of the endoplasmic reticulum (ER) by post-translational C-terminal attachment of PTP1B to the ER membrane network. In our study we investigated the membrane anchor of PTP1B by use of EGFP fusion proteins. We demonstrate that the membrane anchor of PTP1B cannot be narrowed down to a unique amino acid sequence with a defined start and stop point but rather is moveable within several amino acids. Removal of up to seven amino acids from the C-terminus, as well as exchange of single amino acids in the putative transmembrane sequence did not influence subcellular localization of PTP1B. With the method of bimolecular fluorescence complementation we could demonstrate dimerization of PTP1B in vivo. Homodimerization was, in contrast to other tail-anchored proteins, not dependent on the membrane anchor. Our data demonstrate that the C-terminal membrane anchor of PTP1B is formed by a combination of a single stretch transmembrane domain (TMD) followed by a tail. TMD and tail length are variable and there are no sequence-specific features. Our data for PTP1B are consistent with a concept that explains the ER membrane anchor of tail-anchored proteins as a physicochemical structure.

  20. Glycosylphosphatidylinositols: More than just an anchor?

    PubMed Central

    Bate, Clive; Nolan, William; Williams, Alun

    2016-01-01

    ABSTRACT There is increasing interest in the role of glycosylphosphatidylinositol (GPI) anchors that attach some proteins to cell membranes. Far from being biologically inert, GPIs influence the targeting, intracellular trafficking and function of the attached protein. Our recent paper demonstrated the role of sialic acid on the GPI of the cellular prion protein (PrPC). The “prion diseases” arise following the conversion of PrPC to a disease-associated isoform called PrPSc or “prion”. Our paper showed that desialylated PrPC inhibited PrPSc formation. Aggregated PrPSc creates a signaling platform in the cell membrane incorporating and activating cytoplasmic phospholipase A2 (cPLA2), an enzyme that regulates PrPC trafficking and hence PrPSc formation. The presence of desialylated PrPC caused the dissociation of cPLA2 from PrP-containing platforms, reduced the activation of cPLA2 and inhibited PrPSc production. We concluded that sialic acid contained within the GPI attached to PrPC modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrPSc formation. PMID:27195066

  1. Glycosylphosphatidylinositols: More than just an anchor?

    PubMed

    Bate, Clive; Nolan, William; Williams, Alun

    2016-01-01

    There is increasing interest in the role of glycosylphosphatidylinositol (GPI) anchors that attach some proteins to cell membranes. Far from being biologically inert, GPIs influence the targeting, intracellular trafficking and function of the attached protein. Our recent paper demonstrated the role of sialic acid on the GPI of the cellular prion protein (PrP(C)). The "prion diseases" arise following the conversion of PrP(C) to a disease-associated isoform called PrP(Sc) or "prion". Our paper showed that desialylated PrP(C) inhibited PrP(Sc) formation. Aggregated PrP(Sc) creates a signaling platform in the cell membrane incorporating and activating cytoplasmic phospholipase A2 (cPLA2), an enzyme that regulates PrP(C) trafficking and hence PrP(Sc) formation. The presence of desialylated PrP(C) caused the dissociation of cPLA2 from PrP-containing platforms, reduced the activation of cPLA2 and inhibited PrP(Sc) production. We concluded that sialic acid contained within the GPI attached to PrP(C) modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP(Sc) formation. PMID:27195066

  2. Accessorizing and anchoring the LINC complex for multifunctionality

    PubMed Central

    Chang, Wakam; Worman, Howard J.

    2015-01-01

    The linker of nucleoskeleton and cytoskeleton (LINC) complex, composed of outer and inner nuclear membrane Klarsicht, ANC-1, and Syne homology (KASH) and Sad1 and UNC-84 (SUN) proteins, respectively, connects the nucleus to cytoskeletal filaments and performs diverse functions including nuclear positioning, mechanotransduction, and meiotic chromosome movements. Recent studies have shed light on the source of this diversity by identifying factors associated with the complex that endow specific functions as well as those that differentially anchor the complex within the nucleus. Additional diversity may be provided by accessory factors that reorganize the complex into higher-ordered arrays. As core components of the LINC complex are associated with several diseases, understanding the role of accessory and anchoring proteins could provide insights into pathogenic mechanisms. PMID:25559183

  3. A lunar/Martian anchor emplacement system

    NASA Astrophysics Data System (ADS)

    Clinton, Dustin; Holt, Andrew; Jantz, Erik; Kaufman, Teresa; Martin, James; Weber, Reed

    On the Moon or Mars, it is necessary to have an anchor, or a stable, fixed point able to support the forces necessary to rescue a stuck vehicle, act as a stake for a tent in a Martian gale, act as a fulcrum in the erection of general construction poles, or support tent-like regolith shields. The anchor emplacement system must be highly autonomous. It must supply the energy and stability for anchor deployment. The goal of the anchor emplacement system project is to design and build a prototype anchor and to design a conceptual anchor emplacement system. Various anchors were tested in a 1.3 cubic meter test bed containing decomposed granite. A simulated lunar soil was created by adjusting the moisture and compaction characteristics of the soil. We conducted tests on emplacement torque, amount of force the anchor could withstand before failure, anchor pull out force at various angles, and soil disturbances caused by placing the anchor. A single helix auger anchor performed best in this test bed based on energy to emplace, and the ultimate holding capacity. The anchor was optimized for ultimate holding capacity, minimum emplacement torque, and minimum soil disturbance in sandy soils yielding the following dimensions: helix diameter (4.45 cm), pitch (1.27 cm), blade thickness (0.15 cm), total length (35.56 cm), shaft diameter (0.78 cm), and a weight of 212.62 g. The experimental results showed that smaller diameter, single-helix augers held more force than larger diameter augers for a given depth. The emplacement system consists of a flywheel and a motor for power, sealed in a protective box supported by four legs. The flywheel system was chosen over a gear system based on its increased reliability in the lunar environment.

  4. A lunar/Martian anchor emplacement system

    NASA Technical Reports Server (NTRS)

    Clinton, Dustin; Holt, Andrew; Jantz, Erik; Kaufman, Teresa; Martin, James; Weber, Reed

    1993-01-01

    On the Moon or Mars, it is necessary to have an anchor, or a stable, fixed point able to support the forces necessary to rescue a stuck vehicle, act as a stake for a tent in a Martian gale, act as a fulcrum in the erection of general construction poles, or support tent-like regolith shields. The anchor emplacement system must be highly autonomous. It must supply the energy and stability for anchor deployment. The goal of the anchor emplacement system project is to design and build a prototype anchor and to design a conceptual anchor emplacement system. Various anchors were tested in a 1.3 cubic meter test bed containing decomposed granite. A simulated lunar soil was created by adjusting the moisture and compaction characteristics of the soil. We conducted tests on emplacement torque, amount of force the anchor could withstand before failure, anchor pull out force at various angles, and soil disturbances caused by placing the anchor. A single helix auger anchor performed best in this test bed based on energy to emplace, and the ultimate holding capacity. The anchor was optimized for ultimate holding capacity, minimum emplacement torque, and minimum soil disturbance in sandy soils yielding the following dimensions: helix diameter (4.45 cm), pitch (1.27 cm), blade thickness (0.15 cm), total length (35.56 cm), shaft diameter (0.78 cm), and a weight of 212.62 g. The experimental results showed that smaller diameter, single-helix augers held more force than larger diameter augers for a given depth. The emplacement system consists of a flywheel and a motor for power, sealed in a protective box supported by four legs. The flywheel system was chosen over a gear system based on its increased reliability in the lunar environment.

  5. Anchored Instruction and Anchored Assessment: An Ecological Approach to Measuring Situated Learning.

    ERIC Educational Resources Information Center

    Young, Michael F.; Kulikowich, Jonna M.

    Anchored instruction and anchored assessment are described and illustrated through a mathematics problem from the Jasper problem solving series developed at Vanderbilt University in Nashville (Tennessee). Anchored instruction is instruction situated in a context complex enough to provide meaning and reasons for why information is useful. Problems…

  6. Fast Ca2+-induced potentiation of heat-activated ionic currents requires cAMP/PKA signaling and functional AKAP anchoring.

    PubMed

    Distler, C; Rathee, P K; Lips, K S; Obreja, O; Neuhuber, W; Kress, M

    2003-05-01

    Calcium influx and the resulting increase in intracellular calcium concentration ([Ca(2+)](i)) can induce enhanced sensitivity to temperature increases in nociceptive neurons. This sensitization accounts for heat hyperalgesia that is regularly observed following the activation of excitatory inward currents by pain-producing mediators. Here we show that rat sensory neurons express calcium-dependent adenylyl cyclases (AC) using RT-PCR and nonradioactive in situ hybridization. Ionomycin-induced rises in [Ca(2+)](i)-activated calcium-dependent AC and caused translocation of catalytic protein kinase A subunit. Elevation of [Ca(2+)](i) finally resulted in a significant potentiation of heat-activated currents and a drop in heat threshold. This was not prevented in the presence of suramin that nonspecifically uncouples G protein-dependent receptors. The sensitization was, however, inhibited when the specific PKA antagonist PKI(14-22) was added to the pipette solution or when PKA coupling to A kinase anchoring protein (AKAP) was disrupted with InCELLect StHt-31 uncoupling peptide. The results show that heat sensitization in nociceptive neurons can be induced by increases in [Ca(2+)](i) and requires PKA that is functionally coupled to the heat transducer, mostly likely vanilloid receptor VR-1. This calcium-dependent pathway can account for the sensitizing properties of many excitatory mediators that activate cationic membrane currents. PMID:12740405

  7. Anchors of Religious Commitment in Adolescents

    ERIC Educational Resources Information Center

    Layton, Emily; Dollahite, David C.; Hardy, Sam A.

    2011-01-01

    This study explores adolescent religious commitment using qualitative data from a religiously diverse (Jewish, Christian, Muslim) sample of 80 adolescents. A new construct, "anchors of religious commitment," grounded in interview data, is proposed to describe what adolescents commit to as a part of their religious identity. Seven anchors of…

  8. Method of fabrication of anchored nanostructure materials

    DOEpatents

    Seals, Roland D; Menchhofer, Paul A; Howe, Jane Y; Wang, Wei

    2013-11-26

    Methods for fabricating anchored nanostructure materials are described. The methods include heating a nano-catalyst under a protective atmosphere to a temperature ranging from about 450.degree. C. to about 1500.degree. C. and contacting the heated nano-catalysts with an organic vapor to affix carbon nanostructures to the nano-catalysts and form the anchored nanostructure material.

  9. 77 FR 65496 - Commercial Acquisition; Anchor Tenancy

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-29

    ... limited conditions, to issue Anchor Tenancy contracts. Anchor Tenancy means ``an arrangement in which the... . SUPPLEMENTARY INFORMATION: A. Background NASA published a proposed rule in the Federal Register at 76 FR 30301... simplified; it is now discussed only in paragraph (a). The final rule identifies what is meant by an...

  10. 33 CFR 401.15 - Stern anchors.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 3 2010-07-01 2010-07-01 false Stern anchors. 401.15 Section 401.15 Navigation and Navigable Waters SAINT LAWRENCE SEAWAY DEVELOPMENT CORPORATION, DEPARTMENT OF TRANSPORTATION SEAWAY REGULATIONS AND RULES Regulations Condition of Vessels § 401.15 Stern anchors. (a)...

  11. 21 CFR 872.3130 - Preformed anchor.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Preformed anchor. 872.3130 Section 872.3130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES DENTAL DEVICES Prosthetic Devices § 872.3130 Preformed anchor. (a) Identification. A...

  12. 21 CFR 872.3130 - Preformed anchor.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Preformed anchor. 872.3130 Section 872.3130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES DENTAL DEVICES Prosthetic Devices § 872.3130 Preformed anchor. (a) Identification. A...

  13. 21 CFR 872.3130 - Preformed anchor.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Preformed anchor. 872.3130 Section 872.3130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES DENTAL DEVICES Prosthetic Devices § 872.3130 Preformed anchor. (a) Identification. A...

  14. A chemical approach to unraveling the biological function of the glycosylphosphatidylinositol anchor

    PubMed Central

    Paulick, Margot G.; Forstner, Martin B.; Groves, Jay T.; Bertozzi, Carolyn R.

    2007-01-01

    The glycosylphosphatidylinositol (GPI) anchor is a C-terminal posttranslational modification found on many eukaryotic proteins that reside in the outer leaflet of the cell membrane. The complex and diverse structures of GPI anchors suggest a rich spectrum of biological functions, but few have been confirmed experimentally because of the lack of appropriate techniques that allow for structural perturbation in a cellular context. We previously synthesized a series of GPI anchor analogs with systematic deletions within the glycan core and coupled them to the GFP by a combination of expressed protein ligation and native chemical ligation [Paulick MG, Wise AR, Forstner MB, Groves JT, Bertozzi CR (2007) J Am Chem Soc 129:11543–11550]. Here we investigate the behavior of these GPI-protein analogs in living cells. These modified proteins integrated into the plasma membranes of a variety of mammalian cells and were internalized and directed to recycling endosomes similarly to GFP bearing a native GPI anchor. The GPI-protein analogs also diffused freely in cellular membranes. However, changes in the glycan structure significantly affected membrane mobility, with the loss of monosaccharide units correlating to decreased diffusion. Thus, this cellular system provides a platform for dissecting the contributions of various GPI anchor components to their biological function. PMID:18077333

  15. Identification of Ski as a target for Aurora A kinase

    PubMed Central

    Mosquera, Jocelyn; Armisen, Ricardo; Zhao, Hong Ling; Rojas, Diego A.; Maldonado, Edio; Tapia, Julio C; Colombo, Alicia; Hayman, Michael J; Marcelain, Katherine

    2011-01-01

    Ski is a negative regulator of the transforming growth factor-β and other signalling pathways. The absence of SKI in mouse fibroblasts leads to chromosome segregation defects and genomic instability, suggesting a role for Ski during mitosis. At this stage, Ski is phosphorylated but to date little is known about the kinases involved in this process. Here, we show that Aurora A kinase is able to phosphorylate Ski in vitro. In vivo, Aurora A and Ski co-localized at the centrosomes and co-immunoprecipitated. Conversely, a C-terminal truncation mutant of Ski (SkiΔ491–728) lacking a coiled-coil domain, displayed decreased centrosomal localization. This mutant no longer co-immunoprecipitated with Aurora-A in vivo, but was still phosphorylated in vitro, indicating that the Ski-Aurora A interaction takes place at the centrosomes. These data identify Ski as a novel target of Aurora A and contribute to an understanding of the role of these proteins in the mitotic process. PMID:21600873

  16. Anchored nanostructure materials and method of fabrication

    DOEpatents

    Seals, Roland D; Menchhofer, Paul A; Howe, Jane Y; Wang, Wei

    2012-11-27

    Anchored nanostructure materials and methods for their fabrication are described. The anchored nanostructure materials may utilize nano-catalysts that include powder-based or solid-based support materials. The support material may comprise metal, such as NiAl, ceramic, a cermet, or silicon or other metalloid. Typically, nanoparticles are disposed adjacent a surface of the support material. Nanostructures may be formed as anchored to nanoparticles that are adjacent the surface of the support material by heating the nano-catalysts and then exposing the nano-catalysts to an organic vapor. The nanostructures are typically single wall or multi-wall carbon nanotubes.

  17. Auger anchors produce major cost savings

    SciTech Connect

    Webb, B.C.

    1981-10-01

    To meet specific-gravity standards, a 42-in.-diam, 0.598-in.-wall-thickness pipeline being installed in a glacial area full of potholes, peat swamps, and low-lying sections required 425 lb/ft of anchor hold-down. In spots where the underlying soil had an acceptable shear strength, the crew installed auger anchors (costing only $8/ft of pipeline) instead of the concrete set-on weights (at $80/ft) required in areas where the auger anchors would not hold; the savings amounted to about $380,000/mile of line laid.

  18. Anchoring Revisited: The Role of the Comparative Question

    PubMed Central

    Grau, Ina; Bohner, Gerd

    2014-01-01

    When people estimate a numeric value after judging whether it is larger or smaller than a high or low anchor value (comparative question), estimates are biased in the direction of the anchor. One explanation for this anchoring effect is that people selectively access knowledge consistent with the anchor value as part of a positive test strategy. Two studies (total N = 184) supported the alternative explanation that people access knowledge consistent with their own answer to the comparative question. Specifically, anchoring effects emerged when the answer to the comparative question was unexpected (lower than the low anchor or higher than the high anchor). For expected answers (lower than the high anchor or higher than the low anchor), however, anchoring effects were attenuated or reversed. The anchor value itself was almost never reported as an absolute estimate. PMID:24454953

  19. 21 CFR 872.3130 - Preformed anchor.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... DEVICES DENTAL DEVICES Prosthetic Devices § 872.3130 Preformed anchor. (a) Identification. A preformed... the platinum group intended to be incorporated into a dental appliance, such as a denture, to...

  20. Behavior of soil anchors under dynamic loads

    SciTech Connect

    Picornell, M.; Olague, B.

    1997-07-01

    Helical anchors placed in a cohesionless soil in a laboratory setting were tested under static and dynamic loads. The dynamic tests were performed after subjecting the anchors to a seating load. The dynamic load had an intensity that changed in sinusoidal fashion and was superimposed to the static seating loads. Although, the anchors have a static pull-out capacity, when the dynamic loads are applied the anchor experiences additional deformations for each load cycle. The deformations per cycle are initially high but then decrease to a nearly constant rate. Eventually, the constant rate increases suddenly accelerating until failure. This failure can take place even at small fractions of the static pull-out capacity. The rate of deformation per load cycle is found to increase for larger seating loads and for larger dynamic pulsating loads. The results of this study shows that the designer can only adjust loads to decrease the deformation rate to suit the design life of the structure.

  1. Load transfer mechanisms in anchored geosynthetic systems

    NASA Astrophysics Data System (ADS)

    Hryciw, Roman D.

    1990-12-01

    Success of an anchored geosynthetic system (AGS) depends on the satisfactory transfer of load between: the surface-deployed geosynthetic and anchors (typically ribbed reinforcing rods) driven into the slope; the geosynthetic and soil; and the anchors and soil. A study was performed to evaluate the load transfer mechanisms at these interfaces in an AGS. A mathematical model was developed for predicting the pullout resistance of plane ribbed inclusions. The model considered the contribution of both frictional and passive resistance components of pullout resistance. Optical observation of sand around the ribs was made to determine the behavior of soil around the moving ribs during pullout. A theoretical study disclosed that the optimum anchor orientation for stabilization of infinite slopes depends on several factors including slope angle and in-situ stresses. It typically ranges from 20 to 30 degree from the normal to the slope with the anchor driven upslope. An experimental study confirmed that the soil-geosynthetic interface friction angle may be correctly predicted from the residual or critical state friction angle of the sand. Equations were developed for load transfer at curved soil-fabric interfaces. An experimental study verified that the increases in soil stress with distance from the anchor may be predicted by the developed equations.

  2. 46 CFR 28.235 - Anchors and radar reflectors.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 1 2013-10-01 2013-10-01 false Anchors and radar reflectors. 28.235 Section 28.235....235 Anchors and radar reflectors. (a) Each vessel must be fitted with an anchor(s) and chain(s), cable... rigged with gear that provides a radar signature from a distance of 6 miles, each nonmetallic hull...

  3. 46 CFR 28.235 - Anchors and radar reflectors.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 1 2014-10-01 2014-10-01 false Anchors and radar reflectors. 28.235 Section 28.235....235 Anchors and radar reflectors. (a) Each vessel must be fitted with an anchor(s) and chain(s), cable... rigged with gear that provides a radar signature from a distance of 6 miles, each nonmetallic hull...

  4. 46 CFR 28.235 - Anchors and radar reflectors.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 1 2011-10-01 2011-10-01 false Anchors and radar reflectors. 28.235 Section 28.235....235 Anchors and radar reflectors. (a) Each vessel must be fitted with an anchor(s) and chain(s), cable... rigged with gear that provides a radar signature from a distance of 6 miles, each nonmetallic hull...

  5. 46 CFR 28.235 - Anchors and radar reflectors.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Anchors and radar reflectors. 28.235 Section 28.235....235 Anchors and radar reflectors. (a) Each vessel must be fitted with an anchor(s) and chain(s), cable... rigged with gear that provides a radar signature from a distance of 6 miles, each nonmetallic hull...

  6. 46 CFR 28.235 - Anchors and radar reflectors.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 1 2012-10-01 2012-10-01 false Anchors and radar reflectors. 28.235 Section 28.235....235 Anchors and radar reflectors. (a) Each vessel must be fitted with an anchor(s) and chain(s), cable... rigged with gear that provides a radar signature from a distance of 6 miles, each nonmetallic hull...

  7. The Use of Comics-Based Cases in Anchored Instruction

    ERIC Educational Resources Information Center

    Kneller, Matthew F.

    2009-01-01

    The primary purpose of this research was to understand how comics fulfill the role of anchor in an anchored instruction learning environment. Anchored instruction addresses the inert knowledge problem through the use of realistic multimedia stories, or "anchors," that embed a problem and the necessary data to solve it within the narrative. In the…

  8. DEX-1 and DYF-7 establish sensory dendrite length by anchoring dendritic tips during cell migration.

    PubMed

    Heiman, Maxwell G; Shaham, Shai

    2009-04-17

    Cells are devices whose structures delimit function. For example, in the nervous system, neuronal and glial shapes dictate paths of information flow. To understand how cells acquire their shapes, we examined the formation of a sense organ in C. elegans. Using time-lapse imaging, we found that sensory dendrites form by stationary anchoring of dendritic tips during cell-body migration. A genetic screen identified DEX-1 and DYF-7, extracellular proteins required for dendritic tip anchoring, which act cooperatively at the time and place of anchoring. DEX-1 and DYF-7 contain, respectively, zonadhesin and zona pellucida domains, and DYF-7 self-associates into multimers important for anchoring. Thus, unlike other dendrites, amphid dendritic tips are positioned by DEX-1 and DYF-7 without the need for long-range guidance cues. In sequence and function, DEX-1 and DYF-7 resemble tectorins, which anchor stereocilia in the inner ear, suggesting that a sensory dendrite anchor may have evolved into part of a mechanosensor. PMID:19344940

  9. Anchors as Semantic Primes in Value Construction: An EEG Study of the Anchoring Effect

    PubMed Central

    Shen, Qiang; Qiu, Wenwei

    2015-01-01

    Previous research regarding anchoring effects has demonstrated that human judgments are often assimilated to irrelevant information. Studies have demonstrated that anchors influence the economic valuation of various products and experiences; however, the cognitive explanations of this effect remain controversial, and its neural mechanisms have rarely been explored. In the current study, we conducted an electroencephalography (EEG) experiment to investigate the anchoring effect on willingness to accept (WTA) for an aversive hedonic experience and the role of anchors in this judgment heuristic. The behavioral results demonstrated that random numbers affect participants’ WTA for listening to pieces of noise. The participants asked for higher pay after comparing their WTA with higher numbers. The EEG results indicated that anchors also influenced the neural underpinnings of the valuation process. Specifically, when a higher anchor number was drawn, larger P2 and late positive potential amplitudes were elicited, reflecting the anticipation of more intensive pain from the subsequent noise. Moreover, higher anchors induced a stronger theta band power increase compared with lower anchors when subjects listened to the noises, indicating that the participants felt more unpleasant during the actual experience of the noise. The levels of unpleasantness during both anticipation and experience were consistent with the semantic information implied by the anchors. Therefore, these data suggest that a semantic priming process underlies the anchoring effect in WTA. This study provides proof for the robustness of the anchoring effect and neural evidence of the semantic priming model. Our findings indicate that activated contextual information, even seemingly irrelevant, can be embedded in the construction of economic value in the brain. PMID:26439926

  10. Applying a Targeted Label-free Approach using LC-MS AMT Tags to Evaluate Changes in Protein Phosphorylation Following Phosphatase Inhibition

    SciTech Connect

    Yang, Feng; Jaitly, Navdeep; Jayachandran, Hemalatha; Lou, Quanzhou; Monroe, Matthew E.; Du, Xiuxia; Gritsenko, Marina A.; Zhang, Rui; Anderson, David J.; Purvine, Samuel O.; Adkins, Joshua N.; Moore, Ronald J.; Mottaz, Heather M.; Ding, Shi-Jian; Lipton, Mary S.; Camp, David G.; Udseth, Harold R.; Smith, Richard D.; Rossie, Sandra S.

    2007-10-12

    To identify phosphoproteins regulated by the phosphoprotein phosphatase (PPP) family of S/T phosphatases, we performed a large-scale characterization of changes in protein phosphorylation on extracts from HeLa cells treated with or without calyculin A, a potent PPP enzyme inhibitor. A label-free comparative Phosphoproteomics approach using immobilized metal ion affinity chromatography and targeted tandem mass spectrometry was employed to discover and identify signatures based upon distinctive changes in abundance. Overall, 232 proteins were identified as either direct or indirect targets for PPP enzyme regulation. Most of the present identifications represent novel PPP enzyme targets at the level of both phosphorylation site and protein. These include phosphorylation sites within signaling proteins such as p120 Catenin, A Kinase Anchoring Protein 8, JunB, and Type II Phosphatidyl Inositol 4 Kinase. These data can be used to define underlying signaling pathways and events regulated by the PPP family of S/T phosphatases.

  11. Construction of a Highly Active Xylanase Displaying Oleaginous Yeast: Comparison of Anchoring Systems

    PubMed Central

    Duquesne, Sophie; Bozonnet, Sophie; Bordes, Florence; Dumon, Claire; Nicaud, Jean-Marc; Marty, Alain

    2014-01-01

    Three Yarrowia lipolytica cell wall proteins (YlPir, YlCWP1 and YlCBM) were evaluated for their ability to display the xylanase TxXYN from Thermobacillus xylanilyticus on the cell surface of Y. lipolytica. The fusion proteins were produced in Y. lipolytica JMY1212, a strain engineered for mono-copy chromosomal insertion, and enabling accurate comparison of anchoring systems. The construction using YlPir enabled cell bound xylanase activity to be maximised (71.6 U/g). Although 48% of the activity was released in the supernatant, probably due to proteolysis at the fusion zone, this system is three times more efficient for the anchoring of TxXYN than the YlCWP1 system formerly developed for Y. lipolytica. As far as we know it represents the best displayed xylanase activity ever published. It could be an attractive alternative anchoring system to display enzymes in Y. lipolytica. PMID:24743311

  12. Monitoring ground anchor using non-destructive ground anchor integrity test (NDT-GRANIT)

    NASA Astrophysics Data System (ADS)

    Robbany, Z.; Handayani, G.

    2015-09-01

    Monitoring at ground anchor commonly uses a pull out test method, therefor we developing a non-destructive ground anchor integrity testing (NDT-GRANIT). NDT-GRANIT using the principle of seismic waves that have been modified into form of sweep signal, the signal will be demodulated, filtered, and Fourier transformation (inverse discrete Fourier transform) so the data can be interpreted reflected wave from the ground anchor. The method was applied to determine whether the ground anchor still gripped in the subsurface by looking the attenuation of the wave generated sources. From the result we can see that ground anchor does not grip. To validate the results of the comparison method of measurement used pile integrity test.

  13. Monitoring ground anchor using non-destructive ground anchor integrity test (NDT-GRANIT)

    SciTech Connect

    Robbany, Z. Handayani, G.

    2015-09-30

    Monitoring at ground anchor commonly uses a pull out test method, therefor we developing a non-destructive ground anchor integrity testing (NDT-GRANIT). NDT-GRANIT using the principle of seismic waves that have been modified into form of sweep signal, the signal will be demodulated, filtered, and Fourier transformation (inverse discrete Fourier transform) so the data can be interpreted reflected wave from the ground anchor. The method was applied to determine whether the ground anchor still gripped in the subsurface by looking the attenuation of the wave generated sources. From the result we can see that ground anchor does not grip. To validate the results of the comparison method of measurement used pile integrity test.

  14. A human immunoglobulin G receptor exists in both polypeptide-anchored and phosphatidylinositol-glycan-anchored forms.

    PubMed Central

    Scallon, B J; Scigliano, E; Freedman, V H; Miedel, M C; Pan, Y C; Unkeless, J C; Kochan, J P

    1989-01-01

    Several cDNA clones encoding the human immunoglobulin G receptor CD16 were isolated from human lung or peripheral blood leukocyte cDNA libraries. Nucleotide sequence comparisons revealed that the cDNAs could be divided into two groups. cDNA clones in one group encode a protein that terminates 4 amino acids after the putative transmembrane domain. Clones in the second group encode a protein with an extra 21 amino acids that could comprise a cytoplasmic domain. Direct peptide sequencing was used to determine the N terminus of the mature CD16 receptor protein and supported the existence of the two forms of the receptor. Treatment of neutrophils with phosphatidylinositol-specific phospholipase C resulted in the release of a large percentage of the CD16 molecules from the cell surface. In contrast, treatment of natural killer cells with phosphatidylinositol-specific phospholipase C did not release any CD16 from the cell surface. These data demonstrate that both polypeptide-anchored and phosphatidylinositol-glycan-anchored forms of the CD16 molecule exist and that they are differentially expressed on neutrophils and natural killer cells. Images PMID:2525780

  15. Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses.

    PubMed

    Wang, Jimin; Li, Yue; Modis, Yorgo

    2014-04-01

    The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1-E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. PMID:24725935

  16. Characterization of the glycosylphosphatidylinositol-anchor signal sequence of human Cryptic with a hydrophilic extension

    PubMed Central

    Watanabe, Kazuhide; Nagaoka, Tadahiro; Strizzi, Luigi; Mancino, Mario; Gonzales, Monica; Bianco, Caterina; Salomon, David S.

    2012-01-01

    SUMMARY Epidermal Growth Factor-Cripto-1/FRL-1/Cryptic (EGF-CFC) proteins, including human Cripto-1 (hCFC2/hCR-1) and human Cryptic (hCFC1), are membrane-associated Nodal co-receptors, which have critical roles in vertebrate development. Most of the EGF-CFC proteins have been experimentally proven or predicted to be glycosylphosphatidylinositol (GPI)-anchored proteins. However, unlike other EGF-CFC proteins, hCFC1 does not exhibit a typical GPI-signal sequence, containing a 32-amino acid hydrophilic extension in its COOH-terminal end. Here we experimentally demonstrate that the COOH-terminal sequence of hCFC1 functions as a GPI-anchoring signal. Moreover, addition of a hydrophilic epitope tag of 55-amino acids (V5-His) after the GPI signal of hCR-1 interfered with generation of a GPI-anchored form of hCR-1. In contrast, addition of the same epitope tag to the end of GPI signal of hCFC1 did not affect the GPI-attachment of hCFC1. The COOH-terminal signal of hCFC1 could produce two different forms of the protein; a GPI-anchored form and an unprocessed form which was more prone to be secreted into the conditioned medium. The hydrophilic extension of hCFC1 negatively regulates the activity of hCFC1 as a Nodal co-receptor. These results demonstrate the presence of endogenous GPI-signal sequence with a hydrophilic extension, which can generate both GPI-anchored and soluble forms of the protein. PMID:18930707

  17. Monogenean anchor morphometry: systematic value, phylogenetic signal, and evolution

    PubMed Central

    Soo, Oi Yoon Michelle; Tan, Wooi Boon; Lim, Lee Hong Susan

    2016-01-01

    Background. Anchors are one of the important attachment appendages for monogenean parasites. Common descent and evolutionary processes have left their mark on anchor morphometry, in the form of patterns of shape and size variation useful for systematic and evolutionary studies. When combined with morphological and molecular data, analysis of anchor morphometry can potentially answer a wide range of biological questions. Materials and Methods. We used data from anchor morphometry, body size and morphology of 13 Ligophorus (Monogenea: Ancyrocephalidae) species infecting two marine mugilid (Teleostei: Mugilidae) fish hosts: Moolgarda buchanani (Bleeker) and Liza subviridis (Valenciennes) from Malaysia. Anchor shape and size data (n = 530) were generated using methods of geometric morphometrics. We used 28S rRNA, 18S rRNA, and ITS1 sequence data to infer a maximum likelihood phylogeny. We discriminated species using principal component and cluster analysis of shape data. Adams’s Kmult was used to detect phylogenetic signal in anchor shape. Phylogeny-correlated size and shape changes were investigated using continuous character mapping and directional statistics, respectively. We assessed morphological constraints in anchor morphometry using phylogenetic regression of anchor shape against body size and anchor size. Anchor morphological integration was studied using partial least squares method. The association between copulatory organ morphology and anchor shape and size in phylomorphospace was used to test the Rohde-Hobbs hypothesis. We created monogeneaGM, a new R package that integrates analyses of monogenean anchor geometric morphometric data with morphological and phylogenetic data. Results. We discriminated 12 of the 13 Ligophorus species using anchor shape data. Significant phylogenetic signal was detected in anchor shape. Thus, we discovered new morphological characters based on anchor shaft shape, the length between the inner root point and the outer root

  18. Monogenean anchor morphometry: systematic value, phylogenetic signal, and evolution.

    PubMed

    Khang, Tsung Fei; Soo, Oi Yoon Michelle; Tan, Wooi Boon; Lim, Lee Hong Susan

    2016-01-01

    Background. Anchors are one of the important attachment appendages for monogenean parasites. Common descent and evolutionary processes have left their mark on anchor morphometry, in the form of patterns of shape and size variation useful for systematic and evolutionary studies. When combined with morphological and molecular data, analysis of anchor morphometry can potentially answer a wide range of biological questions. Materials and Methods. We used data from anchor morphometry, body size and morphology of 13 Ligophorus (Monogenea: Ancyrocephalidae) species infecting two marine mugilid (Teleostei: Mugilidae) fish hosts: Moolgarda buchanani (Bleeker) and Liza subviridis (Valenciennes) from Malaysia. Anchor shape and size data (n = 530) were generated using methods of geometric morphometrics. We used 28S rRNA, 18S rRNA, and ITS1 sequence data to infer a maximum likelihood phylogeny. We discriminated species using principal component and cluster analysis of shape data. Adams's K mult was used to detect phylogenetic signal in anchor shape. Phylogeny-correlated size and shape changes were investigated using continuous character mapping and directional statistics, respectively. We assessed morphological constraints in anchor morphometry using phylogenetic regression of anchor shape against body size and anchor size. Anchor morphological integration was studied using partial least squares method. The association between copulatory organ morphology and anchor shape and size in phylomorphospace was used to test the Rohde-Hobbs hypothesis. We created monogeneaGM, a new R package that integrates analyses of monogenean anchor geometric morphometric data with morphological and phylogenetic data. Results. We discriminated 12 of the 13 Ligophorus species using anchor shape data. Significant phylogenetic signal was detected in anchor shape. Thus, we discovered new morphological characters based on anchor shaft shape, the length between the inner root point and the outer root

  19. Weighing Anchor in the "Ragged Times"

    ERIC Educational Resources Information Center

    Perry, Tonya B.

    2012-01-01

    In today's middle school classroom, grouping is an essential learning tool that enhances students' ability to collaborate with others and deepen their own thinking. Implementing group work effectively, though, can be a challenge, especially since groups tend to end their work at "ragged" or staggered times. Creating "anchor activities"--respectful…

  20. Finding Chemical Anchors in the Kitchen

    ERIC Educational Resources Information Center

    Haim, Liliana

    2005-01-01

    ''The Chemistry Kitchen'', a unit composed of five activities with kitchen elements for elementary students ages 9-11, introduces the children to the skills and chemical working ideas to be used later as anchors for chemical concepts. These activities include kitchen elements, determining the relative mass and so on.

  1. International Lunar Network (ILN) Anchor Nodes

    NASA Technical Reports Server (NTRS)

    Cohen, Barbara A.

    2009-01-01

    This slide presentation reviews the United States' contribution to the International Lunar Network (ILN) project, the Anchor Nodes project. The ILN is an initiative of 9 national space agencies to establish a set of robotic geophysical monitoring stations on the surface of the Moon. The project is aimed at furthering the understanding of the lunar composition, and interior structure.

  2. Expectations of Behaviorally Anchored Rating Scales.

    ERIC Educational Resources Information Center

    Jacobs, Rick; And Others

    1980-01-01

    Behaviorally anchored rating scales (BARS) are no better or worse than other methods when assessed on a quantitative basis but have greater potential when assessed on use and qualitative criteria. Suggestions are offered for extending BARS research to process questions and domains other than performance appraisal. (Author)

  3. Simple suture and anchor in rabbit hips

    PubMed Central

    Garcia Filho, Fernando Cal; Guarniero, Roberto; de Godoy Júnior, Rui Maciel; Pereira, César Augusto Martins; Matos, Marcos Almeida; Garcia, Lucas Cortizo

    2012-01-01

    Objective Using biomechanical studies, this research aims to compare hip capsulorrhaphy in rabbits, carried out with two different techniques: capsulorrhaphy with simple sutures and with anchors. Method Thirteen New Zealand Albino (Oryctolaguscuniculus) male rabbits, twenty-six hip joints, were used. First, a pilot project was performed with three rabbits (six hip joints). This experiment consisted of ten rabbits divided into two groups: group 1 underwent capsulorrhaphy on both right and left hips with simple suture using polyglycolic acid absorbable thread, and group 2 underwent capsulorrhaphy with titanium anchors. After a four-week postoperative period, the animals were euthanized and the hip joints were frozen. On the same day of the biomechanical studies, after the hip joints were previously unfrozen, the following parameters were evaluated: rigidity, maximum force, maximum deformity and energy. Results There was no relevant statistical difference in rigidity, maximum force, maximum deformity and energy between the simple suture and anchor groups. Conclusion Through biomechanical analyses, using parameters of rigidity, maximum force, maximum deformity and energy, it has been shown that capsulorrhaphy with simple suture and with anchors has similar results in rabbit hip joints. Level of Evidence II, Prospective Comparative Study. PMID:24453618

  4. 50 CFR 622.432 - Anchoring restriction.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Anchoring restriction. 622.432 Section 622.432 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE FISHERIES OF THE CARIBBEAN, GULF OF MEXICO, AND SOUTH ATLANTIC Reef...

  5. 50 CFR 622.432 - Anchoring restriction.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Anchoring restriction. 622.432 Section 622.432 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE FISHERIES OF THE CARIBBEAN, GULF OF MEXICO, AND SOUTH ATLANTIC Reef...

  6. 24 CFR 3285.401 - Anchoring instructions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... URBAN DEVELOPMENT MODEL MANUFACTURED HOME INSTALLATION STANDARDS Anchorage Against Wind § 3285.401... wind by use of anchor assembly type installations or by connecting the home to an alternative... must require the home to be secured against the wind, as described in this section. The...

  7. 24 CFR 3285.401 - Anchoring instructions.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... URBAN DEVELOPMENT MODEL MANUFACTURED HOME INSTALLATION STANDARDS Anchorage Against Wind § 3285.401... wind by use of anchor assembly type installations or by connecting the home to an alternative... must require the home to be secured against the wind, as described in this section. The...

  8. Anchoring the Panic Disorder Severity Scale

    ERIC Educational Resources Information Center

    Keough, Meghan E.; Porter, Eliora; Kredlow, M. Alexandra; Worthington, John J.; Hoge, Elizabeth A.; Pollack, Mark H.; Shear, M. Katherine; Simon, Naomi M.

    2012-01-01

    The Panic Disorder Severity Scale (PDSS) is a clinician-administered measure of panic disorder symptom severity widely used in clinical research. This investigation sought to provide clinically meaningful anchor points for the PDSS both in terms of clinical severity as measured by the Clinical Global Impression-Severity Scale (CGI-S) and to extend…

  9. Influence of Anchoring on Burial Depth of Submarine Pipelines

    PubMed Central

    Zhuang, Yuan; Li, Yang; Su, Wei

    2016-01-01

    Since the beginning of the twenty-first century, there has been widespread construction of submarine oil-gas transmission pipelines due to an increase in offshore oil exploration. Vessel anchoring operations are causing more damage to submarine pipelines due to shipping transportation also increasing. Therefore, it is essential that the influence of anchoring on the required burial depth of submarine pipelines is determined. In this paper, mathematical models for ordinary anchoring and emergency anchoring have been established to derive an anchor impact energy equation for each condition. The required effective burial depth for submarine pipelines has then been calculated via an energy absorption equation for the protection layer covering the submarine pipelines. Finally, the results of the model calculation have been verified by accident case analysis, and the impact of the anchoring height, anchoring water depth and the anchor weight on the required burial depth of submarine pipelines has been further analyzed. PMID:27166952

  10. 63. ANCHOR WINDLASS ROOM AFT LOOKING FORWARD ON CENTERLINE ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    63. ANCHOR WINDLASS ROOM - AFT LOOKING FORWARD ON CENTERLINE SHOWING LINE REELS, MAIN ANCHOR CHAIN, CHAIN STOPPERS, CAPSTAN AND CONTROLS. - U.S.S. HORNET, Puget Sound Naval Shipyard, Sinclair Inlet, Bremerton, Kitsap County, WA

  11. 9. CABLE ANCHORAGE DETAIL, NORTHWEST ABUTMENT (NOTE MOSSCOVERED CONCRETE ANCHOR ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    9. CABLE ANCHORAGE DETAIL, NORTHWEST ABUTMENT (NOTE MOSS-COVERED CONCRETE ANCHOR LEFT OF ANCHOR BOLTS) - Nisqually Suspension Bridge, Spanning Nisqually River on Service Road, Longmire, Pierce County, WA

  12. 345. Caltrans, Photographer September 20, 1935 "WEST ANCHOR ARM"; DETAIL ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    345. Caltrans, Photographer September 20, 1935 "WEST ANCHOR ARM"; DETAIL VIEW OF CANTILEVER TRUSS WEST ANCHOR ARM UNDER CONSTRUCTION. 7-1023 - San Francisco Oakland Bay Bridge, Spanning San Francisco Bay, San Francisco, San Francisco County, CA

  13. Influence of Anchoring on Burial Depth of Submarine Pipelines.

    PubMed

    Zhuang, Yuan; Li, Yang; Su, Wei

    2016-01-01

    Since the beginning of the twenty-first century, there has been widespread construction of submarine oil-gas transmission pipelines due to an increase in offshore oil exploration. Vessel anchoring operations are causing more damage to submarine pipelines due to shipping transportation also increasing. Therefore, it is essential that the influence of anchoring on the required burial depth of submarine pipelines is determined. In this paper, mathematical models for ordinary anchoring and emergency anchoring have been established to derive an anchor impact energy equation for each condition. The required effective burial depth for submarine pipelines has then been calculated via an energy absorption equation for the protection layer covering the submarine pipelines. Finally, the results of the model calculation have been verified by accident case analysis, and the impact of the anchoring height, anchoring water depth and the anchor weight on the required burial depth of submarine pipelines has been further analyzed. PMID:27166952

  14. Inositolphosphoglycan mediators structurally related to glycosyl phosphatidylinositol anchors: synthesis, structure and biological activity.

    PubMed

    Martín-Lomas, M; Khiar, N; García, S; Koessler, J L; Nieto, P M; Rademacher, T W

    2000-10-01

    The preparation of the pseudopentasaccharide 1a, an inositol-phosphoglycan (IPG) that contains the conserved linear structure of glycosyl phosphatidylinositol anchors (GPI anchors), was carried out by using a highly convergent 2+3-block synthesis approach which involves imidate and sulfoxide glycosylation reactions. The preferred solution conformation of this structure was determined by using NMR spectroscopy and molecular dynamics simulations prior to carrying out quantitative structure--activity relationship studies in connection with the insulin signalling process. The ability of 1a to stimulate lipogenesis in rat adipocytes as well as to inhibit cAMP dependent protein kinase and to activate pyruvate dehydrogenase phosphatase was investigated. Compound 1a did not show any significant activity, which may be taken as a strong indication that the GPI anchors are not the precursors of the IPG mediators. PMID:11072827

  15. Maleimide-functionalized lipids that anchor polypeptides to lipid bilayers and membranes.

    PubMed

    Elliott, J T; Prestwich, G D

    2000-01-01

    Two maleimide-containing diacylglycerol derivatives were synthesized to permit the anchoring of short peptides and longer polypeptides to phospholipid bilayers and membranes. The maleimide was introduced at the site normally occupied by a phospholipid headgroup. The first lipid, the dipalmitoyl ester of 1-maleimido-2,3-propanediol, was developed as a membrane anchor for extracellular domains of transmembrane proteins. The second anchoring lipid, in which the 3-position contained a 6-aminohexanoate, was designed for convenient modification with amine-reactive reporter groups. Specifically, the NBD fluorophore, 7-nitrobenzo-2-oxa-1, 3-diazole-aminohexanoic-N-hydroxysuccinimide ester, was attached to give an fluorescent anchoring reagent. Next, these reagents were applied to the anchoring of a C-terminally cysteamine-modified 8 kDa polypeptide that comprises the extracellular N-terminal domain of the human thrombin receptor, a transmembrane protease-activated receptor (PAR-1). Gel filtration and fluorescence analysis showed that the fluorescent lipopolypeptide spontaneously inserted into preformed phospholipid vesicles, but it did not insert into whole cell membranes. In contrast, the dipalmitoyl derivative could only be reconstituted into artificial membranes by mixing the lipopolypeptide and phospholipid before vesicle formation. These results suggest that biophysical interactions governing the lipopolypeptide insertion into artificial and cellular membranes may differ. The thiol-reactive lipidating reagents should be valuable materials for studying the structure and function of peptides and polypeptides at phospholipid bilayer surfaces. PMID:11087332

  16. Career Paths, Images and Anchors: A Study with Brazilian Professionals

    ERIC Educational Resources Information Center

    Kilimnik, Zelia Miranda; de Oliveira, Luiz Claudio Vieira; Sant'anna, Anderson De Souza; Barros, Delba Teixeira Rodrigues

    2011-01-01

    This article analyses career anchors changes associated to images and professionals trajectories. Its main question: Do anchors careers change through time? We conducted twelve interviews involving professionals from the Administration Area, applying Schein's Career Anchors Inventory (1993). We did the same two years later. In both of them, the…

  17. Students' Anchoring Predisposition: An Illustration from Spring Training Baseball

    ERIC Educational Resources Information Center

    Mohrweis, Lawrence C.

    2014-01-01

    The anchoring tendency results when decision makers anchor on initial values and then make final assessments that are adjusted insufficiently away from the initial values. The professional literature recognizes that auditors often risk falling into the judgment trap of anchoring and adjusting (Ranzilla et al., 2011). Students may also be unaware…

  18. Removal of the glycosylphosphatidylinositol anchor from PrP(Sc) by cathepsin D does not reduce prion infectivity.

    PubMed

    Lewis, Patrick A; Properzi, Francesca; Prodromidou, Kanella; Clarke, Anthony R; Collinge, John; Jackson, Graham S

    2006-04-15

    According to the protein-only hypothesis of prion propagation, prions are composed principally of PrP(Sc), an abnormal conformational isoform of the prion protein, which, like its normal cellular precursor (PrP(C)), has a GPI (glycosylphosphatidylinositol) anchor at the C-terminus. To date, elucidating the role of this anchor on the infectivity of prion preparations has not been possible because of the resistance of PrP(Sc) to the activity of PI-PLC (phosphoinositide-specific phospholipase C), an enzyme which removes the GPI moiety from PrP(C). Removal of the GPI anchor from PrP(Sc) requires denaturation before treatment with PI-PLC, a process that also abolishes infectivity. To circumvent this problem, we have removed the GPI anchor from PrP(Sc) in RML (Rocky Mountain Laboratory)-prion-infected murine brain homogenate using the aspartic endoprotease cathepsin D. This enzyme eliminates a short sequence at the C-terminal end of PrP to which the GPI anchor is attached. We found that this modification has no effect (i) on an in vitro amplification model of PrP(Sc), (ii) on the prion titre as determined by a highly sensitive N2a-cell based bioassay, or (iii) in a mouse bioassay. These results show that the GPI anchor has little or no role in either the propagation of PrP(Sc) or on prion infectivity. PMID:16441239

  19. Removal of the glycosylphosphatidylinositol anchor from PrPSc by cathepsin D does not reduce prion infectivity

    PubMed Central

    Lewis, Patrick A.; Properzi, Francesca; Prodromidou, Kanella; Clarke, Anthony R.; Collinge, John; Jackson, Graham S.

    2006-01-01

    According to the protein-only hypothesis of prion propagation, prions are composed principally of PrPSc, an abnormal conformational isoform of the prion protein, which, like its normal cellular precursor (PrPC), has a GPI (glycosylphosphatidylinositol) anchor at the C-terminus. To date, elucidating the role of this anchor on the infectivity of prion preparations has not been possible because of the resistance of PrPSc to the activity of PI-PLC (phosphoinositide-specific phospholipase C), an enzyme which removes the GPI moiety from PrPC. Removal of the GPI anchor from PrPSc requires denaturation before treatment with PI-PLC, a process that also abolishes infectivity. To circumvent this problem, we have removed the GPI anchor from PrPSc in RML (Rocky Mountain Laboratory)-prion-infected murine brain homogenate using the aspartic endoprotease cathepsin D. This enzyme eliminates a short sequence at the C-terminal end of PrP to which the GPI anchor is attached. We found that this modification has no effect (i) on an in vitro amplification model of PrPSc, (ii) on the prion titre as determined by a highly sensitive N2a-cell based bioassay, or (iii) in a mouse bioassay. These results show that the GPI anchor has little or no role in either the propagation of PrPSc or on prion infectivity. PMID:16441239

  20. Composite materials formed with anchored nanostructures

    DOEpatents

    Seals, Roland D; Menchhofer, Paul A; Howe, Jane Y; Wang, Wei

    2015-03-10

    A method of forming nano-structure composite materials that have a binder material and a nanostructure fiber material is described. A precursor material may be formed using a mixture of at least one metal powder and anchored nanostructure materials. The metal powder mixture may be (a) Ni powder and (b) NiAl powder. The anchored nanostructure materials may comprise (i) NiAl powder as a support material and (ii) carbon nanotubes attached to nanoparticles adjacent to a surface of the support material. The process of forming nano-structure composite materials typically involves sintering the mixture under vacuum in a die. When Ni and NiAl are used in the metal powder mixture Ni.sub.3Al may form as the binder material after sintering. The mixture is sintered until it consolidates to form the nano-structure composite material.

  1. Geodesic defect anchoring on nematic shells.

    PubMed

    Mirantsev, Leonid V; Sonnet, André M; Virga, Epifanio G

    2012-08-01

    Nematic shells are colloidal particles coated with nematic liquid crystal molecules, which may freely glide and rotate on the colloid's surface while keeping their long axis on the local tangent plane. Molecular dynamics simulations on a nanoscopic spherical shell indicate that under appropriate adhesion conditions for the molecules on the equator, the equilibrium nematic texture exhibits at each pole a pair of +1/2 defects so close to one another to be treated as one +1 defect. Spirals connect the polar defects, though the continuum limit of the interaction potential would not feature any elastic anisotropy. A molecular averaging justifies an anchoring defect energy that feels the geodesics emanating from the defect. All our observations are explained by such a geodesic anchoring, which vanishes on flat manifolds. PMID:23005713

  2. Partial credit model and pivot anchoring.

    PubMed

    Bode, R K

    2001-01-01

    This article contains information on the Rasch measurement partial credit model: what it is, how it differs from other Rasch models, when to use it, and how to use it. The calibration of instruments with increasingly complex items is described, starting with dichotomous items and moving on to polychotomous items using a single rating scale, and mixed polychotomous items using multiple rating scales, and instruments in which each item has its own rating scale. It also introduces a procedure for aligning rating scale categories to be used when more than one rating scale is used in a single instrument. Pivot anchoring is defined and an illustration of its use with the mental health scale of the SF-36 that contains positive and negative worded items is provided. It finally describes the effect of pivot anchoring on step calibrations, the item hierarchy, and person measures. PMID:12000858

  3. International Lunar Network (ILN) Anchor Nodes

    NASA Technical Reports Server (NTRS)

    Cohen, Barbara A.

    2008-01-01

    This slide presentation reviews what we know about the interior and surface of the moon and the need to establish a robotic set of geophysical monitoring stations on the surface of the Moon for the purpose of providing significant scientific value to the exploration of the Moon. The ILN Anchor Nodes will provide the backbone of the network in a way that accomplishes new science and allows other nodes to be flexible contributors to the network.

  4. Anchoring in a novel bimanual coordination pattern.

    PubMed

    Maslovat, Dana; Lam, Melanie Y; Brunke, Kirstin M; Chua, Romeo; Franks, Ian M

    2009-02-01

    Anchoring in cyclical movements has been defined as regions of reduced spatial or temporal variability [Beek, P. J. (1989). Juggling dynamics. PhD thesis. Amsterdam: Free University Press] that are typically found at movement reversal points. For in-phase and anti-phase movements, synchronizing reversal points with a metronome pulse has resulted in decreased anchor point variability and increased pattern stability [Byblow, W. D., Carson, R. G., & Goodman, D. (1994). Expressions of asymmetries and anchoring in bimanual coordination. Human Movement Science, 13, 3-28; Fink, P. W., Foo, P., Jirsa, V. K., & Kelso, J. A. S. (2000). Local and global stabilization of coordination by sensory information. Experimental Brain Research, 134, 9-20]. The present experiment examined anchoring during acquisition, retention, and transfer of a 90 degrees phase-offset continuous bimanual coordination pattern (whereby the right limb lags the left limb by one quarter cycle), involving horizontal flexion about the elbow. Three metronome synchronization strategies were imposed: participants either synchronized maximal flexion of the right arm (i.e., single metronome), both flexion and extension of the right arm (i.e., double metronome within-limb), or flexion of each arm (i.e., double metronome between-limb) to an auditory metronome. In contrast to simpler in-phase and anti-phase movements, synchronization of additional reversal points to the metronome did not reduce reversal point variability or increase pattern stability. Furthermore, practicing under different metronome synchronization strategies did not appear to have a significant effect on the rate of acquisition of the pattern. PMID:18842313

  5. Does the tail wag the dog? How the structure of a glycosylphosphatidylinositol anchor affects prion formation.

    PubMed

    Bate, Clive; Nolan, William; Williams, Alun

    2016-03-01

    There is increasing interest in the role of the glycosylphosphatidylinositol (GPI) anchor attached to the cellular prion protein (PrP(C)). Since GPI anchors can alter protein targeting, trafficking and cell signaling, our recent study examined how the structure of the GPI anchor affected prion formation. PrP(C) containing a GPI anchor from which the sialic acid had been removed (desialylated PrP(C)) was not converted to PrP(Sc) in prion-infected neuronal cell lines and in scrapie-infected primary cortical neurons. In uninfected neurons desialylated PrP(C) was associated with greater concentrations of gangliosides and cholesterol than PrP(C). In addition, the targeting of desialylated PrP(C) to lipid rafts showed greater resistance to cholesterol depletion than PrP(C). The presence of desialylated PrP(C) caused the dissociation of cytoplasmic phospholipase A2 (cPLA2) from PrP-containing lipid rafts, reduced the activation of cPLA2 and inhibited PrP(Sc) production. We conclude that the sialic acid moiety of the GPI attached to PrP(C) modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP(Sc) formation. PMID:26901126

  6. Test Score Equating Using a Mini-Version Anchor and a Midi Anchor: A Case Study Using SAT[R] Data

    ERIC Educational Resources Information Center

    Liu, Jinghua; Sinharay, Sandip; Holland, Paul W.; Curley, Edward; Feigenbaum, Miriam

    2011-01-01

    This study explores an anchor that is different from the traditional miniature anchor in test score equating. In contrast to a traditional "mini" anchor that has the same spread of item difficulties as the tests to be equated, the studied anchor, referred to as a "midi" anchor (Sinharay & Holland), has a smaller spread of item difficulties than…

  7. Electropermanent magnetic anchoring for surgery and endoscopy.

    PubMed

    Tugwell, Josef; Brennan, Philip; O'Shea, Conor; O'Donoghue, Kilian; Power, Timothy; O'Shea, Michael; Griffiths, James; Cahill, Ronan; Cantillon-Murphy, Padraig

    2015-03-01

    The use of magnets for anchoring of instrumentation in minimally invasive surgery and endoscopy has become of increased interest in recent years. Permanent magnets have significant advantages over electromagnets for these applications; larger anchoring and retraction force for comparable size and volume without the need for any external power supply. However, permanent magnets represent a potential hazard in the operating field where inadvertent attraction to surgical instrumentation is often undesirable. The current work proposes an interesting hybrid approach which marries the high forces of permanent magnets with the control of electromagnetic technology including the ability to turn the magnet OFF when necessary. This is achieved through the use of an electropermanent magnet, which is designed for surgical retraction across the abdominal and gastric walls. Our electropermanent magnet, which is hand-held and does not require continuous power, is designed with a center lumen which may be used for trocar or needle insertion. The device in this application has been demonstrated successfully in the porcine model where coupling between an intraluminal ring magnet and our electropermanent magnet facilitated guided insertion of an 18 Fr Tuohy needle for guidewire placement. Subsequent investigations have demonstrated the ability to control the coupling distance of the system alleviating shortcomings with current methods of magnetic coupling due to variation in transabdominal wall thicknesses. With further refinement, the magnet may find application in the anchoring of endoscopic and surgical instrumentation for minimally invasive interventions in the gastrointestinal tract. PMID:25361499

  8. Short read DNA fragment anchoring algorithm

    PubMed Central

    Wang, Wendi; Zhang, Peiheng; Liu, Xinchun

    2009-01-01

    Background The emerging next-generation sequencing method based on PCR technology boosts genome sequencing speed considerably, the expense is also get decreased. It has been utilized to address a broad range of bioinformatics problems. Limited by reliable output sequence length of next-generation sequencing technologies, we are confined to study gene fragments with 30~50 bps in general and it is relatively shorter than traditional gene fragment length. Anchoring gene fragments in long reference sequence is an essential and prerequisite step for further assembly and analysis works. Due to the sheer number of fragments produced by next-generation sequencing technologies and the huge size of reference sequences, anchoring would rapidly becoming a computational bottleneck. Results and discussion We compared algorithm efficiency on BLAT, SOAP and EMBF. The efficiency is defined as the count of total output results divided by time consumed to retrieve them. The data show that our algorithm EMBF have 3~4 times efficiency advantage over SOAP, and at least 150 times over BLAT. Moreover, when the reference sequence size is increased, the efficiency of SOAP will get degraded as far as 30%, while EMBF have preferable increasing tendency. Conclusion In conclusion, we deem that EMBF is more suitable for short fragment anchoring problem where result completeness and accuracy is predominant and the reference sequences are relatively large. PMID:19208116

  9. Coracoclavicular stabilization using a suture anchor technique.

    PubMed

    Friedman, Darren J; Barron, O Alton; Catalano, Louis; Donahue, Joseph P; Zambetti, George

    2008-06-01

    Multiple fixation options exist for coracoclavicular stabilization, but many are technically demanding and require hardware removal. In the study reported here, we reviewed a specific fixation technique that includes suture anchors moored in the base of the coracoid process. We retrospectively reviewed 24 consecutive cases of patients who underwent coracoclavicular stabilization with a suture anchor for a type III or type V acromioclavicular (AC) joint separation or a group II, type II or type V distal clavicle fracture. Eighteen of the 22 patients had full strength and painless range of motion (ROM) in the affected extremity by 3 months and at final follow-up (minimum, 24 months; mean, 39 months). Two patients were lost to follow-up. Four patients had early complications likely secondary to documented noncompliance. Two of these 4 patients underwent reoperation with a similar procedure and remained asymptomatic at a minimum follow-up of 15 months. One patient underwent osteophyte and knot excision 7 months after surgery and remained asymptomatic at 30 months. Our results suggest that coracoclavicular stabilization using a suture anchor technique is a safe and reliable method of treating acromioclavicular joint separations and certain distal clavicle fractures in the compliant patient. PMID:18716693

  10. Structural basis for membrane anchoring of HIV-1 envelope spike.

    PubMed

    Dev, Jyoti; Park, Donghyun; Fu, Qingshan; Chen, Jia; Ha, Heather Jiwon; Ghantous, Fadi; Herrmann, Tobias; Chang, Weiting; Liu, Zhijun; Frey, Gary; Seaman, Michael S; Chen, Bing; Chou, James J

    2016-07-01

    HIV-1 envelope spike (Env) is a type I membrane protein that mediates viral entry. We used nuclear magnetic resonance to determine an atomic structure of the transmembrane (TM) domain of HIV-1 Env reconstituted in bicelles that mimic a lipid bilayer. The TM forms a well-ordered trimer that protects a conserved membrane-embedded arginine. An amino-terminal coiled-coil and a carboxyl-terminal hydrophilic core stabilize the trimer. Individual mutations of conserved residues did not disrupt the TM trimer and minimally affected membrane fusion and infectivity. Major changes in the hydrophilic core, however, altered the antibody sensitivity of Env. These results show how a TM domain anchors, stabilizes, and modulates a viral envelope spike and suggest that its influence on Env conformation is an important consideration for HIV-1 immunogen design. PMID:27338706

  11. Intrinsic disorder within an AKAP-protein kinase A complex guides local substrate phosphorylation

    PubMed Central

    Smith, F Donelson; Reichow, Steve L; Esseltine, Jessica L; Shi, Dan; Langeberg, Lorene K; Scott, John D; Gonen, Tamir

    2013-01-01

    Anchoring proteins sequester kinases with their substrates to locally disseminate intracellular signals and avert indiscriminate transmission of these responses throughout the cell. Mechanistic understanding of this process is hampered by limited structural information on these macromolecular complexes. A-kinase anchoring proteins (AKAPs) spatially constrain phosphorylation by cAMP-dependent protein kinases (PKA). Electron microscopy and three-dimensional reconstructions of type-II PKA-AKAP18γ complexes reveal hetero-pentameric assemblies that adopt a range of flexible tripartite configurations. Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an ∼16 nanometer radius of motion to the associated catalytic subunits. Manipulating flexibility within the PKA holoenzyme augmented basal and cAMP responsive phosphorylation of AKAP-associated substrates. Cell-based analyses suggest that the catalytic subunit remains within type-II PKA-AKAP18γ complexes upon cAMP elevation. We propose that the dynamic movement of kinase sub-structures, in concert with the static AKAP-regulatory subunit interface, generates a solid-state signaling microenvironment for substrate phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.01319.001 PMID:24192038

  12. Robotic Ankle for Omnidirectional Rock Anchors

    NASA Technical Reports Server (NTRS)

    Parness, Aaron; Frost, Matthew; Thatte, Nitish

    2013-01-01

    Future robotic exploration of near-Earth asteroids and the vertical and inverted rock walls of lava caves and cliff faces on Mars and other planetary bodies would require a method of gripping their rocky surfaces to allow mobility without gravitational assistance. In order to successfully navigate this terrain and drill for samples, the grippers must be able to produce anchoring forces in excess of 100 N. Additionally, the grippers must be able to support the inertial forces of a moving robot, as well gravitational forces for demonstrations on Earth. One possible solution would be to use microspine arrays to anchor to rock surfaces and provide the necessary load-bearing abilities for robotic exploration of asteroids. Microspine arrays comprise dozens of small steel hooks supported on individual suspensions. When these arrays are dragged along a rock surface, the steel hooks engage with asperities and holes on the surface. The suspensions allow for individual hooks to engage with asperities while the remaining hooks continue to drag along the surface. This ensures that the maximum possible number of hooks engage with the surface, thereby increasing the load-bearing abilities of the gripper. Using the microspine array grippers described above as the end-effectors of a robot would allow it to traverse terrain previously unreachable by traditional wheeled robots. Furthermore, microspine-gripping robots that can perch on cliffs or rocky walls could enable a new class of persistent surveillance devices for military applications. In order to interface these microspine grippers with a legged robot, an ankle is needed that can robotically actuate the gripper, as well as allow it to conform to the large-scale irregularities in the rock. The anchor serves three main purposes: deploy and release the anchor, conform to roughness or misalignment with the surface, and cancel out any moments about the anchor that could cause unintentional detachment. The ankle design contains a

  13. Binding constants of membrane-anchored receptors and ligands: A general theory corroborated by Monte Carlo simulations.

    PubMed

    Xu, Guang-Kui; Hu, Jinglei; Lipowsky, Reinhard; Weikl, Thomas R

    2015-12-28

    Adhesion processes of biological membranes that enclose cells and cellular organelles are essential for immune responses, tissue formation, and signaling. These processes depend sensitively on the binding constant K2D of the membrane-anchored receptor and ligand proteins that mediate adhesion, which is difficult to measure in the "two-dimensional" (2D) membrane environment of the proteins. An important problem therefore is to relate K2D to the binding constant K3D of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in three dimensions (3D). In this article, we present a general theory for the binding constants K2D and K3D of rather stiff proteins whose main degrees of freedom are translation and rotation, along membranes and around anchor points "in 2D," or unconstrained "in 3D." The theory generalizes previous results by describing how K2D depends both on the average separation and thermal nanoscale roughness of the apposing membranes, and on the length and anchoring flexibility of the receptors and ligands. Our theoretical results for the ratio K2D/K3D of the binding constants agree with detailed results from Monte Carlo simulations without any data fitting, which indicates that the theory captures the essential features of the "dimensionality reduction" due to membrane anchoring. In our Monte Carlo simulations, we consider a novel coarse-grained model of biomembrane adhesion in which the membranes are represented as discretized elastic surfaces, and the receptors and ligands as anchored molecules that diffuse continuously along the membranes and rotate at their anchor points. PMID:26723621

  14. Allosteric inhibition of Aurora-A kinase by a synthetic vNAR domain.

    PubMed

    Burgess, Selena G; Oleksy, Arkadiusz; Cavazza, Tommaso; Richards, Mark W; Vernos, Isabelle; Matthews, David; Bayliss, Richard

    2016-07-01

    The vast majority of clinically approved protein kinase inhibitors target the ATP-binding pocket directly. Consequently, many inhibitors have broad selectivity profiles and most have significant off-target effects. Allosteric inhibitors are generally more selective, but are difficult to identify because allosteric binding sites are often unknown or poorly characterized. Aurora-A is activated through binding of TPX2 to an allosteric site on the kinase catalytic domain, and this knowledge could be exploited to generate an inhibitor. Here, we generated an allosteric inhibitor of Aurora-A kinase based on a synthetic, vNAR single domain scaffold, vNAR-D01. Biochemical studies and a crystal structure of the Aurora-A/vNAR-D01 complex show that the vNAR domain overlaps with the TPX2 binding site. In contrast with the binding of TPX2, which stabilizes an active conformation of the kinase, binding of the vNAR domain stabilizes an inactive conformation, in which the αC-helix is distorted, the canonical Lys-Glu salt bridge is broken and the regulatory (R-) spine is disrupted by an additional hydrophobic side chain from the activation loop. These studies illustrate how single domain antibodies can be used to characterize the regulatory mechanisms of kinases and provide a rational basis for structure-guided design of allosteric Aurora-A kinase inhibitors. PMID:27411893

  15. Allosteric inhibition of Aurora-A kinase by a synthetic vNAR domain

    PubMed Central

    Burgess, Selena G.; Oleksy, Arkadiusz; Cavazza, Tommaso; Richards, Mark W.; Vernos, Isabelle; Matthews, David

    2016-01-01

    The vast majority of clinically approved protein kinase inhibitors target the ATP-binding pocket directly. Consequently, many inhibitors have broad selectivity profiles and most have significant off-target effects. Allosteric inhibitors are generally more selective, but are difficult to identify because allosteric binding sites are often unknown or poorly characterized. Aurora-A is activated through binding of TPX2 to an allosteric site on the kinase catalytic domain, and this knowledge could be exploited to generate an inhibitor. Here, we generated an allosteric inhibitor of Aurora-A kinase based on a synthetic, vNAR single domain scaffold, vNAR-D01. Biochemical studies and a crystal structure of the Aurora-A/vNAR-D01 complex show that the vNAR domain overlaps with the TPX2 binding site. In contrast with the binding of TPX2, which stabilizes an active conformation of the kinase, binding of the vNAR domain stabilizes an inactive conformation, in which the αC-helix is distorted, the canonical Lys-Glu salt bridge is broken and the regulatory (R-) spine is disrupted by an additional hydrophobic side chain from the activation loop. These studies illustrate how single domain antibodies can be used to characterize the regulatory mechanisms of kinases and provide a rational basis for structure-guided design of allosteric Aurora-A kinase inhibitors. PMID:27411893

  16. Histologic and morphologic evaluation of explanted bone anchors from bone-anchored hearing aids.

    PubMed

    Mlynski, Robert; Goldberg, Eva; Ebmeyer, Joerg; Scheich, Matthias; Gattenlöhner, Stefan; Schwager, Konrad; Hagen, Rudolf; Shehata-Dieler, Wafaa

    2009-05-01

    Bone-anchored hearing aids are a standard option in rehabilitation of patients with conductive or mixed hearing loss, and also CROS fitting. However, the skin-penetrating bone anchor repeatedly gives reason for discussion about the risk of infection of surrounding tissues as a major cause of malfunction. In the present study, explanted bone anchors with surrounding bone and soft tissue were examined and compared with the morphology of lost implants. The anchors originated from five patients. Two needed explantation due to deafness with the need of cochlea implantation. A third patient underwent explantation due to meningeal irritation by the bone anchor. Another patient lost the implant due to mechanical stress shortly after implantation. The last implant was lost in a child without apparent reason. All implants were clinically free of infection and had been stable for a median implantation period of 12 months. During the explantation procedure, the fixtures were recovered together with the attached soft tissue and bone. The specimens were examined by light microscopy or scanning electron microscopy (SEM). Sectioning for light microscopy was performed with a diamond-coated saw microtome. Histopathologic examination of the surrounding skin and subcutaneous soft tissue showed slight inflammation in one case only. The bone was regularly vital, presenting no signs of inflammation. The threads of the fixtures were filled with bone, with particularly strong attachment to the flank of traction. The SEM investigation exposed the ultrastructural interaction of bone with the implant surface. Filiform- and podocyte-like processes of osteocytes attach to the implant; lost implants did not reflect these features. Implant integration involves both osseointegration as well as soft tissue integration. Titanium oxide as the active implant surface promotes this integration even in unstable implants. The morphologic analysis exposed structural areas of the implant with weak bone

  17. Analysis of Glenoid Inter-anchor Distance with an All-Suture Anchor System

    PubMed Central

    Kramer, Jonathan; Robinson, Sean; Dutton, Pascual; Dickinson, Ephraim; Rodriguez, John Paul; Camisa, William; Leasure, Jeremi M.; Montgomery, William H.

    2016-01-01

    Objectives: Arthroscopic shoulder stabilization using suture anchors are commonly used techniques. More recently developed all-suture systems employ smaller diameter anchors, which increase repair contact area and allow greater placement density on narrow surfaces such as the glenoid. Our goal is investigate the strength characteristics of various inter-anchor distances in a human glenoid model. Methods: Twelve fresh-frozen human cadaveric glenoids were potted after the labrum was excised. The glenoids were then implanted with 1.4 mm all-suture anchors (Juggerknot, Biomet, Warsaw, IN) at varying inter-anchor distances. Anchors were implanted adjacent to one another or at 2 mm, 3 mm, or 5 mm distances using a template with pre-drilled holes. The glenoids were then underwent single cycle pullout testing using a test frame (Instron 8521, Instron Inc., Norwood, MA). A 5 N preload was applied to the construct and the actuator was driven away from the shoulder at a rate of 12.5mm/s as seen in Figure 1. Force and displacement were collected from the test frame actuator at a rate of 500 Hz. The primary outcomes were failure strength and stiffness. Stiffness was calculated from the initial linear region of the force displacement curve. Failure strength was defined as the first local maximum inflection point in the force displacement curve. Results: During load to fail testing, all but three of the specimens had both anchors pull out of the glenoid. The other mode of failure included one or both of the sutures failing. Stiffness was 13.52 ± 3.8, 17.97 ± 5.02, 17.59 ± 4.65 and 18.95 ± 4.67 N/mm for the adjacent, 2 mm, 3 mm and 5 mm treatment groups as shown Table 1. The adjacent group had a significantly lower stiffness compared to the other treatment groups. Failure strength was 48.68 ± 20.64, 76.16 ± 23.78, 73.19 ± 35.83 and 87.04 ± 34.67 N for the adjacent, 2 mm, 3 mm and 5 mm treatment groups as shown in Table 1. The adjacent group had a significantly lower

  18. AKAP79 Selectively Enhances Protein Kinase C Regulation of GluR1 at a Ca2+-Calmodulin-dependent Protein Kinase II/Protein Kinase C Site*

    PubMed Central

    Tavalin, Steven J.

    2008-01-01

    Enhancement of AMPA receptor activity in response to synaptic plasticity inducing stimuli may arise, in part, through phosphorylation of the GluR1 AMPA receptor subunit at Ser-831. This site is a substrate for both Ca2+-calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC). However, neuronal protein levels of CaMKII may exceed those of PKC by an order of magnitude. Thus, it is unclear how PKC could effectively regulate this common target site. The multivalent neuronal scaffold A-kinase-anchoring protein 79 (AKAP79) is known to bind PKC and is linked to GluR1 by synapse-associated protein 97 (SAP97). Here, biochemical studies demonstrate that AKAP79 localizes PKC activity near the receptor, thus accelerating Ser-831 phosphorylation. Complementary electrophysiological studies indicate that AKAP79 selectively shifts the dose-dependence for PKC modulation of GluR1 receptor currents ∼20-fold, such that low concentrations of PKC are as effective as much higher CaMKII concentrations. By boosting PKC activity near a target substrate, AKAP79 provides a mechanism to overcome limitations in kinase abundance thereby ensuring faithful signal propagation and efficient modification of AMPA receptor-mediated responses. PMID:18305116

  19. PAQR3 modulates cholesterol homeostasis by anchoring Scap/SREBP complex to the Golgi apparatus

    PubMed Central

    Xu, Daqian; Wang, Zheng; Zhang, Yuxue; Jiang, Wei; Pan, Yi; Song, Bao-Liang; Chen, Yan

    2015-01-01

    Cholesterol biosynthesis is regulated by transcription factors SREBPs and their escort protein Scap. On sterol depletion, Scap/SREBP complex is transported from endoplasmic reticulum (ER) to the Golgi apparatus where SREBP is activated. Under cholesterol sufficient condition, Insigs act as anchor proteins to retain Scap/SREBP in the ER. However, the anchor protein of Scap/SREBP in the Golgi is unknown. Here we report that a Golgi-localized membrane protein progestin and adipoQ receptors 3 (PAQR3) interacts with Scap and SREBP and tethers them to the Golgi. PAQR3 promotes Scap/SREBP complex formation, potentiates SREBP processing and enhances lipid synthesis. The mutually exclusive interaction between Scap and PAQR3 or Insig-1 is regulated by cholesterol level. PAQR3 knockdown in liver blunts SREBP pathway and decreases hepatic cholesterol content. Disrupting the interaction of PAQR3 with Scap/SREBP by a synthetic peptide inhibits SREBP processing and activation. Thus, PAQR3 regulates cholesterol homeostasis by anchoring Scap/SREBP to the Golgi and disruption of such function reduces cholesterol biosynthesis. PMID:26311497

  20. Anchor Toolkit - a secure mobile agent system

    SciTech Connect

    Mudumbai, Srilekha S.; Johnston, William; Essiari, Abdelilah

    1999-05-19

    Mobile agent technology facilitates intelligent operation insoftware systems with less human interaction. Major challenge todeployment of mobile agents include secure transmission of agents andpreventing unauthorized access to resources between interacting systems,as either hosts, or agents, or both can act maliciously. The Anchortoolkit, designed by LBNL, handles the transmission and secure managementof mobile agents in a heterogeneous distributed computing environment. Itprovides users with the option of incorporating their security managers.This paper concentrates on the architecture, features, access control anddeployment of Anchor toolkit. Application of this toolkit in a securedistributed CVS environment is discussed as a case study.

  1. BAHA: Bone-Anchored Hearing Aid

    PubMed Central

    Hagr, Abdulrahman

    2007-01-01

    Bone-Anchored Hearing Aid (BAHA) has proven performance and advantages for patients with aural atresia or chronic ear drainage who cannot wear air-conduction hearing aids. The BAHA has both cosmetic and acoustic advantages over most conventional hearing aids and hence is becoming increasingly popular. Moreover, BAHA improves the quality of life and has also significantly reduces ear discharge. This extensive review of the literature pertaining to BAHA discus the history, the indications, the advantages, the prediction of the outcome and the complications of this device as well as comparing it to the conventional hearing aids. PMID:21475438

  2. Minireview: Steroid/Nuclear Receptor-Regulated Dynamics of Occluding and Anchoring Junctions

    PubMed Central

    Kapadia, Bhumika J.

    2014-01-01

    A diverse set of physiological signals control intercellular interactions by regulating the structure and function of occluding junctions (tight junctions) and anchoring junctions (adherens junctions and desmosomes). These plasma membrane junctions are comprised of multiprotein complexes of transmembrane and cytoplasmic peripheral plasma membrane proteins. Evidence from many hormone-responsive tissues has shown that expression, modification, molecular interactions, stability, and localization of junctional complex-associated proteins can be targeted by nuclear hormone receptors and their ligands through transcriptional and nontranscriptional mechanisms. The focus of this minireview is to discuss molecular, cellular, and physiological studies that directly link nuclear receptor- and ligand-triggered signaling pathways to the regulation of occluding and anchoring junction dynamics. PMID:25203673

  3. Beam and pump currents for a MARS anchor

    SciTech Connect

    Stroud, P.D.; Devoto, R.S.

    1982-01-01

    The MARS anchor has been modeled and analyzed with a numerical bounce-average Fokker-Planck treatment. The interactions between the various ion classes (passing, spanning and trapped) are treated in detail. Consistent solutions have been obtained for the complete asymmetric anchor. For baseline MARS parameters, 5.7 MW per end must be delivered by the anchor neutral beams, and 6.5 A per end must be pumped to maintain the sloshing ion axial density profiles.

  4. Anchor-induced chondral damage in the hip

    PubMed Central

    Matsuda, Dean K.; Bharam, Srino; White, Brian J.; Matsuda, Nicole A.; Safran, Marc

    2015-01-01

    The purpose of this study is to investigate the outcomes from anchor-induced chondral damage of the hip, both with and without frank chondral penetration. A multicenter retrospective case series was performed of patients with chondral deformation or penetration during initial hip arthroscopic surgery. Intra-operative findings, post-surgical clinical courses, hip outcome scores and descriptions of arthroscopic treatment in cases requiring revision surgery and anchor removal are reported. Five patients (three females) of mean age 32 years (range, 16–41 years) had documented anchor-induced chondral damage with mean 3.5 years (range, 1.5–6.0 years) follow-up. The 1 o'clock position (four cases) and anterior and mid-anterior portals (two cases each) were most commonly implicated. Two cases of anchor-induced acetabular chondral deformation without frank penetration had successful clinical and radiographic outcomes, while one case progressed from deformation to chondral penetration with clinical worsening. Of the cases that underwent revision hip arthroscopy, all three had confirmed exposed hard anchors which were removed. Two patients have had clinical improvement and one patient underwent early total hip arthroplasty. Anchor-induced chondral deformation without frank chondral penetration may be treated with close clinical and radiographic monitoring with a low threshold for revision surgery and anchor removal. Chondral penetration should be treated with immediate removal of offending hard anchor implants. Preventative measures include distal-based portals, small diameter and short anchors, removable hard anchors, soft suture-based anchors, curved drill and anchor insertion instrumentation and attention to safe trajectories while visualizing the acetabular articular surface. PMID:27011815

  5. Anchor-induced chondral damage in the hip.

    PubMed

    Matsuda, Dean K; Bharam, Srino; White, Brian J; Matsuda, Nicole A; Safran, Marc

    2015-01-01

    The purpose of this study is to investigate the outcomes from anchor-induced chondral damage of the hip, both with and without frank chondral penetration. A multicenter retrospective case series was performed of patients with chondral deformation or penetration during initial hip arthroscopic surgery. Intra-operative findings, post-surgical clinical courses, hip outcome scores and descriptions of arthroscopic treatment in cases requiring revision surgery and anchor removal are reported. Five patients (three females) of mean age 32 years (range, 16-41 years) had documented anchor-induced chondral damage with mean 3.5 years (range, 1.5-6.0 years) follow-up. The 1 o'clock position (four cases) and anterior and mid-anterior portals (two cases each) were most commonly implicated. Two cases of anchor-induced acetabular chondral deformation without frank penetration had successful clinical and radiographic outcomes, while one case progressed from deformation to chondral penetration with clinical worsening. Of the cases that underwent revision hip arthroscopy, all three had confirmed exposed hard anchors which were removed. Two patients have had clinical improvement and one patient underwent early total hip arthroplasty. Anchor-induced chondral deformation without frank chondral penetration may be treated with close clinical and radiographic monitoring with a low threshold for revision surgery and anchor removal. Chondral penetration should be treated with immediate removal of offending hard anchor implants. Preventative measures include distal-based portals, small diameter and short anchors, removable hard anchors, soft suture-based anchors, curved drill and anchor insertion instrumentation and attention to safe trajectories while visualizing the acetabular articular surface. PMID:27011815

  6. Decoding Cytoskeleton-Anchored and Non-Anchored Receptors from Single-Cell Adhesion Force Data.

    PubMed

    Sariisik, Ediz; Popov, Cvetan; Müller, Jochen P; Docheva, Denitsa; Clausen-Schaumann, Hauke; Benoit, Martin

    2015-10-01

    Complementary to parameters established for cell-adhesion force curve analysis, we evaluated the slope before a force step together with the distance from the surface at which the step occurs and visualized the result in a two-dimensional density plot. This new tool allows detachment steps of long membrane tethers to be distinguished from shorter jumplike force steps, which are typical for cytoskeleton-anchored bonds. A prostate cancer cell line (PC3) immobilized on an atomic-force-microscopy sensor interacted with three different substrates: collagen-I (Col-I), bovine serum albumin, and a monolayer of bone marrow-derived stem cells (SCP1). To address PC3 cells' predominant Col-I binding molecules, an antibody-blocking β1-integrin was used. Untreated PC3 cells on Col-I or SCP1 cells, which express Col-I, predominantly showed jumps in their force curves, while PC3 cells on bovine-serum-albumin- and antibody-treated PC3 cells showed long membrane tethers. The probability density plots thus revealed that β1-integrin-specific interactions are predominately anchored to the cytoskeleton, while the nonspecific interactions are mainly membrane-anchored. Experiments with latrunculin-A-treated PC3 cells corroborated these observations. The plots thus reveal details of the anchoring of bonds to the cell and provide a better understanding of receptor-ligand interactions. PMID:26445433

  7. Nonrotating, self-centering anchor assembly for anchoring a bolt in a borehole

    DOEpatents

    Bevan, John E.; King, Grant W.

    1998-01-01

    An expandable anchor assembly is provided for anchoring the threaded end portion of an elongated roof bolt in a borehole. The anchoring assembly includes a hollow outer sleeve in the form of a plurality of symmetrically arranged, longitudinal segmented wall portions with exterior gripping teeth and an inner expander sleeve in the form of a corresponding plurality of longitudinal wall portions symmetrically arranged about a central axis to define an inner threaded cylindrical section. The inner sleeve is captured within and moveable axially relative to the outer sleeve. As the threaded end portion of the elongated bolt is inserted into the inner threaded cylindrical section of the inner sleeve from the trailing end to the leading end thereof, the inner sleeve expands over and clamps around the threaded end portion of the elongated bolt. Thereafter, partial withdrawal of the elongated bolt from the borehole causes the inner sleeve to axially move relative to the outer sleeve from the leading end toward the trailing end of the outer sleeve in a wedging action to cause the outer sleeve to radially expand and force engagement of the gripping teeth against the sidewall of the borehole to thereby secure the expandable anchor assembly and therewith the threaded end portion of the elongated bolt within the borehole.

  8. Nonrotating, self-centering anchor assembly for anchoring a bolt in a borehole

    DOEpatents

    Bevan, J.E.; King, G.W.

    1998-12-08

    An expandable anchor assembly is provided for anchoring the threaded end portion of an elongated roof bolt in a borehole. The anchoring assembly includes a hollow outer sleeve in the form of a plurality of symmetrically arranged, longitudinal segmented wall portions with exterior gripping teeth and an inner expander sleeve in the form of a corresponding plurality of longitudinal wall portions symmetrically arranged about a central axis to define an inner threaded cylindrical section. The inner sleeve is captured within and moveable axially relative to the outer sleeve. As the threaded end portion of the elongated bolt is inserted into the inner threaded cylindrical section of the inner sleeve from the trailing end to the leading end thereof, the inner sleeve expands over and clamps around the threaded end portion of the elongated bolt. Thereafter, partial withdrawal of the elongated bolt from the borehole causes the inner sleeve to axially move relative to the outer sleeve from the leading end toward the trailing end of the outer sleeve in a wedging action to cause the outer sleeve to radially expand and force engagement of the gripping teeth against the sidewall of the borehole to thereby secure the expandable anchor assembly and therewith the threaded end portion of the elongated bolt within the borehole. 8 figs.

  9. Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

    SciTech Connect

    Jemmerson, R.; Low, M.G.

    1987-09-08

    Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either /sup 3/H-fatty acids or (/sup 3/H)ethanolamine as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material. Phosphatidylinositol-specific phospholipase C (PI-PLC) released a substantial proportion of the /sup 3/H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of (/sup 3/H)ethanolamine-labeled material. PI-PLC also liberated catalytically active alkaline phosphatase from viable cells, and this could be selectively blocked by monoclonal antibodies to alkaline phosphatase. However, the alkaline phosphatase released from /sup 3/H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast, treatment with bromelain removed both the /sup 3/H-fatty acid and the (/sup 3/H)ethanolamine label from purified alkaline phosphatase. Subtilisin was also able to remove the (/sup 3/H)ethanolamine label from the purified alkaline phosphatase. The /sup 3/H radioactivity in alkaline phosphatase purified from (/sup 3/H)ethanolamine-labeled cells comigrated with authentic (/sup 3/H)ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the /sup 3/H-fatty acid and (/sup 3/H)ethanolamine are covalently attached to the carboxyl-terminal segment since bromelain and subtilisin both release alkaline phosphatase from the membrane by cleavage at that end of the polypeptide chain. The data are consistent with findings for other proteins recently shown to be anchored in the membrane through a glycosylphosphatidylinositol structure and indicate that a similar structure contributes to the membrane anchoring of alkaline phosphatase.

  10. Transosseous Acetabular Labral Repair as an Alternative to Anchors

    PubMed Central

    Pérez-Carro, Luis; Cabello, Andres Gonzalez; Rakha, Mohamed Ibrahim; Patnaik, Sarthak; Centeno, Elias; Miranda, Victor; Fernández, Ana Alfonso

    2015-01-01

    Labral tears are the most common pathology in patients undergoing hip arthroscopy and the most common cause of mechanical hip symptoms. Labral repair techniques have been described in the literature using suture anchors placed as close as possible to the acetabular rim without penetrating the articular surface. Optimal surgical technique for labral repair is very important, and an inappropriate entry point and guide angulation may lead to intra-articular penetration of the anchor, chondral damage, anchor loosening, or inadequate fixation. A shallow dysplastic hip, the drilling trajectory, the narrow width of the acetabular rim, or some specific anatomic variations may generate difficulty during anchor placement. Suture anchors themselves have been associated with several significant complications, including rim fracture, osteolysis, enlargement of drill holes, and infection. The treatment of labral lesions with transosseous suture is an alternative to anchor use, eliminating the need for anchors and avoiding anchor-associated complications. This technique offers versatility to surgeons and is more cost-effective for patients and health services. We aim to describe the indications and technique for transosseous labral repair without anchors. PMID:26697295

  11. New Retrievable Coil Anchors: Preliminary In Vivo Experiences in Swine

    SciTech Connect

    Konya, A. Wright, K.C.

    2005-04-15

    Purpose. To design and test retrievable coil anchors to improve the safety and efficacy of coil embolization. Methods. Fifty-two 0.038-inch homemade retrievable stainless steel coils were equipped with one of four different pre-shaped nitinol anchors and tested in 38 pigs. All coils with the anchor were completely retrieved and redeployed 3-18 times (median 7 times) prior to release. Types 1 and 2 anchored coils were acutely deployed in the external iliac arteries (n = 10 each), and chronically tested (1 week) in the common carotid arteries (n = 6 each). Larger type 1 (n = 4), type 3 (n = 6), and type 4 (n = 4) anchored coils were acutely deployed in the abdominal aorta. The largest type 1 anchors (n = 6) were acutely tested in the inferior vena cava. Results. All anchored coils were successfully retrieved and repositioned several times. All but two coils formed a compact plug and there was no coil migration except with two mechanically defective type 3 anchors. Conclusion. The use of retrievable anchors allowed the coils to be retrieved and repositioned, prevented coil migration, and enabled compact coil configuration.

  12. The Ponto bone-anchored hearing system.

    PubMed

    Westerkull, Patrik

    2011-01-01

    Based on a foundation of a well-documented Brånemark implant experience and the most advanced sound processing technology from Oticon, Oticon Medical introduced in 2009 a new bone-anchored hearing system, the Ponto system. Although the principle of percutaneous direct bone conduction is established, new technology aiming at improving speech perception and sound comfort in noisy environments is introduced with the new Ponto system. This has been possible by using the sound-processing platform from the most advanced conventional hearing aids from Oticon. Furthermore, specific steps have been taken aiming at improving the implant as well as the skin-penetrating abutment. The Ponto sound processor coupling has been developed to withstand long-term wear and tear. With the Ponto system, a new alternative is offered to the various groups of both new and existing users of bone-anchored hearing systems. In clinical studies of the Ponto Pro sound processor, the objective testing verified the high performance of the adaptive directionality system. The subjective questionnaires on speech perception and device handling also verified that the Ponto system offers efficient hearing rehabilitation as well as other user benefits. The Ponto system also includes possibilities to fit the sound processor 2 weeks after surgery. Referred clinical studies with the Ponto system have, however, not yet been published in the literature (autumn 2010).Clinical results have this far been presented at scientific conferences. PMID:21389702

  13. Expressed Glycosylphosphatidylinositol-Anchored Horseradish Peroxidase Identifies Co-Clustering Molecules in Individual Lipid Raft Domains

    PubMed Central

    Miyagawa-Yamaguchi, Arisa; Kotani, Norihiro; Honke, Koichi

    2014-01-01

    Lipid rafts that are enriched in glycosylphosphatidylinositol (GPI)-anchored proteins serve as a platform for important biological events. To elucidate the molecular mechanisms of these events, identification of co-clustering molecules in individual raft domains is required. Here we describe an approach to this issue using the recently developed method termed enzyme-mediated activation of radical source (EMARS), by which molecules in the vicinity within 300 nm from horseradish peroxidase (HRP) set on the probed molecule are labeled. GPI-anchored HRP fusion proteins (HRP-GPIs), in which the GPI attachment signals derived from human decay accelerating factor and Thy-1 were separately connected to the C-terminus of HRP, were expressed in HeLa S3 cells, and the EMARS reaction was catalyzed by these expressed HRP-GPIs under a living condition. As a result, these different HRP-GPIs had differences in glycosylation and localization and formed distinct clusters. This novel approach distinguished molecular clusters associated with individual GPI-anchored proteins, suggesting that it can identify co-clustering molecules in individual raft domains. PMID:24671047

  14. Defective lipid remodeling of GPI anchors in peroxisomal disorders, Zellweger syndrome, and rhizomelic chondrodysplasia punctata

    PubMed Central

    Kanzawa, Noriyuki; Shimozawa, Nobuyuki; Wanders, Ronald J. A.; Ikeda, Kazutaka; Murakami, Yoshiko; Waterham, Hans R.; Mukai, Satoru; Fujita, Morihisa; Maeda, Yusuke; Taguchi, Ryo; Fujiki, Yukio; Kinoshita, Taroh

    2012-01-01

    Many cell surface proteins in mammalian cells are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). The predominant form of mammalian GPI contains 1-alkyl-2-acyl phosphatidylinositol (PI), which is generated by lipid remodeling from diacyl PI. The conversion of diacyl PI to 1-alkyl-2-acyl PI occurs in the ER at the third intermediate in the GPI biosynthetic pathway. This lipid remodeling requires the alkyl-phospholipid biosynthetic pathway in peroxisome. Indeed, cells defective in dihydroxyacetone phosphate acyltransferase (DHAP-AT) or alkyl-DHAP synthase express only the diacyl form of GPI-anchored proteins. A defect in the alkyl-phospholipid biosynthetic pathway causes a peroxisomal disorder, rhizomelic chondrodysplasia punctata (RCDP), and defective biogenesis of peroxisomes causes Zellweger syndrome, both of which are lethal genetic diseases with multiple clinical phenotypes such as psychomotor defects, mental retardation, and skeletal abnormalities. Here, we report that GPI lipid remodeling is defective in cells from patients with Zellweger syndrome having mutations in the peroxisomal biogenesis factors PEX5, PEX16, and PEX19 and in cells from patients with RCDP types 1, 2, and 3 caused by mutations in PEX7, DHAP-AT, and alkyl-DHAP synthase, respectively. Absence of the 1-alkyl-2-acyl form of GPI-anchored proteins might account for some of the complex phenotypes of these two major peroxisomal disorders. PMID:22253471

  15. An androgenic gland membrane-anchored gene associated with the crustacean insulin-like androgenic gland hormone.

    PubMed

    Rosen, Ohad; Manor, Rivka; Weil, Simy; Aflalo, Eliahu D; Bakhrat, Anna; Abdu, Uri; Sagi, Amir

    2013-06-01

    Crustacean male sexual differentiation is governed by the androgenic gland (AG) and specifically by the secreted insulin-like AG hormone (IAG), thus far identified in several decapod species including the Australian red claw crayfish Cherax quadricarinatus (termed Cq-IAG). While a few insulin-like AG genes have been identified in crustaceans, other AG-specific genes have not been documented until now. In the present study, we describe the recent identification of a non-IAG AG-specific transcript obtained from the C. quadricarinatus AG cDNA library. This transcript, termed C. quadricarinatus membrane-anchored AG-specific factor (Cq-MAG), was fully sequenced and found to encode a putative product of 189 amino acids including a signal anchoring peptide. Expression of a recombinant GFP fusion protein lacking the signal anchor encoding sequence dramatically affected recombinant protein localization pattern. While the expression of the deleterious fusion protein was observed throughout most of the cell, the native GFP::Cq-MAG fusion protein was observed mainly surrounding the periphery of the nucleus, demonstrating an endoplasmic reticulum (ER)-like localization pattern. Moreover, co-expression of the wild-type Cq-MAG (fused to GFP) and the Cq-IAG hormone revealed that these peptides indeed co-localize. This study is the first to report a protein specifically associated with the insulin-like AG hormone in addition to the finding of another AG-specific transcript in crustaceans. Previous knowledge suggests that insulin/insulin-like factor secretion involves tissue-specific transcripts and membrane-anchored proteins. In this regard, Cq-MAG's tissue specificity, anchoring properties and intracellular co-localization with Cq-IAG suggest that it may play a role in the processing and secretion of this insulin-like AG hormone. PMID:23470660

  16. A localized multimeric anchor attaches the Caulobacter holdfast to the cell pole

    PubMed Central

    Hardy, Gail G.; Allen, Rebecca C.; Toh, Evelyn; Long, Maria; Brown, Pamela J. B.; Cole-Tobian, Jennifer L.; Brun, Yves V.

    2010-01-01

    Summary Caulobacter crescentus attachment is mediated by the holdfast, a complex of polysaccharide anchored to the cell by HfaA, HfaB and HfaD. We show that all three proteins are surface-exposed outer membrane (OM) proteins. HfaA is similar to fimbrial proteins and assembles into a high molecular weight (HMW) form requiring HfaD, but not holdfast polysaccharide. The HfaD HMW form is dependent on HfaA but not on holdfast polysaccharide. We show that HfaA and HfaD form homomultimers and that they require HfaB for stability and OM translocation. All three proteins localize to the late predivisional flagellar pole, remain at this pole in swarmer cells, and localize at the stalk tip after the stalk is synthesized at the same pole. Hfa protein localization requires the holdfast polysaccharide secretion proteins and the polar localization factor PodJ. A hfaB mutant is much more severely deficient in adherence and holdfast attachment than hfaA and hfaD mutants. A hfaA, hfaD double mutant phenocopies either single mutant, suggesting that HfaB is involved in holdfast attachment beyond secretion of HfaA and HfaD. We hypothesize HfaB secretes HfaA and HfaD across the outer membrane, and the three proteins form a complex anchoring the holdfast to the stalk. PMID:20233308

  17. Structure of a CGI-58 motif provides the molecular basis of lipid droplet anchoring.

    PubMed

    Boeszoermenyi, Andras; Nagy, Harald Manuel; Arthanari, Haribabu; Pillip, Christoph Jens; Lindermuth, Hanna; Luna, Rafael Eulogio; Wagner, Gerhard; Zechner, Rudolf; Zangger, Klaus; Oberer, Monika

    2015-10-30

    Triacylglycerols (TGs) stored in lipid droplets (LDs) are hydrolyzed in a highly regulated metabolic process called lipolysis to free fatty acids that serve as energy substrates for β-oxidation, precursors for membrane lipids and signaling molecules. Comparative gene identification-58 (CGI-58) stimulates the enzymatic activity of adipose triglyceride lipase (ATGL), which catalyzes the hydrolysis of TGs to diacylglycerols and free fatty acids. In adipose tissue, protein-protein interactions between CGI-58 and the LD coating protein perilipin 1 restrain the ability of CGI-58 to activate ATGL under basal conditions. Phosphorylation of perilipin 1 disrupts these interactions and mobilizes CGI-58 for the activation of ATGL. We have previously demonstrated that the removal of a peptide at the N terminus (residues 10-31) of CGI-58 abrogates CGI-58 localization to LDs and CGI-58-mediated activation of ATGL. Here, we show that this tryptophan-rich N-terminal peptide serves as an independent LD anchor, with its three tryptophans serving as focal points of the left (harboring Trp(21) and Trp(25)) and right (harboring Trp(29)) anchor arms. The solution state NMR structure of a peptide comprising the LD anchor bound to dodecylphosphocholine micelles as LD mimic reveals that the left arm forms a concise hydrophobic core comprising tryptophans Trp(21) and Trp(25) and two adjacent leucines. Trp(29) serves as the core of a functionally independent anchor arm. Consequently, simultaneous tryptophan alanine permutations in both arms abolish localization and activity of CGI-58 as opposed to tryptophan substitutions that occur in only one arm. PMID:26350461

  18. Structure of a CGI-58 Motif Provides the Molecular Basis of Lipid Droplet Anchoring*

    PubMed Central

    Boeszoermenyi, Andras; Nagy, Harald Manuel; Arthanari, Haribabu; Pillip, Christoph Jens; Lindermuth, Hanna; Luna, Rafael Eulogio; Wagner, Gerhard; Zechner, Rudolf; Zangger, Klaus; Oberer, Monika

    2015-01-01

    Triacylglycerols (TGs) stored in lipid droplets (LDs) are hydrolyzed in a highly regulated metabolic process called lipolysis to free fatty acids that serve as energy substrates for β-oxidation, precursors for membrane lipids and signaling molecules. Comparative gene identification-58 (CGI-58) stimulates the enzymatic activity of adipose triglyceride lipase (ATGL), which catalyzes the hydrolysis of TGs to diacylglycerols and free fatty acids. In adipose tissue, protein-protein interactions between CGI-58 and the LD coating protein perilipin 1 restrain the ability of CGI-58 to activate ATGL under basal conditions. Phosphorylation of perilipin 1 disrupts these interactions and mobilizes CGI-58 for the activation of ATGL. We have previously demonstrated that the removal of a peptide at the N terminus (residues 10–31) of CGI-58 abrogates CGI-58 localization to LDs and CGI-58-mediated activation of ATGL. Here, we show that this tryptophan-rich N-terminal peptide serves as an independent LD anchor, with its three tryptophans serving as focal points of the left (harboring Trp21 and Trp25) and right (harboring Trp29) anchor arms. The solution state NMR structure of a peptide comprising the LD anchor bound to dodecylphosphocholine micelles as LD mimic reveals that the left arm forms a concise hydrophobic core comprising tryptophans Trp21 and Trp25 and two adjacent leucines. Trp29 serves as the core of a functionally independent anchor arm. Consequently, simultaneous tryptophan alanine permutations in both arms abolish localization and activity of CGI-58 as opposed to tryptophan substitutions that occur in only one arm. PMID:26350461

  19. Expanding the clinical and molecular characteristics of PIGT-CDG, a disorder of glycosylphosphatidylinositol anchors.

    PubMed

    Lam, Christina; Golas, Gretchen A; Davids, Mariska; Huizing, Marjan; Kane, Megan S; Krasnewich, Donna M; Malicdan, May Christine V; Adams, David R; Markello, Thomas C; Zein, Wadih M; Gropman, Andrea L; Lodish, Maya B; Stratakis, Constantine A; Maric, Irina; Rosenzweig, Sergio D; Baker, Eva H; Ferreira, Carlos R; Danylchuk, Noelle R; Kahler, Stephen; Garnica, Adolfo D; Bradley Schaefer, G; Boerkoel, Cornelius F; Gahl, William A; Wolfe, Lynne A

    2015-01-01

    PIGT-CDG, an autosomal recessive syndromic intellectual disability disorder of glycosylphosphatidylinositol (GPI) anchors, was recently described in two independent kindreds [Multiple Congenital Anomalies-Hypotonia-Seizures Syndrome 3 (OMIM, #615398)]. PIGT encodes phosphatidylinositol-glycan biosynthesis class T, a subunit of the heteropentameric transamidase complex that facilitates the transfer of GPI to proteins. GPI facilitates attachment (anchoring) of proteins to cell membranes. We describe, at ages 7 and 6 years, two children of non-consanguineous parents; they had hypotonia, severe global developmental delay, and intractable seizures along with endocrine, ophthalmologic, skeletal, hearing, and cardiac anomalies. Exome sequencing revealed that both siblings had compound heterozygous variants in PIGT (NM_015937.5), i.e., c.918dupC, a novel duplication leading to a frameshift, and c.1342C > T encoding a previously described missense variant. Flow cytometry studies showed decreased surface expression of GPI-anchored proteins on granulocytes, consistent with findings in previous cases. These siblings further delineate the clinical spectrum of PIGT-CDG, reemphasize the neuro-ophthalmologic presentation, clarify the endocrine features, and add hypermobility, low CSF albumin quotient, and hearing loss to the phenotypic spectrum. Our results emphasize that GPI anchor-related congenital disorders of glycosylation (CDGs) should be considered in subjects with early onset severe seizure disorders and dysmorphic facial features, even in the presence of a normal carbohydrate-deficient transferrin pattern and N-glycan profiling. Currently available screening for CDGs will not reliably detect this family of disorders, and our case reaffirms that the use of flow cytometry and genetic testing is essential for diagnosis in this group of disorders. PMID:25943031

  20. VASP Governs Actin Dynamics by Modulating Filament Anchoring

    PubMed Central

    Trichet, Léa; Campàs, Otger; Sykes, Cécile; Plastino, Julie

    2007-01-01

    Actin filament dynamics at the cell membrane are important for cell-matrix and cell-cell adhesions and the protrusion of the leading edge. Since actin filaments must be connected to the cell membrane to exert forces but must also detach from the membrane to allow it to move and evolve, the balance between actin filament tethering and detachment at adhesion sites and the leading edge is key for cell shape changes and motility. How this fine tuning is performed in cells remains an open question, but possible candidates are the Drosophila enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins, which localize to dynamic actin structures in the cell. Here we study VASP-mediated actin-related proteins 2/3 (Arp2/3) complex-dependent actin dynamics using a substrate that mimics the fluid properties of the cell membrane: an oil-water interface. We show evidence that polymerization activators undergo diffusion and convection on the fluid surface, due to continual attachment and detachment to the actin network. These dynamics are enhanced in the presence of VASP, and we observe cycles of catastrophic detachment of the actin network from the surface, resulting in stop-and-go motion. These results point to a role for VASP in the modulation of filament anchoring, with implications for actin dynamics at cell adhesions and at the leading edge of the cell. PMID:17098798

  1. Targeting the membrane-anchored serine protease testisin with a novel engineered anthrax toxin prodrug to kill tumor cells and reduce tumor burden

    PubMed Central

    Martin, Erik W.; Buzza, Marguerite S.; Driesbaugh, Kathryn H.; Liu, Shihui; Fortenberry, Yolanda M.; Leppla, Stephen H.; Antalis, Toni M.

    2015-01-01

    The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. Here, we sought to engineer anthrax toxin protective antigen (PrAg), which is proteolytically activated on the cell surface by the proprotein convertase furin to instead be activated by tumor cell-expressed membrane-anchored serine proteases to function as a tumoricidal agent. PrAg's native activation sequence was mutated to a sequence derived from protein C inhibitor (PCI) that can be cleaved by membrane-anchored serine proteases, to generate the mutant protein PrAg-PCIS. PrAg-PCIS was resistant to furin cleavage in vitro, yet cytotoxic to multiple human tumor cell lines when combined with FP59, a chimeric anthrax toxin lethal factor-Pseudomonas exotoxin fusion protein. Molecular analyses showed that PrAg-PCIS can be cleaved in vitro by several serine proteases including the membrane-anchored serine protease testisin, and mediates increased killing of testisin-expressing tumor cells. Treatment with PrAg-PCIS also potently attenuated the growth of testisin-expressing xenograft tumors in mice. The data indicates PrAg can be engineered to target tumor cell-expressed membrane-anchored serine proteases to function as a potent tumoricidal agent. PMID:26392335

  2. 30 CFR 56.9311 - Anchoring stationary sizing devices.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Anchoring stationary sizing devices. 56.9311 Section 56.9311 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND... Dumping Sites § 56.9311 Anchoring stationary sizing devices. Grizzlies and other stationary sizing...

  3. Memory for Dialogue: Recalling an Anchor through Talk and Response.

    ERIC Educational Resources Information Center

    Beaver, Pam

    This paper reports on a project involving student recall of the dialogue in a movie and retention of the "anchor," which in this case refers to a videotape recording of "To Kill a Mockingbird." The project looked at how students retained knowledge over a few days and what kind of activities resulted from expertise with an anchor. The goal of…

  4. Anchored Instruction in Preservice Educational Technology Classes: A Research Project.

    ERIC Educational Resources Information Center

    Bauer, Jeffrey W.

    This study examined the effectiveness of the anchored instruction (i.e., using a theme or anchor around which various learning activities take place) approach in preparing preservice teachers to integrate technology. Participants were 48 students enrolled in the three sections of a preservice educational technology class during the summer of 1997.…

  5. 30 CFR 56.9311 - Anchoring stationary sizing devices.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Anchoring stationary sizing devices. 56.9311 Section 56.9311 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND... Dumping Sites § 56.9311 Anchoring stationary sizing devices. Grizzlies and other stationary sizing...

  6. Using Anchored Instruction to Evaluate Mathematical Growth and Understanding

    ERIC Educational Resources Information Center

    Kurz, Terri L.; Batarelo, Ivana

    2005-01-01

    Anchored instruction is designed to present problems in a meaningful context to allow for investigations into real life environments. The Jasper Project was created to allow students to investigate mathematical dilemmas using anchored instruction techniques. This study uses case study methods to examine the perceptions that preservice teachers…

  7. Bearing area: a new indication for suture anchor pullout strength?

    PubMed

    Yakacki, Christopher Michael; Griffis, Jack; Poukalova, Mariya; Gall, Ken

    2009-08-01

    Studies performed to quantify the pullout strength of suture anchors have not adequately defined the basic device parameters that control monotonic pullout. The bearing area of a suture anchor can be used to understand and predict anchor pullout strength in a soft-bone model. First, conical-shaped test samples were varied in size and shape and tested for pullout in 5, 8, and 10 pcf sawbone models. Next, bearing area and pullout strength relationships developed from the test samples were validated against nine commercially available suture anchors, including the Mitek QuickAnchor and SpiraLok, Opus Magnum(2), ArthroCare ParaSorb, and Arthrex BioCorkscrew. The samples showed a direct correlation between bearing area and pullout strength. Increased insertion depth was a secondary condition that also increased pullout strength. The pullout strength for the suture anchors followed the predicted trends of conical devices based on their individual bearing areas. For the 5 and 8 pcf models, only two and three devices, respectively, fell outside the predicted pullout strength range by more than a standard deviation. The use of a synthetic sawbone model was validated against the pullout strength of an Arthrex Corkscrew in five fresh-frozen cadaver humeral heads. The bearing area of a suture anchor can be used to predict the pullout strength independent of design in a soft-bone model. This work helps provide a foundation to understand the principles that affect the pullout strength of suture anchors. PMID:19226593

  8. Understanding Rasch Measurement: Partial Credit Model and Pivot Anchoring.

    ERIC Educational Resources Information Center

    Bode, Rita K.

    2001-01-01

    Describes the Rasch measurement partial credit model, what it is, how it differs from other Rasch models, and when and how to use it. Also describes the calibration of instruments with increasingly complex items. Explains pivot anchoring and illustrates its use and describes the effect of pivot anchoring on step calibrations, item hierarchy, and…

  9. 107. View showing open caisson Pier 4 with anchor bolts ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    107. View showing open caisson Pier 4 with anchor bolts placed ready for last pour of concrete. Also pile driver driving falsework piles for south anchor arm. Located at end of the old ferry landing slip at Crockett side of straits. - Carquinez Bridge, Spanning Carquinez Strait at Interstate 80, Vallejo, Solano County, CA

  10. Software Note: Using BILOG for Fixed-Anchor Item Calibration

    ERIC Educational Resources Information Center

    DeMars, Christine E.; Jurich, Daniel P.

    2012-01-01

    The nonequivalent groups anchor test (NEAT) design is often used to scale item parameters from two different test forms. A subset of items, called the anchor items or common items, are administered as part of both test forms. These items are used to adjust the item calibrations for any differences in the ability distributions of the groups taking…

  11. Electrically insulated MLI and thermal anchor

    SciTech Connect

    Kamiya, Koji; Furukawa, Masato; Murakami, Haruyuki; Kizu, Kaname; Tsuchiya, Katsuhiko; Koidea, Yoshihiko; Yoshida, Kiyoshi; Hatakenaka, Ryuta; Miyakita, Takeshi

    2014-01-29

    The thermal shield of JT-60SA is kept at 80 K and will use the multilayer insulation (MLI) to reduce radiation heat load to the superconducting coils at 4.4 K from the cryostat at 300 K. Due to plasma pulse operation, the MLI is affected by eddy current in toroidal direction. The MLI is designed to suppress the current by electrically insulating every 20 degree in the toroidal direction by covering the MLI with polyimide films. In this paper, two kinds of designs for the MLI system are proposed, focusing on a way to overlap the layers. A boil-off calorimeter method and temperature measurement has been performed to determine the thermal performance of the MLI system. The design of the electrical insulated thermal anchor between the toroidal field (TF) coil and the thermal shield is also explained.

  12. Anchoring submersible ultrasonic receivers in river channels with stable substrate

    USGS Publications Warehouse

    Bettoli, Phillip William; Scholten, G.D.; Hubbs, D.

    2010-01-01

    We developed an anchoring system for submersible ultrasonic receivers (SURs) that we placed on the bottom of the riverine reaches of three main-stem reservoirs in the upper Tennessee River. Each anchor consisted of a steel tube (8.9 x 35.6 cm) welded vertically to a round plate of steel (5.1 x 40.6 cm). All seven SURs and their 57-kg anchors were successfully deployed and retrieved three times over 547 d by a dive team employing surface air-breathing equipment and a davit-equipped boat. All of the anchors and their SURs remained stationary over two consecutive winters on the hard-bottom, thalweg sites where they were deployed. The SUR and its anchor at the most downriver site experienced flows that exceeded 2,100 m(3)/s and mean water column velocities of about 0.9 m/s.

  13. The Role of Plasmalemmal-Cortical Anchoring on the Stability of Transmembrane Electropores

    PubMed Central

    Kennedy, S. M.; Ji, Z.; Rockweiler, N. B.; Hahn, A. R.; Booske, J. H.; Hagness, S. C.

    2009-01-01

    The structure of eukaryotic cells is maintained by a network of filamentous actin anchored subjacently to the plasma membrane. This structure is referred to as the actin cortex. We present a locally constrained surface tension model for electroporation in order to address the influence of plasmalemmal-cortical anchoring on electropore dynamics. This model predicts that stable electropores are possible under certain conditions. The existence of stable electropores has been suggested in several experimental studies. The electropore radius at which stability is achieved is a function of the characteristic radii of locally constrained regions about the plasma membrane. This model opens the possibility of using actin-modifying compounds to physically manipulate cortical density, thereby manipulating electroporation dynamics. It also underscores the need to improve electroporation models further by incorporating the influence of trans-electropore ionic and aqueous flow, cortical flexibility, transmembrane protein mobility, and active cellular wound healing mechanisms. PMID:20490371

  14. Deletion of Mitochondrial Anchoring Protects Dysmyelinating Shiverer: Implications for Progressive MS

    PubMed Central

    Joshi, Dinesh C.; Zhang, Chuan-Li; Lin, Tien-Min; Gusain, Anchal; Harris, Melissa G.; Tree, Esther; Yin, Yewin; Wu, Connie; Sheng, Zu-Hang; Dempsey, Robert J; Fabry, Zsuzsanna

    2015-01-01

    The demyelinating disease multiple sclerosis (MS) has an early inflammatory phase followed by an incurable progressive phase with subdued inflammation and poorly understood neurodegenerative mechanism. In this study, we identified various parallelisms between progressive MS and the dysmyelinating mouse model Shiverer and then genetically deleted a major neuron-specific mitochondrial anchoring protein Syntaphilin (SNPH) from the mouse. Prevailing evidence suggests that deletion of SNPH is harmful in demyelination. Surprisingly, SNPH deletion produces striking benefits in the Shiverer by prolonging survival, reducing cerebellar damage, suppressing oxidative stress, and improving mitochondrial health. In contrast, SNPH deletion does not benefit clinical symptoms in experimental autoimmune encephalomyelitis (EAE), a model for early-phase MS. We propose that deleting mitochondrial anchoring is a novel, specific treatment for progressive MS. PMID:25834054

  15. Streptolysin-O induces release of glycosylphosphatidylinositol-anchored alkaline phosphatase from ROS cells by vesiculation independently of phospholipase action.

    PubMed Central

    Xie, M; Low, M G

    1995-01-01

    Streptolysin-O (SLO), a cholesterol-binding agent, was used for studies on the release of glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase (AP) from ROS cells. Treatment of cells with SLO resulted in a time- and concentration-dependent release of AP into the extracellular medium. This release was potentiated by Ca2+ and bovine serum, but not by GPI-specific phospholipase D (GPI-PLD) purified from bovine serum. The released AP distributed to the detergent phase after Triton X-114 phase separation. This result suggested that the released AP contained an intact GPI anchor, and thus both proteolysis and anchor degradation by anchor-specific hydrolases, including GPI-PLD, as the potential mechanisms for SLO-mediated AP release were ruled out. The released AP sedimented at 100,000 g. A substantial amount of lipids was detected in the 100,000 g pellet. Cholesterol and sphingomyelin were enriched in SLO-released material, compared with intact cells. These results were consistent with vesiculation as the mechanism for SLO induction of AP release. Two other cholesterol-binding agents, saponin and digitonin, were also able to release AP, possibly by a similar vesiculation mechanism, whereas others, including nystatin, filipin and beta-escin, failed to elicit any AP release. Eight GPI-anchored proteins were identified in ROS cells, and all were substantially enriched in the vesicles released by SLO. Taken together, these results do not provide any support for the hypothesis that the clustering of GPI-anchored proteins in the plasma membrane is responsible for their resistance to GPI-PLD cleavage. Images Figure 5 Figure 6 PMID:7832771

  16. Expression of a kinase-dead form of CPK33 involved in florigen complex formation causes delayed flowering

    PubMed Central

    Kawamoto, Nozomi; Endo, Motomu; Araki, Takashi

    2015-01-01

    Regulation of flowering time is crucial for reproductive success of plants. FLOWERING LOCUS T (FT) protein is a central component of florigen and forms a ternary complex with 14-3-3 and FD, a basic leucine zipper transcription factor, in the shoot apex and promotes flowering. This complex formation requires phosphorylation of threonine residue at position 282 of FD. A calcium-dependent protein kinase CPK33 is responsible for the phosphorylation. However, possibly due to functional redundancy among calcium-dependent protein kinases, impact of the loss of CPK33 reported in the previous study was rather limited. Here, we report that expression of a kinase-dead form of CPK33 caused a clear delayed-flowering phenotype, supporting for an important role of CPK33 in florigen function through FD phosphorylation. PMID:26440648

  17. Peptide Anchor for Folate-Targeted Liposomal Delivery.

    PubMed

    Nogueira, Eugénia; Mangialavori, Irene C; Loureiro, Ana; Azoia, Nuno G; Sárria, Marisa P; Nogueira, Patrícia; Freitas, Jaime; Härmark, Johan; Shimanovich, Ulyana; Rollett, Alexandra; Lacroix, Ghislaine; Bernardes, Gonçalo J L; Guebitz, Georg; Hebert, Hans; Moreira, Alexandra; Carmo, Alexandre M; Rossi, Juan Pablo F C; Gomes, Andreia C; Preto, Ana; Cavaco-Paulo, Artur

    2015-09-14

    Specific folate receptors are abundantly overexpressed in chronically activated macrophages and in most cancer cells. Directed folate receptor targeting using liposomes is usually achieved using folate linked to a phospholipid or cholesterol anchor. This link is formed using a large spacer like polyethylene glycol. Here, we report an innovative strategy for targeted liposome delivery that uses a hydrophobic fragment of surfactant protein D linked to folate. Our proposed spacer is a small 4 amino acid residue linker. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity, with high stability and specificity. To compare the drug delivery potential of both liposomal targeting systems, we encapsulated the nuclear dye Hoechst 34580. The eventual increase in blue fluorescence would only be detectable upon liposome disruption, leading to specific binding of this dye to DNA. Our delivery system was proven to be more efficient (2-fold) in Caco-2 cells than classic systems where the folate moiety is linked to liposomes by polyethylene glycol. PMID:26241560

  18. Aurora-A kinase (AURKA) in normal and pathological cell growth

    PubMed Central

    Nikonova, Anna S.; Astsaturov, Igor; Serebriiskii, Ilya G.; Dunbrack, Roland L.; Golemis, Erica A.

    2013-01-01

    Temporally and spatially controlled activation of the Aurora-A kinase (AURKA) is regulates centrosome maturation, entry into mitosis, formation and function of the bipolar spindle, and cytokinesis. Genetic amplification, and mRNA and protein overexpression of Aurora-A are common in many types of solid tumor, and associated with aneuploidy, supernumerary centrosomes, defective mitotic spindles, and resistance to apoptosis. These properties have led Aurora-A to be considered a high value target for development of cancer therapeutics, with multiple agents currently in early phase clinical trials. More recently, identification of additional, non-mitotic functions and means of activation of Aurora-A during interphase neurite elongation and ciliary resorption have significantly expanded understanding of its function, and may offer insights into clinical performance of Aurora-A inhibitors. We here review mitotic and non-mitotic functions of Aurora-A, discuss Aurora-A regulation in the context of protein structural information, and evaluate progress in understanding and inhibiting Aurora-A in cancer. PMID:22864622

  19. Wave propagation in damage assessment of ground anchors

    NASA Astrophysics Data System (ADS)

    Zima, B.; Rucka, M.

    2015-07-01

    The inspection possibilities of ground anchors are limited to destructive test such as pull-out test. Guided wave propagation gives an opportunity to develop an inspection system dedicated to determine the condition of inspected element without violation of their integrity. In this paper the experimental study on wave propagation in laboratory models of ground anchors are presented. Experiments were conducted for different bonding lengths and different frequencies of excitation. Waves were generated by a piezoelectric actuator and the laser vibrometry technique was used to register velocity signals. For all tested anchors it was possible to identify the boundary between steel and concrete based on the registered reflections in wave propagation signals.

  20. Ideals as Anchors for Relationship Experiences

    PubMed Central

    Frye, Margaret; Trinitapoli, Jenny

    2016-01-01

    Research on young-adult sexuality in sub-Saharan Africa typically conceptualizes sex as an individual-level risk behavior. We introduce a new approach that connects the conditions surrounding the initiation of sex with subsequent relationship well-being, examines relationships as sequences of interdependent events, and indexes relationship experiences to individually held ideals. New card-sort data from southern Malawi capture young women’s relationship experiences and their ideals in a sequential framework. Using optimal matching, we measure the distance between ideal and experienced relationship sequences to (1) assess the associations between ideological congruence and perceived relationship well-being, (2) compare this ideal-based approach to other experience-based alternatives, and (3) identify individual- and couple-level correlates of congruence between ideals and experiences in the romantic realm. We show that congruence between ideals and experiences conveys relationship well-being along four dimensions: expressions of love and support, robust communication habits, perceived biological safety, and perceived relationship stability. We further show that congruence is patterned by socioeconomic status and supported by shared ideals within romantic dyads. We argue that conceiving of ideals as anchors for how sexual experiences are manifest advances current understandings of romantic relationships, and we suggest that this approach has applications for other domains of life. PMID:27110031

  1. Photoinduced anchoring on a chalcogenide surface

    NASA Astrophysics Data System (ADS)

    Sheremet, N.; Kurioz, Yu.; Klebanov, M.; Lyubin, V.; Slyusarenko, K.; Reznikov, Yu.

    2012-05-01

    We present basic characteristics and a model of photoinduced anchoring of liquid crystals (LCs) on a chalcogenide surface. It was found that characteristics of the alignment strongly depend on the LC material for the same chalcogenide glass. The photoalignment is partially reversible and can be controlled by changing the light polarization direction. We propose a model in which the existence of dichroic units on the chalcogenide surface and competition between two mechanisms of the photoalignment is suggested. The first mechanism is related to the light-induced anisotropy on the chalcogenide surface and results in an easy orientation axes of a LC parallel to the polarization of the incident light. The second mechanism is attributed to an energy transfer from the dichroic units after the light absorption to the LC molecules adsorbed on the chalcogenide surface. The transferred energy causes polarization-sensitive desorption of the LC molecules from the chalcogenide surface and the light-induced easy orientation axis of a LC perpendicular to the incident light polarization. The competition between these mechanisms leads to the observed change of the direction of the easy axis with the exposure.

  2. Anchored boundary conditions for locally isostatic networks

    NASA Astrophysics Data System (ADS)

    Theran, Louis; Nixon, Anthony; Ross, Elissa; Sadjadi, Mahdi; Servatius, Brigitte; Thorpe, M. F.

    2015-11-01

    Finite pieces of locally isostatic networks have a large number of floppy modes because of missing constraints at the surface. Here we show that by imposing suitable boundary conditions at the surface the network can be rendered effectively isostatic. We refer to these as anchored boundary conditions. An important example is formed by a two-dimensional network of corner sharing triangles, which is the focus of this paper. Another way of rendering such networks isostatic is by adding an external wire along which all unpinned vertices can slide (sliding boundary conditions). This approach also allows for the incorporation of boundaries associated with internal holes and complex sample geometries, which are illustrated with examples. The recent synthesis of bilayers of vitreous silica has provided impetus for this work. Experimental results from the imaging of finite pieces at the atomic level need such boundary conditions, if the observed structure is to be computer refined so that the interior atoms have the perception of being in an infinite isostatic environment.

  3. Material Testing for Robotic Omnidirectional Anchor

    NASA Technical Reports Server (NTRS)

    Witkoe, Kevin S.

    2012-01-01

    To successfully explore near-Earth Asteroids the question of mobility emerges as the key issue for any robotic mission. When small bodies have extremely low escape velocities, traditional methods, such as wheels, would send the robot hurtling off of the asteroid's surface. To solve this problem, JPL has developed an omni-directional anchoring mechanism for use in microgravity that utilizes microspine technology. These microspines are placed in circular arrays with 16 independent carriages biasing the surface of the rock. The asperities in the surface allow the gripper to hold nearly 150N in all directions. While the gripper has been proven successful on consolidated rocks, it had yet to be tested on a variety of other surfaces that are suspected to separate the large boulders on an asteroid. Since asteroid surfaces vary widely, from friable rocks to lose ponds of regolith, the gripper was tested in a large variety of materials such as, bonded pumice, sand, gravel, and loose rocks. The forces are applied tangent, at 45 degrees, and normal to the surface of the material. The immediate results from this experiment will give insight into the gripper's effectiveness across the wide spectrum of materials found on asteroids.

  4. Knotless anchors with sutures external to the anchor body may be at risk for suture cutting through osteopenic bone

    PubMed Central

    Ono, Y.; Woodmass, J. M.; Nelson, A. A.; Boorman, R. S.; Thornton, G. M.

    2016-01-01

    Objectives This study evaluated the mechanical performance, under low-load cyclic loading, of two different knotless suture anchor designs: sutures completely internal to the anchor body (SpeedScrew) and sutures external to the anchor body and adjacent to bone (MultiFIX P). Methods Using standard suture loops pulled in-line with the rotator cuff (approximately 60°), anchors were tested in cadaveric bone and foam blocks representing normal to osteopenic bone. Mechanical testing included preloading to 10 N and cyclic loading for 500 cycles from 10 N to 60 N at 60 mm/min. The parameters evaluated were initial displacement, cyclic displacement and number of cycles and load at 3 mm displacement relative to preload. Video recording throughout testing documented the predominant source of suture displacement and the distance of ‘suture cutting through bone’. Results In cadaveric bone and foam blocks, MultiFIX P anchors had significantly greater initial displacement, and lower number of cycles and lower load at 3 mm displacement than SpeedScrew anchors. Video analysis revealed ‘suture cutting through bone’ as the predominant source of suture displacement in cadaveric bone (qualitative) and greater ‘suture cutting through bone’ comparing MultiFIX P with SpeedScrew anchors in foam blocks (quantitative). The greater suture displacement in MultiFIX P anchors was predominantly from suture cutting through bone, which was enhanced in an osteopenic bone model. Conclusions Anchors with sutures external to the anchor body are at risk for suture cutting through bone since the suture eyelet is at the distal tip of the implant and the suture directly abrades against the bone edge during cyclic loading. Suture cutting through bone may be a significant source of fixation failure, particularly in osteopenic bone. Cite this article: Y. Ono, J. M. Woodmass, A. A. Nelson, R. S. Boorman, G. M. Thornton, I. K. Y. Lo. Knotless anchors with sutures external to the anchor body may be

  5. High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation

    PubMed Central

    Anandapadamanaban, Madhanagopal; Andresen, Cecilia; Helander, Sara; Ohyama, Yoshifumi; Siponen, Marina I.; Lundström, Patrik; Kokubo, Tetsuro; Ikura, Mitsuhiko; Moche, Martin; Sunnerhagen, Maria

    2016-01-01

    The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 Å) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 when bound to yeast TBP, together with mutational data. The yTAF1-TAND1, which in itself acts as a transcriptional activator, binds into the DNA-binding TBP concave surface by presenting similar anchor residues to TBP as E. coli Mot1 but from a distinct structural scaffold. Furthermore, we show how yTAF1-TAND2 employs an aromatic and acidic anchoring pattern to bind a conserved yTBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides compelling insight into the competitive multiprotein TBP interplay critical to transcriptional regulation. PMID:23851461

  6. Detail on west elevation of anchor above door; camera facing ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Detail on west elevation of anchor above door; camera facing east. - Mare Island Naval Shipyard, Smithery, California Avenue, west side at California Avenue & Eighth Street, Vallejo, Solano County, CA

  7. 19. WEST ANCHOR SPAN OF THROUGH TRUSS AND PIERS NO. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    19. WEST ANCHOR SPAN OF THROUGH TRUSS AND PIERS NO. 2 AND 3, FROM WEST RIVERBANK. VIEW TO NORTH. - MacArthur Bridge, Spanning Mississippi River on Highway 34 between IA & IL, Burlington, Des Moines County, IA

  8. 20. DETAIL OF WEST ANCHOR SPAN, CANTILEVER ARMS AND WEST ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    20. DETAIL OF WEST ANCHOR SPAN, CANTILEVER ARMS AND WEST HALF OF SUSPENDED SPAN OF THROUGH TRUSS. VIEW TO NORTHEAST. - MacArthur Bridge, Spanning Mississippi River on Highway 34 between IA & IL, Burlington, Des Moines County, IA

  9. Visual implant elastomer and anchor tag retention in largemouth bass

    USGS Publications Warehouse

    Hartman, K.J.; Janney, E.C.

    2006-01-01

    We double-marked largemouth bass Micropterus salmoides with Floy FD-68B anchor tags and visible implant elastomer (VIE) marks before stocking to compare retention of the two marks for age-0 (178 mm total length [TL]) and age-1 (273 mm TL) largemouth bass. In a short-term (31-d) evaluation, retention rate of anchor tags was over 94% for each age-class and retention of VIE marks was 98% in both age-classes. In a longer-term comparison of fish stocked into the Ohio River, retention was substantially higher for VIE marks (92.9%) than for anchor tags (42.9%) after 403 d (ages combined). Although anchor tags had high retention in two sizes of largemouth bass during the short-term experiment, they should not be used in situations where accurate identification of marked fish is required for periods longer than 123 d. ?? Copyright by the American Fisheries Society 2006.

  10. 21. DETAIL OF WEST (AMERICAN) CANTILEVER AND ANCHOR ARMS OF ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    21. DETAIL OF WEST (AMERICAN) CANTILEVER AND ANCHOR ARMS OF MAIN SPAN, SHOWING PIER M. VIEW TO NORTH. - Blue Water Bridge, Spanning St. Clair River at I-69, I-94, & Canadian Route 402, Port Huron, St. Clair County, MI

  11. 22. DETAIL OF EAST (CANADIAN) CANTILEVER AND ANCHOR ARMS OF ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    22. DETAIL OF EAST (CANADIAN) CANTILEVER AND ANCHOR ARMS OF MAIN SPAN, SHOWING PIER C. VIEW TO NORTH. - Blue Water Bridge, Spanning St. Clair River at I-69, I-94, & Canadian Route 402, Port Huron, St. Clair County, MI

  12. 34. View of pier 3, showing supporting main anchor arm ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    34. View of pier 3, showing supporting main anchor arm and cantilever arm spans, as seen from shore near pier 4, looking north - Williamstown-Marietta Bridge, Spanning Ohio River between Williamstown & Marietta, Williamstown, Wood County, WV

  13. 22. View showing main anchor arm, as viewed from main ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    22. View showing main anchor arm, as viewed from main cantilever arm looking south. Note upper chord eyebar arrangement. - Williamstown-Marietta Bridge, Spanning Ohio River between Williamstown & Marietta, Williamstown, Wood County, WV

  14. 43. DETAIL OF PINNED UPPER CHORD CONNECTION BETWEEN ANCHOR ARM ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    43. DETAIL OF PINNED UPPER CHORD CONNECTION BETWEEN ANCHOR ARM AND SUSPENDED (PANEL 67). VIEW TO NORTH. - Blue Water Bridge, Spanning St. Clair River at I-69, I-94, & Canadian Route 402, Port Huron, St. Clair County, MI

  15. ANCHOR SETTING PLAN FOR TYPICAL APPROACH SPANS AND ELEVATION OF ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    ANCHOR SETTING PLAN FOR TYPICAL APPROACH SPANS AND ELEVATION OF BENT CAPS FOR APALACHICOLA RIVER BRIDGE, SHEET 5509-M1 - Apalachicola River Bridge, State Route 20 spanning the Apalachicola River, Blountstown, Calhoun County, FL

  16. ANCHOR SETTING PLAN FOR PIERS 1, 2, 3, 4, 5 ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    ANCHOR SETTING PLAN FOR PIERS 1, 2, 3, 4, 5 AND 6, APALACHICOLA RIVER BRIDGE, SHEET 5505 TO 8-M1 - Apalachicola River Bridge, State Route 20 spanning the Apalachicola River, Blountstown, Calhoun County, FL

  17. ANCHOR SETTING PLAN, WITH DETAILS, FOR STEEL BENTS AT APPROACH ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    ANCHOR SETTING PLAN, WITH DETAILS, FOR STEEL BENTS AT APPROACH SPANS FOR APALACHICOLA RIVER BRIDGE, SHEET 5509-M2 - Apalachicola River Bridge, State Route 20 spanning the Apalachicola River, Blountstown, Calhoun County, FL

  18. Theory of anchoring on a two-dimensionally grooved surface

    NASA Astrophysics Data System (ADS)

    Fukuda, Jun-Ichi; Gwag, Jin Seog; Yoneya, Makoto; Yokoyama, Hiroshi

    2008-01-01

    We investigate analytically the anchoring of a nematic liquid crystal on a two-dimensionally grooved surface of arbitrary shape, induced by the elastic distortions of a liquid crystal adjacent to the surface. Our theoretical framework applied to a surface with square grooves reveals that such a surface can exhibit bistable anchoring, while a direct extension of a well-known theory of Berreman [Phys. Rev. Lett. 28, 1683 (1972)] results in no azimuthal anchoring in the so-called one-constant case ( K1=K2=K3 , with K1 , K2 , and K3 being the splay, twist, and bend elastic constants, respectively). We show under the assumption of K1=K2=K that the direction of the bistable easy axes and the anchoring strength crucially depend on the ratios K3/K and K24/K , where K24 is the saddle-splay surface elastic constant. To demonstrate the applicability of our theory to general cases and to elucidate the effect of surface shape and the elastic constants on the properties of surface anchoring, we also consider several specific cases of interest; one-dimensional grooves of arbitrary shape, rhombic grooves, and surfaces possessing 2N -fold symmetry, including hexagonal grooves, and show the following: (i) The rescaled anchoring energy f(ϕ)/f(π/2) of one-dimensional grooves, with ϕ being the angle between the director n and the groove direction, is independent of the groove shape. (ii) Whether two diagonal axes of rhombic grooves can become easy axes depends sensitively on K3/K , K24/K and the angle α between the grooves. The angle α yielding the maximum anchoring strength for given groove pitch and amplitude depends again on K3/K and K24/K ; in some cases α=0 (one-dimensional grooves), and in other cases α≠0 , gives the maximum anchoring strength. Square grooves (α=π/2) do not necessarily exhibit the largest anchoring strength. (iii) A surface possessing 2N -fold symmetry can yield N -stable azimuthal anchoring. However, when K1=K2=K3 and N≥3 , azimuthal anchoring is

  19. Tripolin A, a Novel Small-Molecule Inhibitor of Aurora A Kinase, Reveals New Regulation of HURP's Distribution on Microtubules

    PubMed Central

    Kesisova, Iliana A.; Nakos, Konstantinos C.; Tsolou, Avgi; Angelis, Dimitrios; Lewis, Joe; Chatzaki, Aikaterini; Agianian, Bogos; Giannis, Athanassios; Koffa, Maria D.

    2013-01-01

    Mitotic regulators exhibiting gain of function in tumor cells are considered useful cancer therapeutic targets for the development of small-molecule inhibitors. The human Aurora kinases are a family of such targets. In this study, from a panel of 105 potential small-molecule inhibitors, two compounds Tripolin A and Tripolin B, inhibited Aurora A kinase activity in vitro. In human cells however, only Tripolin A acted as an Aurora A inhibitor. We combined in vitro, in vivo single cell and in silico studies to demonstrate the biological action of Tripolin A, a non-ATP competitive inhibitor. Tripolin A reduced the localization of pAurora A on spindle microtubules (MTs), affected centrosome integrity, spindle formation and length, as well as MT dynamics in interphase, consistent with Aurora A inhibition by RNAi or other specific inhibitors, such as MLN8054 or MLN8237. Interestingly, Tripolin A affected the gradient distribution towards the chromosomes, but not the MT binding of HURP (Hepatoma Up-Regulated Protein), a MT-associated protein (MAP) and substrate of the Aurora A kinase. Therefore Tripolin A reveals a new way of regulating mitotic MT stabilizers through Aurora A phosphorylation. Tripolin A is predicted to bind Aurora A similarly but not identical to MLN8054, therefore it could be used to dissect pathways orchestrated by Aurora kinases as well as a scaffold for further inhibitor development. PMID:23516487

  20. Lentiviral Engineered Fibroblasts Expressing Codon-Optimized COL7A1 Restore Anchoring Fibrils in RDEB

    PubMed Central

    Georgiadis, Christos; Syed, Farhatullah; Petrova, Anastasia; Abdul-Wahab, Alya; Lwin, Su M.; Farzaneh, Farzin; Chan, Lucas; Ghani, Sumera; Fleck, Roland A.; Glover, Leanne; McMillan, James R.; Chen, Mei; Thrasher, Adrian J.; McGrath, John A.; Di, Wei-Li; Qasim, Waseem

    2016-01-01

    Cells therapies, engineered to secrete replacement proteins, are being developed to ameliorate otherwise debilitating diseases. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by defects of type VII collagen, a protein essential for anchoring fibril formation at the dermal-epidermal junction. Whereas allogeneic fibroblasts injected directly into the dermis can mediate transient disease modulation, autologous gene-modified fibroblasts should evade immunological rejection and support sustained delivery of type VII collagen at the dermal-epidermal junction. We demonstrate the feasibility of such an approach using a therapeutic grade, self-inactivating-lentiviral vector, encoding codon-optimized COL7A1, to transduce RDEB fibroblasts under conditions suitable for clinical application. Expression and secretion of type VII collagen was confirmed with transduced cells exhibiting supranormal levels of protein expression, and ex vivo migration of fibroblasts was restored in functional assays. Gene-modified RDEB fibroblasts also deposited type VII collagen at the dermal-epidermal junction of human RDEB skin xenografts placed on NOD-scid IL2Rgammanull recipients, with reconstruction of human epidermal structure and regeneration of anchoring fibrils at the dermal-epidermal junction. Fibroblast-mediated restoration of protein and structural defects in this RDEB model strongly supports proposed therapeutic applications in man. PMID:26763448

  1. Lentiviral Engineered Fibroblasts Expressing Codon-Optimized COL7A1 Restore Anchoring Fibrils in RDEB.

    PubMed

    Georgiadis, Christos; Syed, Farhatullah; Petrova, Anastasia; Abdul-Wahab, Alya; Lwin, Su M; Farzaneh, Farzin; Chan, Lucas; Ghani, Sumera; Fleck, Roland A; Glover, Leanne; McMillan, James R; Chen, Mei; Thrasher, Adrian J; McGrath, John A; Di, Wei-Li; Qasim, Waseem

    2016-01-01

    Cells therapies, engineered to secrete replacement proteins, are being developed to ameliorate otherwise debilitating diseases. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by defects of type VII collagen, a protein essential for anchoring fibril formation at the dermal-epidermal junction. Whereas allogeneic fibroblasts injected directly into the dermis can mediate transient disease modulation, autologous gene-modified fibroblasts should evade immunological rejection and support sustained delivery of type VII collagen at the dermal-epidermal junction. We demonstrate the feasibility of such an approach using a therapeutic grade, self-inactivating-lentiviral vector, encoding codon-optimized COL7A1, to transduce RDEB fibroblasts under conditions suitable for clinical application. Expression and secretion of type VII collagen was confirmed with transduced cells exhibiting supranormal levels of protein expression, and ex vivo migration of fibroblasts was restored in functional assays. Gene-modified RDEB fibroblasts also deposited type VII collagen at the dermal-epidermal junction of human RDEB skin xenografts placed on NOD-scid IL2Rgamma(null) recipients, with reconstruction of human epidermal structure and regeneration of anchoring fibrils at the dermal-epidermal junction. Fibroblast-mediated restoration of protein and structural defects in this RDEB model strongly supports proposed therapeutic applications in man. PMID:26763448

  2. Genome mapping by random anchoring: A discrete theoretical analysis

    NASA Astrophysics Data System (ADS)

    Zhang, M. Q.; Marr, T. G.

    1993-11-01

    As a part of the international human genome project, large-scale genomic maps of human and other model organisms are being generated. More recently, mapping using various anchoring (as opposed to the traditional "fingerprinting") strategies have been proposed based largely on mathematical models. In all of the theoretical work dealing with anchoring, an anchor has been idealized as a point on a continuous, infinite-length genome. In general, it is not desirable to make these assumptions, since in practice they may be violated under a variety of actual biological situations. Here we analyze a discrete model that can be used to predict the expected progress made when mapping by random anchoring. By virtue of keeping all three length scales (genome length, clone length, and probe length) finite, our results for the random anchoring strategy are derived in full generality, which contain previous results as special cases and hence can have broad application for planning mapping experiments or assessing the accuracy of the continuum models. Finally, we pose a challenging nonrandom anchoring model corresponding to a more efficient mapping scheme.

  3. Filamentous structures in skeletal muscle: anchors for the subsarcolemmal space.

    PubMed

    Khairani, Astrid Feinisa; Tajika, Yuki; Takahashi, Maiko; Ueno, Hitoshi; Murakami, Tohru; Soenggono, Arifin; Yorifuji, Hiroshi

    2015-03-01

    In skeletal muscle fibers, intermediate filaments and actin filaments provide structural support to the myofibrils and the sarcolemma. For many years, it was poorly understood from ultrastructural observations that how these filamentous structures were kept anchored. The present study was conducted to determine the architecture of filamentous anchoring structures in the subsarcolemmal space and the intermyofibrils. The diaphragms (Dp) of adult wild type and mdx mice (mdx is a model for Duchenne muscular dystrophy) were subjected to tension applied perpendicular to the long axis of the muscle fibers, with or without treatment with 1% Triton X-100 or 0.03% saponin. These experiments were conducted to confirm the presence and integrity of the filamentous anchoring structures. Transmission electron microscopy revealed that these structures provide firm transverse connections between the sarcolemma and peripheral myofibrils. Most of the filamentous structures appeared to be inserted into subsarcolemmal densities, forming anchoring connections between the sarcolemma and peripheral myofibrils. In some cases, actin filaments were found to run longitudinally in the subsarcolemmal space to connect to the sarcolemma or in some cases to connect to the intermyofibrils as elongated thin filaments. These filamentous anchoring structures were less common in the mdx Dp. Our data suggest that the transverse and longitudinal filamentous structures form an anchoring system in the subsarcolemmal space and the intermyofibrils. PMID:24519712

  4. Epoxy-coated rock anchors for upper Occoquan Dam

    SciTech Connect

    Bruen, M.P.; Pansic, N.; Schwartz, M.I.

    1995-12-31

    High-capacity, epoxy-coated anchors were installed at Upper Occoquan Dam to increase the stability of the 70-foot-high concrete gravity dam and powerhouse under revised Probable Maximum Flood (PMF) conditions. The post-tensioned anchorage system consisted of 56 multi-strand rock anchors with design loads of 700 to 1855 kips, averaging 1500 kips per tendon. A double corrosion protection system was specified to provide protection throughout the entire anchor length. During anchor stressing and testing, significant creep movement under constant loads equivalent to 133% of the design load was experienced and exceeded the requisite Post-Tensioning Institute (PTI) criteria. In addition to the creep phenomena, seating losses during transfer of the load to the end anchorage are at least 2 to 3 times greater than that which has been experienced with bare-wire strand tendons. On the basis of anchor test results, modifications were made to the anchor testing protocol, acceptance criteria, and the approach used for assessment of the long-term performance of the anchorage system.

  5. Free versus anchored numerical estimation: A unified approach.

    PubMed

    Opfer, John E; Thompson, Clarissa A; Kim, Dan

    2016-04-01

    Children's number-line estimation has produced a lively debate about representational change, supported by apparently incompatible data regarding descriptive adequacy of logarithmic (Opfer, Siegler, & Young, 2011) and cyclic power models (Slusser, Santiago, & Barth, 2013). To test whether methodological differences might explain discrepant findings, we created a fully crossed 2×2 design and assigned 96 children to one of four cells. In the design, we crossed anchoring (free, anchored) and sampling (over-, even-), which were candidate factors to explain discrepant findings. In three conditions (free/over-sampling, free/even-sampling, and anchored/over-sampling), the majority of children provided estimates better fit by the logarithmic than cyclic power function. In the last condition (anchored/even-sampling), the reverse was found. Results suggest that logarithmically-compressed numerical estimates do not depend on sampling, that the fit of cyclic power functions to children's estimates is likely an effect of anchors, and that a mixed log/linear model provides a useful model for both free and anchored numerical estimation. PMID:26774104

  6. Evaluation of mitral valve replacement anchoring in a phantom

    NASA Astrophysics Data System (ADS)

    McLeod, A. Jonathan; Moore, John; Lang, Pencilla; Bainbridge, Dan; Campbell, Gordon; Jones, Doug L.; Guiraudon, Gerard M.; Peters, Terry M.

    2012-02-01

    Conventional mitral valve replacement requires a median sternotomy and cardio-pulmonary bypass with aortic crossclamping and is associated with significant mortality and morbidity which could be reduced by performing the procedure off-pump. Replacing the mitral valve in the closed, off-pump, beating heart requires extensive development and validation of surgical and imaging techniques. Image guidance systems and surgical access for off-pump mitral valve replacement have been previously developed, allowing the prosthetic valve to be safely introduced into the left atrium and inserted into the mitral annulus. The major remaining challenge is to design a method of securely anchoring the prosthetic valve inside the beating heart. The development of anchoring techniques has been hampered by the expense and difficulty in conducting large animal studies. In this paper, we demonstrate how prosthetic valve anchoring may be evaluated in a dynamic phantom. The phantom provides a consistent testing environment where pressure measurements and Doppler ultrasound can be used to monitor and assess the valve anchoring procedures, detecting pararvalvular leak when valve anchoring is inadequate. Minimally invasive anchoring techniques may be directly compared to the current gold standard of valves sutured under direct vision, providing a useful tool for the validation of new surgical instruments.

  7. The Dynamics of Scaling: A Memory-Based Anchor Model of Category Rating and Absolute Identification

    ERIC Educational Resources Information Center

    Petrov, Alexander A.; Anderson, John R.

    2005-01-01

    A memory-based scaling model--ANCHOR--is proposed and tested. The perceived magnitude of the target stimulus is compared with a set of anchors in memory. Anchor selection is probabilistic and sensitive to similarity, base-level strength, and recency. The winning anchor provides a reference point near the target and thereby converts the global…

  8. 46 CFR 130.240 - Anchors and chains for OSVs of 100 or more gross tons.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Miscellaneous Equipment and...— (1) One anchor of the tabular weight and one-half the tabulated length of anchor chain listed in the applicable standard; or (2) Two anchors of one-half the tabular weight with the total length of anchor...

  9. 46 CFR 130.240 - Anchors and chains for OSVs of 100 or more gross tons.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Miscellaneous Equipment and...— (1) One anchor of the tabular weight and one-half the tabulated length of anchor chain listed in the applicable standard; or (2) Two anchors of one-half the tabular weight with the total length of anchor...

  10. 46 CFR 130.240 - Anchors and chains for OSVs of 100 or more gross tons.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Miscellaneous Equipment and...— (1) One anchor of the tabular weight and one-half the tabulated length of anchor chain listed in the applicable standard; or (2) Two anchors of one-half the tabular weight with the total length of anchor...

  11. 46 CFR 130.240 - Anchors and chains for OSVs of 100 or more gross tons.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Miscellaneous Equipment and...— (1) One anchor of the tabular weight and one-half the tabulated length of anchor chain listed in the applicable standard; or (2) Two anchors of one-half the tabular weight with the total length of anchor...

  12. Adsorption-induced anchoring transitions at nematic-liquid-crystal-crystal interfaces

    NASA Astrophysics Data System (ADS)

    Pieranski, P.; Jérôme, B.

    1989-07-01

    Discontinuous (first-order) anchoring transitions are shown to occur as a function of water adsorption at the nematic-liquid-crystal(E9)-gypsum and E9-mica interfaces. The transitions involve growth of domains with an anchoring a' making a finite angle with the direction a of the parent anchoring. The growth proceeds by motion of walls separating the domains with the new anchoring a' from the matrix with the parental anchoring a. A Landau-type expression for the interfacial energy is proposed and used to classify the anchoring transitions and to explain the behavior of the domains during the first-order anchoring transitions.

  13. Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting

    PubMed Central

    Kooijmans, Sander A. A.; Aleza, Clara Gómez; Roffler, Steve R.; van Solinge, Wouter W.; Vader, Pieter; Schiffelers, Raymond M.

    2016-01-01

    Background Extracellular vesicles (EVs) are attractive candidate drug delivery systems due to their ability to functionally transport biological cargo to recipient cells. However, the apparent lack of target cell specificity of exogenously administered EVs limits their therapeutic applicability. In this study, we propose a novel method to equip EVs with targeting properties, in order to improve their interaction with tumour cells. Methods EV producing cells were transfected with vectors encoding for anti-epidermal growth factor receptor (EGFR) nanobodies, which served as targeting ligands for tumour cells, fused to glycosylphosphatidylinositol (GPI) anchor signal peptides derived from decay-accelerating factor (DAF). EVs were isolated using ultrafiltration/size-exclusion liquid chromatography and characterized using western blotting, Nanoparticle Tracking Analysis, and electron microscopy. EV–tumour cell interactions were analyzed under static conditions using flow cytometry and under flow conditions using a live-cell fluorescence microscopy-coupled perfusion system. Results EV analysis showed that GPI-linked nanobodies were successfully displayed on EV surfaces and were highly enriched in EVs compared with parent cells. Display of GPI-linked nanobodies on EVs did not alter general EV characteristics (i.e. morphology, size distribution and protein marker expression), but greatly improved EV binding to tumour cells dependent on EGFR density under static conditions. Moreover, nanobody-displaying EVs showed a significantly improved cell association to EGFR-expressing tumour cells under flow conditions. Conclusions We show that nanobodies can be anchored on the surface of EVs via GPI, which alters their cell targeting behaviour. Furthermore, this study highlights GPI-anchoring as a new tool in the EV toolbox, which may be applied for EV display of a variety of proteins, such as antibodies, reporter proteins and signaling molecules. PMID:26979463

  14. Influence of Trifluoroethanol on Membrane Interfacial Anchoring Interactions of Transmembrane α-Helical Peptides

    PubMed Central

    Özdirekcan, Suat; Nyholm, Thomas K. M.; Raja, Mobeen; Rijkers, Dirk T. S.; Liskamp, Rob M. J.; Killian, J. Antoinette

    2008-01-01

    Interfacial anchoring interactions between aromatic amino acid residues and the lipid-water interface are believed to be important determinants for membrane protein structure and function. Thus, it is possible that molecules that partition into the lipid-water interface can influence membrane protein activity simply by interfering with these anchoring interactions. Here we tested this hypothesis by investigating the effects of 2,2,2-trifluoroethanol (TFE) on the interaction of a Trp-flanked synthetic transmembrane peptide (acetyl-GW2(LA)8LW2A-NH2) with model membranes of dimyristoylphosphatidylcholine. Two striking observations were made. First, using 2H nuclear magnetic resonance on acyl chain deuterated lipids, we found that addition of 4 or 8 vol % of TFE completely abolishes the ability of the peptide to order and stretch the lipid acyl chains in these relatively thin bilayers. Second, we observed that addition of 8 vol % TFE reduces the tilt angle of the peptide from 5.3° to 2.5°, as measured by 2H NMR on Ala-d4 labeled peptides. The “straightening” of the peptide was accompanied by an increased exposure of Trp to the aqueous phase, as shown by Trp-fluorescence quenching experiments using acrylamide. The observation of a reduced tilt angle was surprising because we also found that TFE partioning results in a significant thinning of the membrane, which would increase the extent of hydrophobic mismatch. In contrast to the Trp-flanked peptide, no effect of TFE was observed on the interaction of a Lys-flanked analog (acetyl-GK2(LA)8LK2A-NH2) with the lipid bilayer. These results emphasize the importance of interfacial anchoring interactions for membrane organization and provide new insights into how molecules such as TFE that can act as anesthetics may affect the behavior of membrane proteins that are enriched in aromatic amino acids at the lipid-water interface. PMID:17905843

  15. Sustained load performance of adhesive anchor systems in concrete

    NASA Astrophysics Data System (ADS)

    Davis, Todd Marshall

    Stemming from a tragic failure of an adhesive anchor system, this research project investigated the sustained load performance of adhesive anchors in concrete under different installation and in-service conditions. The literature review investigated the current state of art of adhesive anchors. Extensive discussion was devoted to the behavior of adhesive anchors in concrete as well as the many factors that can affect their short-term and sustained load strength. Existing standards and specifications for the testing, design, construction, and inspection of adhesive anchors were covered. Based on the results of the literature review and the experience of the research group, a triage was conducted on many parameters identified as possibly affecting the sustained load performance of adhesive anchors and the highest priority parameters were investigated in this project. A stress versus time-to-failure approach was used to evaluate sensitivity of three ICC-ES AC 308 approved adhesive anchor systems. Of the various parameters investigated, only elevated in-service temperature and manufacturer's cure time was shown to exhibit adverse effects on sustained loads more than that predicted by short-term tests of fully cured adhesive over a reasonable structure lifetime of 75 years. In a related study, various tests were conducted on the adhesive alone (time-temperature superposition, time-stress superposition, and dogbone tensile tests). The results of that study were used to investigate the existence of a correlation with long-term anchor pullout testing in concrete. No consistent correlations were detected for the adhesives in the study. Tests were also conducted on the effect of early-age concrete on adhesive anchor bond strength. On the basis of confined test bond-strength alone, adhesive A (vinyl ester) did not show any significant increase after 14 days (102% of 28 day strength at 14 days), and adhesive B and C (epoxies) did not show any significant increase after 7 days

  16. Highly Polymorphic Family of Glycosylphosphatidylinositol-Anchored Surface Antigens with Evidence of Developmental Regulation in Toxoplasma gondii▿

    PubMed Central

    Pollard, Angela M.; Onatolu, Krystal N.; Hiller, Luisa; Haldar, Kasturi; Knoll, Laura J.

    2008-01-01

    The life cycle of the apicomplexan parasite Toxoplasma gondii requires that an infectious cyst develop and be maintained throughout the life of the host. The molecules displayed on the parasite surface are important in controlling the immune response to the parasite. T. gondii has a superfamily of glycosylphosphatidylinositol (GPI)-anchored surface antigens, termed the surface antigen (SAG) and SAG-related surface antigens, that are developmentally regulated during infection. Using a clustering algorithm, we identified a new family of 31 surface proteins that are predicted to be GPI anchored but are unrelated to the SAG proteins, and thus we named these proteins SAG-unrelated surface antigens (SUSA). Analysis of the single nucleotide polymorphism density showed that the members of this family are the most polymorphic genes within the T. gondii genome. Immunofluorescence of SUSA1 and SUSA2, two members of the family, revealed that they are found on the parasite surface. We confirmed that SUSA1 and SUSA2 are GPI anchored by phospholipase cleavage. Analysis of expressed sequence tags (ESTs) revealed that SUSA1 had 22 of 23 ESTs from chronic infection. Analysis of mRNA and protein confirmed that SUSA1 is highly expressed in the chronic form of the parasite. Sera from mice with chronic T. gondii infection reacted to SUSA1, indicating that SUSA1 interacts with the host immune system during infection. This group of proteins likely represents a new family of polymorphic GPI-anchored surface antigens that are recognized by the host's immune system and whose expression is regulated during infection. PMID:17938221

  17. Transfer of the glycosylphosphatidylinositol-anchored 5′-nucleotidase CD73 from adiposomes into rat adipocytes stimulates lipid synthesis

    PubMed Central

    Müller, G; Jung, C; Wied, S; Biemer-Daub, G; Frick, W

    2010-01-01

    Background and purpose: In addition to predominant localization at detergent-insoluble, glycolipid-enriched plasma membrane microdomains (DIGs), glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-proteins) have been found associated with lipid droplets (LDs) and adiposomes. Adiposomes are vesicles that are released from adipocytes in response to anti-lipolytic and lipogenic signals, such as H2O2, palmitate and the antidiabetic sulfonylurea drug, glimepiride, and harbour (c)AMP-degrading GPI-proteins, among them the 5-nucleotidase CD73. Here the role of adiposomes in GPI-protein-mediated information transfer was studied. Experimental approach: Adiposomes were incubated with isolated rat adipocytes under various conditions. Trafficking of CD73 and lipid synthesis were analysed. Key results: Upon blockade of GPI-protein trafficking, CD73 specifically associated with DIGs of small, and to a lower degree, large, adipocytes. On reversal of the blockade, CD73 appeared at cytosolic LD in time- adiposome concentration- and signal (H2O2 > glimepiride > palmitate)-dependent fashion. The salt- and carbonate-resistant association of CD73 with structurally intact DIGs and LD was dependent on its intact GPI anchor. Upon incubation with small and to a lower degree, large adipocytes, adiposomes increased lipid synthesis in the absence or presence of H2O2, glimepiride and palmitate and improved the sensitivity toward these signals. Upregulation of lipid synthesis by adiposomes was dependent on the translocation of CD73 with intact GPI anchors from DIGs to LD. Conclusions: The signal-induced transfer of GPI-anchored CD73 from adiposomes via DIGs to LD of adipocytes mediates paracrine upregulation of lipid synthesis within the adipose tissue. PMID:20590586

  18. Biogenesis of the mitochondrial TOM complex: Mim1 promotes insertion and assembly of signal-anchored receptors.

    PubMed

    Becker, Thomas; Pfannschmidt, Sylvia; Guiard, Bernard; Stojanovski, Diana; Milenkovic, Dusanka; Kutik, Stephan; Pfanner, Nikolaus; Meisinger, Chris; Wiedemann, Nils

    2008-01-01

    The translocase of the outer membrane (TOM complex) is the central entry gate for nuclear-encoded mitochondrial precursor proteins. All Tom proteins are also encoded by nuclear genes and synthesized as precursors in the cytosol. The channel-forming beta-barrel protein Tom40 is targeted to mitochondria via Tom receptors and inserted into the outer membrane by the sorting and assembly machinery (SAM complex). A further outer membrane protein, Mim1, plays a less defined role in assembly of Tom40 into the TOM complex. The three receptors Tom20, Tom22, and Tom70 are anchored in the outer membrane by a single transmembrane alpha-helix, located at the N terminus in the case of Tom20 and Tom70 (signal-anchored) or in the C-terminal portion in the case of Tom22 (tail-anchored). Insertion of the precursor of Tom22 into the outer membrane requires pre-existing Tom receptors while the import pathway of the precursors of Tom20 and Tom70 is only poorly understood. We report that Mim1 is required for efficient membrane insertion and assembly of Tom20 and Tom70, but not Tom22. We show that Mim1 associates with SAM(core) components to a large SAM complex, explaining its role in late steps of the assembly pathway of Tom40. We conclude that Mim1 is not only required for biogenesis of the beta-barrel protein Tom40 but also for membrane insertion and assembly of signal-anchored Tom receptors. Thus, Mim1 plays an important role in the efficient assembly of the mitochondrial TOM complex. PMID:17974559

  19. Membrane anchoring stabilizes and favors secretion of New Delhi metallo-β-lactamase.

    PubMed

    González, Lisandro J; Bahr, Guillermo; Nakashige, Toshiki G; Nolan, Elizabeth M; Bonomo, Robert A; Vila, Alejandro J

    2016-07-01

    Carbapenems, 'last-resort' β-lactam antibiotics, are inactivated by zinc-dependent metallo-β-lactamases (MBLs). The host innate immune response withholds nutrient metal ions from microbial pathogens by releasing metal-chelating proteins such as calprotectin. We show that metal sequestration is detrimental for the accumulation of MBLs in the bacterial periplasm, because those enzymes are readily degraded in their nonmetallated form. However, the New Delhi metallo-β-lactamase (NDM-1) can persist under conditions of metal depletion. NDM-1 is a lipidated protein that anchors to the outer membrane of Gram-negative bacteria. Membrane anchoring contributes to the unusual stability of NDM-1 and favors secretion of this enzyme in outer-membrane vesicles (OMVs). OMVs containing NDM-1 can protect nearby populations of bacteria from otherwise lethal antibiotic levels, and OMVs from clinical pathogens expressing NDM-1 can carry this MBL and the blaNDM gene. We show that protein export into OMVs can be targeted, providing possibilities of new antibacterial therapeutic strategies. PMID:27182662

  20. Glycosylphosphatidylinositol Anchor Analogues Sequester Cholesterol and Reduce Prion Formation*

    PubMed Central

    Bate, Clive; Tayebi, Mourad; Williams, Alun

    2010-01-01

    A hallmark of prion diseases is the conversion of the host-encoded prion protein (PrPC where C is cellular) into an alternatively folded, disease-related isoform (PrPSc, where Sc is scrapie), the accumulation of which is associated with synapse degeneration and ultimately neuronal death. The formation of PrPSc is dependent upon the presence of PrPC in specific, cholesterol-sensitive membrane microdomains, commonly called lipid rafts. PrPC is targeted to these lipid rafts because it is attached to membranes via a glycosylphosphatidylinositol anchor. Here, we show that treatment of prion-infected neuronal cell lines (ScN2a, ScGT1, or SMB cells) with synthetic glycosylphosphatidylinositol analogues, glucosamine-phosphatidylinositol (glucosamine-PI) or glucosamine 2-O-methyl inositol octadecyl phosphate, reduced the PrPSc content of these cells in a dose-dependent manner. In addition, ScGT1 cells treated with glucosamine-PI did not transmit infection following intracerebral injection to mice. Treatment with glucosamine-PI increased the cholesterol content of ScGT1 cell membranes and reduced activation of cytoplasmic phospholipase A2 (PLA2), consistent with the hypothesis that the composition of cell membranes affects key PLA2-dependent signaling pathways involved in PrPSc formation. The effect of glucosamine-PI on PrPSc formation was also reversed by the addition of platelet-activating factor. Glucosamine-PI caused the displacement of PrPC from lipid rafts and reduced expression of PrPC at the cell surface, putative sites for PrPSc formation. We propose that treatment with glucosamine-PI modifies local micro-environments that control PrPC expression and activation of PLA2 and subsequently inhibits PrPSc formation. PMID:20427265

  1. Nuclear localization and molecular partners of BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP-ribosylation factors.

    PubMed

    Padilla, Philip Ian; Pacheco-Rodriguez, Gustavo; Moss, Joel; Vaughan, Martha

    2004-03-01

    Brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) is an approximately 200-kDa brefeldin A-inhibited guanine nucleotide-exchange protein that preferentially activates ADP-ribosylation factor 1 (ARF1) and ARF3. BIG1 was found in cytosol in a multiprotein complex with a similar ARF-activating protein, BIG2, which is also an A kinase-anchoring protein. In HepG2 cells growing with serum, BIG1 was primarily cytosolic and Golgi-associated. After incubation overnight without serum, a large fraction of endogenous BIG1 was in the nuclei. By confocal immunofluorescence microscopy, BIG1 was localized with nucleoporin p62 at the nuclear envelope (probably during nucleocytoplasmic transport) and also in nucleoli, clearly visible against the less concentrated overall matrix staining. BIG1 was also identified by Western blot analyses in purified subnuclear fractions (e.g., nucleoli and nuclear matrix). Antibodies against BIG1, nucleoporin, or nucleolin coimmunoprecipitated the other two proteins from purified nuclei. In contrast, BIG2 was not associated with nuclear BIG1. Also of note, ARF was never detected among proteins precipitated from purified nuclei by anti-BIG1 antibodies, although microscopically the two proteins do appear sometimes to be colocalized in the nucleus. These data are consistent with independent intracellular movements and actions of BIG1 and BIG2, and they are also evidence of the participation of BIG1 in both Golgi and nuclear functions. PMID:14973189

  2. Protein complex analysis of native brain potassium channels by proteomics.

    PubMed

    Sandoz, Guillaume; Lesage, Florian

    2008-01-01

    TREK potassium channels belong to a family of channel subunits with two-pore domains (K(2P)). TREK1 knockout mice display impaired polyunsaturated fatty acid-mediated protection against brain ischemia, reduced sensitivity to volatile anesthetics, resistance to depression and altered perception of pain. Recently, we isolated native TREK1 channels from mouse brain and identified their specific components by mass spectrometry. Among the identified partners, the A-Kinase Anchoring Protein AKAP150 binds to a regulatory domain of TREK1 and acts as a molecular switch. It transforms low activity, outwardly rectifying TREK1 currents into robust leak conductances resistant to stimulation by arachidonic acid, membrane stretch and acidification. Inhibition of the TREK1/AKAP150 channel by Gs-coupled receptors is as extensive as for TREK1 alone (but faster) whereas inhibition of TREK1/AKAP150 by Gq-coupled receptors is reduced. Furthermore, the association of AKAP150 with TREK1 channels integrates them into postsynaptic scaffolds where G protein-coupled membrane receptors and channels dock simultaneously. This chapter describes the proteomic approach used to study the composition of native TREK1 channels and point out its advantages and limitations over more classical methods (two-hybrid screenings in the yeast and bacteria or GST-pull down). PMID:18998088

  3. Perinuclear Anchoring of H3K9-Methylated Chromatin Stabilizes Induced Cell Fate in C. elegans Embryos.

    PubMed

    Gonzalez-Sandoval, Adriana; Towbin, Benjamin D; Kalck, Veronique; Cabianca, Daphne S; Gaidatzis, Dimos; Hauer, Michael H; Geng, Liqing; Wang, Li; Yang, Teddy; Wang, Xinghao; Zhao, Kehao; Gasser, Susan M

    2015-12-01

    Interphase chromatin is organized in distinct nuclear sub-compartments, reflecting its degree of compaction and transcriptional status. In Caenorhabditis elegans embryos, H3K9 methylation is necessary to silence and to anchor repeat-rich heterochromatin at the nuclear periphery. In a screen for perinuclear anchors of heterochromatin, we identified a previously uncharacterized C. elegans chromodomain protein, CEC-4. CEC-4 binds preferentially mono-, di-, or tri-methylated H3K9 and localizes at the nuclear envelope independently of H3K9 methylation and nuclear lamin. CEC-4 is necessary for endogenous heterochromatin anchoring, but not for transcriptional repression, in contrast to other known H3K9 methyl-binders in worms, which mediate gene repression but not perinuclear anchoring. When we ectopically induce a muscle differentiation program in embryos, cec-4 mutants fail to commit fully to muscle cell fate. This suggests that perinuclear sequestration of chromatin during development helps restrict cell differentiation programs by stabilizing commitment to a specific cell fate. PAPERCLIP. PMID:26607792

  4. A novel human erythrocyte glycosylphosphatidylinositol (GPI)-anchored glycoprotein ACA. Isolation, purification, primary structure determination, and molecular parameters of its lipid structure.

    PubMed

    Becker Kojić, Zorica A; Terness, Peter

    2002-10-25

    A method has been elaborated to isolate and purify up to homogeneity a novel membrane glycoprotein containing a glycosyl-phosphatidylinositol (GPI) anchor by means of salting out with ammonium sulfate (40-80% saturation), followed by preparative SDS-PAGE, chromatography and acetone precipitation. The preparation obtained was homogeneous upon electrophoresis in the presence of 0.1% SDS after reduction with 2-mercaptoethanol. It is protein-soluble at its isoelectrical point (pH 5.5) with molecular mass of 65,000 daltons. The isolated protein is linked to the membrane via glycosyl-phosphatidylinositol susceptible to cleavage by purified phospholipase C. The hydrophobic portion of the glycolipid membrane anchor of the protein was radiolabeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine and hydrolyzed with glycosyl-phosphatidylinositol-specific phospholipase C, followed by enzymatic deacetylation of the remaining lipid. Thin-layer chromatography showed that the generated radiolabeled fragment migrates with the same mobility as that of variant surface glycoprotein (VSG), obtained in the same manner. In this study we describe a novel erythrocyte membrane GPI-linked protein with the structural feature of an anchor that, in contrast to other GPI-linked erythrocyte proteins, has a non-acetylated inositol ring and diacylglycerol rather than alkyl-acyl glycerol as a lipid tail of the anchor. PMID:12167612

  5. Behavior of Giant Vesicles with Anchored DNA Molecules

    PubMed Central

    Nikolov, Vesselin; Lipowsky, Reinhard; Dimova, Rumiana

    2007-01-01

    We study changes in curvature and elastic properties of lipid membranes induced by anchoring of long hydrophilic polymers at low polymer surface concentrations (corresponding to the mushroom regime). The effect of anchored polymers on the membrane spontaneous curvature is characterized by monitoring the changes in the fluctuation spectra and the morphology of giant unilamellar vesicles. The polymers used in our study are fluorescently labeled and biotinylated λ-phage DNA molecules which bind to biotinylated giant unilamellar vesicles via a biotin-avidin-biotin linkage. By varying the amount of biotinylated lipid in the membrane, we control the surface concentration of anchors. At low anchor concentrations, the spontaneous curvature of the membrane increases linearly with the DNA concentration. The linear increase is consistent with theoretical predictions for polymer surface concentrations in the mushroom regime. At higher anchor concentrations, which should still belong to the mushroom regime, the vesicles undergo budding transitions. In this latter regime, the bud size is used to estimate the polymer-induced membrane curvature. PMID:17384074

  6. The "loop with anchor" technique to repair mitral valve prolapse.

    PubMed

    Isoda, Susumu; Osako, Motohiko; Kimura, Tamizo; Mashiko, Yuji; Yamanaka, Nozomu; Nakamura, Shingo; Maehara, Tadaaki

    2012-01-01

    The current surgical technique of using an artificial chord (composed of expanded polytetrafluoroethylene [ePTFE] sutures) to repair mitral prolapse is technically difficult to perform. Slippery knot tying and the difficulty of changing the chordae length after the hydrostatic test are frustrating problems. The loop technique solves the problem of slippery knot tying but not the problem of changing the chordae length. Our "loop with anchor" technique consists of the following elements: construction of an anchor at the papillary muscle; determining the loop length; tying the loop to the anchor; suturing the loop to the mitral valve; the hydrostatic test; and re-suturing or changing the loop, if needed. Adjustments can be made for the entire procedure or for a portion of the procedure. PMID:22156285

  7. Expression of GPI anchored human recombinant erythropoietin in CHO cells is devoid of glycosylation heterogeneity.

    PubMed

    Singh, Pankaj Kumar; Devasahayam, Mercy; Devi, Sobita

    2015-04-01

    Erythropoietin is a glycohormone involved in the regulation of the blood cell levels. It is a 166 amino acid protein having 3 N-glycosylation and one O-linked glycosylation sites, and is used to treat anaemia related illness. Though human recombinant erythropoietin (rEPO) is produced in CHO cells, the loss in quality control is 80% due to incomplete glycosylation of the rEPO with low levels of fully glycosylated active rEPO. Here, we describe the expression from CHO cells of fully glycosylated human rEPO when expressed as a GPI anchored molecule (rEPO-g). The results demonstrated the production of a homogenous completely glycosylated human rEPO-g as a 42 kD band without any low molecular weight glycoform variants as shown by affinity chromatography followed by SDS-PAGE and anti-human EPO specific western blot. The western blot using specific monoclonal antibody is the available biochemical technique to prove the presence of homogeneity in the expressed recombinant protein. The GPI anchor can be removed during the purification process to yield a therapeutically relevant recombinant erythropoietin molecule cells with a higher in vivo biological activity due to its high molecular weight of 40 kD. This is possibly the first report on the production of a homogenous and completely glycosylated human rEPO from CHO cells for efficient therapy. PMID:26011979

  8. Modeling and Simulation of Anchoring Processess for Small Body Exploration

    NASA Technical Reports Server (NTRS)

    Quadrelli, Marco B.; Mazahar, Hammad; Negrut, Dan

    2012-01-01

    This paper describes recent work done in modeling and simulation of anchoring processes in granular media, with applications to anchoring on a Near Earth Object (NEO), where the forces due to interactions with the regolith are much stronger than the local surface gravity field. This effort is part of a larger systems engineering capability developed at JPL to answer key questions, validate requirements, conduct key system and mission trades,and evaluate performance and risk related to NEO operations for any proposed human or robotic missions to a NEO.

  9. 17. VIEW OF ANCHOR BRIDGE NUMBER 310 LOOKING EAST ALONG ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    17. VIEW OF ANCHOR BRIDGE NUMBER 310 LOOKING EAST ALONG THE MAIN LINE TRACK LOCATED TO THE NORTH OF THE COS COB POWER PLANT. ANCHOR BRIDGES LOCATED AT TWO MILE INTERVALS WITHSTAND CATENARY TENSION AND PROVIDE A PLATFORM FOR MOUNTING OIL FILLED CIRCUIT BREAKERS, LIGHTNING ARRESTORS AND OTHER ELECTRICAL EQUIPMENT. THE ROOF OF THE LOAD DISPATCHER'S TOWER CAN BE SEEN DIRECTLY BEHIND THE RIGHT SIDE OF THE BRIDGE. - New York, New Haven & Hartford Railroad, Cos Cob Power Plant, Sound Shore Drive, Greenwich, Fairfield County, CT

  10. Probing the Huntingtin 1-17 Membrane Anchor on a Phospholipid Bilayer by Using All-Atom Simulations

    PubMed Central

    Côté, Sébastien; Binette, Vincent; Salnikov, Evgeniy S.; Bechinger, Burkhard; Mousseau, Normand

    2015-01-01

    Mislocalization and aggregation of the huntingtin protein are related to Huntington’s disease. Its first exon—more specifically the first 17 amino acids (Htt17)—is crucial for the physiological and pathological functions of huntingtin. It regulates huntingtin’s activity through posttranslational modifications and serves as an anchor to membrane-containing organelles of the cell. Recently, structure and orientation of the Htt17 membrane anchor were determined using a combined solution and solid-state NMR approach. This prompted us to refine this model by investigating the dynamics and thermodynamics of this membrane anchor on a POPC bilayer using all-atom, explicit solvent molecular dynamics and Hamiltonian replica exchange. Our simulations are combined with various experimental measurements to generate a high-resolution atomistic model for the huntingtin Htt17 membrane anchor on a POPC bilayer. More precisely, we observe that the single α-helix structure is more stable in the phospholipid membrane than the NMR model obtained in the presence of dodecylphosphocholine detergent micelles. The resulting Htt17 monomer has its hydrophobic plane oriented parallel to the bilayer surface. Our results further unveil the key residues interacting with the membrane in terms of hydrogen bonds, salt-bridges, and nonpolar contributions. We also observe that Htt17 equilibrates at a well-defined insertion depth and that it perturbs the physical properties—order parameter, thickness, and area per lipid—of the bilayer in a manner that could favor its dimerization. Overall, our observations reinforce and refine the NMR measurements on the Htt17 membrane anchor segment of huntingtin that is of fundamental importance to its biological functions. PMID:25762330

  11. Current status of frameless anchored IUD for immediate intracesarean insertion.

    PubMed

    Wildemeersch, Dirk; Goldstuck, Norman D; Hasskamp, Thomas

    2016-01-01

    Immediate postpartum intrauterine device (IUD) insertion deserves great attention as it can provide immediate, timely and convenient contraception plus the added benefit of preventing repeat unintended pregnancies. Although women post vaginal delivery can benefit from immediate post-placenta contraception, women undergoing Cesarean section clearly need contraception, as an inter-delivery interval shorter than 18 months places them at a high risk for uterine rupture. The main drawback of currently available framed IUD devices for immediate postpartum insertion of an IUD is their high expulsion and displacement rates when inserted immediately postpartum after both vaginal and Cesarean delivery. Current research suggests that a brief window of opportunity exists of 10 minutes for insertion of conventional IUDs after which time expulsion rates both immediately and over time are greatly enhanced. This paper summarizes the current research conducted to overcome the expulsion problems associated with conventional T-shaped devices as well as through the use of an anchored frameless device. In the 1970s and 1980s, attempts were made to solve the expulsion problem by modifying existing devices, such as adding absorbable sutures (Delta-T) or additional appendages. These attempts proved to be clinically unsuccessful as the catgut suture added to the transverse arms did not provide sufficient resistance to prevent downward displacement and expulsion. An anchoring technique to suspend a copper IUD to the fundus of the uterus was developed in Belgium in the 1980s and has been the subject of extensive ongoing clinical research since 1985. Recently the frameless copper releasing anchor IUD, GyneFix, has been tested for postplacental insertion. Initially, the anchor was modified by the inclusion of a biodegradable cone which was added below the anchoring knot. Clinical studies confirmed the adequacy of this approach suggesting that it was technically possible to anchor an IUD

  12. [Dactylogyrids (Monogenea: Dactylogyridea) with an unusual number of anchors, their origin and phylogenetic significance. Reference data].

    PubMed

    Gerasev, P I

    2002-01-01

    It is considered that in Anacanthorinae, Markewitschiana and Pseudacolpenteron pavlovskii (Dactylogyridae s. s.) the anchors are absent originally. In the other dactylogyrids (s. l.) without anchors (Pseudacolpenteron ignotus; Acolpenteron; Anonchohaptor-Icelanchohaptor; Kritskyia-Telethecium-Pavanelliela) these structures are lost. In related genera Rhinoxenus and Nanotrema the dorsal pair of anchors is transformed into the "spikes"; in Heteronchocleidinae there is one anchor; in Trinigyrus and Schilbetrematoides are two anchors; and in Urogyrus three anchors are reduced. But in Rhinonaster, Cacatyocotyle, Callceostomella, Neocalseostoma elongatum and Pseudodactylogyridae the presence of one pair of ventral "peduncular" or "haptoral" anchors appear to be the original state. For Anacanthorinae and Dactylogyridae s. s. the presence of 18-16 hooks, original lacking of anchors, appearance of one pair of them, evolutionary development and subsequent reduction of these structures are characteristic. In the dactylogyrids (s. l.) having two pairs of anchors and 14 hooks, one pair of the anchors is present originally, then usually the second dorsal pair grows up, and finally, the reduction of a part of anchors or all the anchors is possible. In the 16-hooked dactylogyrids (s. l.) having two pairs of anchors (Tetraonchinea, Amphibdellainea, Neodactylodiscinea) the original lacking or reduction of the anchors is not reported. The author considers, that it is unwarranted to unite all dactylogyrids (s. l.) without anchors into the superorder Ananchorea Malmberg, 1990 or to postulate the original presence of the two pairs of ventral anchors in this group. It is also impossible to unit all dactylogyrids (s. l.) having 0-4 anchors and 18-14 hooks into Dactylogyridae sensu Boeger et Kritsky, 1993. PMID:12070963

  13. Activation of c-fos gene expression by a kinase-deficient epidermal growth factor receptor.

    PubMed Central

    Eldredge, E R; Korf, G M; Christensen, T A; Connolly, D C; Getz, M J; Maihle, N J

    1994-01-01

    The intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGFR) has been shown to be responsible for many of the pleiotropic intracellular effects resulting from ligand stimulation [W.S. Chen, C.S. Lazar, M. Poenie, R.Y. Tsien, G.N. Gill, and M.G. Rosenfeld, Nature (London) 328:820-823, 1987; A.M. Honegger, D. Szapary, A. Schmidt, R. Lyall, E. Van Obberghen, T.J. Dull, A. Ulrich, and J. Schlessinger, Mol. Cell. Biol. 7:4568-4571, 1987]. Recently, however, it has been shown that addition of ligand to cells expressing kinase-defective EGFR mutants can result in the phosphorylation of mitogen-activated protein kinase (R. Campos-González and J.R. Glenney, Jr., J. Biol. Chem. 267:14535-14538, 1992; E. Selva, D.L. Raden, and R.J. Davis, J. Biol. Chem. 268:2250-2254, 1993), as well as stimulation of DNA synthesis (K.J. Coker, J.V. Staros, and C.A. Guyer, Proc. Natl. Acad. Sci. USA 91:6967-6971, 1994). Moreover, mitogen-activated protein kinase has been shown to phosphorylate the transcription factor p62TCF in vitro, leading to enhanced ternary complex formation between p62TCF, p67SRF, and the c-fos serum response element (SRE) [H. Gille, A.D. Sharrocks, and P.E. Shaw, Nature (London) 358:414-417, 1992]. On the basis of these observations, we have investigated the possibility that the intrinsic tyrosine kinase activity of the EGFR may not be necessary for transcriptional activation mediated via p62TCF. Here, we demonstrate that a kinase-defective EGFR mutant can signal ligand-induced expression of c-fos protein and that a significant component of this induction appears to be mediated at the transcriptional level. Investigation of transcriptional activation mediated by the c-fos SRE shows that this response is impaired by mutations in the SRE which eliminate binding of p62(TCF). These data indicate that information inherent in the structure of the EGFR can be accessed by ligand stimulation independent of the receptor's catalytic kinase function

  14. The induction of polyploidy or apoptosis by the Aurora A kinase inhibitor MK8745 is p53-dependent

    PubMed Central

    Ho, Alan L; Schwartz, Gary K

    2012-01-01

    Aurora kinases are mitotic serine/threonine protein kinases and are attractive novel targets for anticancer therapy. Many small-molecule inhibitors of Aurora kinases are currently undergoing clinical trials. Aurora A kinase is essential for successful mitotic transition. MK8745 is a novel and selective small-molecule inhibitor of Aurora A kinase. MK8745 induced apoptotic cell death in a p53-dependent manner when tested in vitro in cell lines of multiple lineages. Cells expressing wild-type p53 showed a short delay in mitosis followed by cytokinesis, resulting in 2N cells along with apoptosis. However, cells lacking or with mutant p53 resulted in a prolonged arrest in mitosis followed by endoreduplication and polyploidy. Cytokinesis was completely inhibited in p53-deficient cells, as observed by the absence of 2N cell population. The induction of apoptosis in p53-proficient cells was associated with activation of caspase 3 and release of cytochrome c but was independent of p21. Exposure of p53 wild-type cells to MK8745 resulted in the induction of p53 phosphorylation (ser15) and an increase in p53 protein expression. p53-dependent apoptosis by MK8745 was further confirmed in HCT 116 p53−/− cells transfected with wild-type p53. Transient knockdown of Aurora A by specific siRNA recapitulated these p53-dependent effects, with greater percent induction of apoptosis in p53 wild-type cells. In conclusion, our studies show p53 as a determining factor for induction of apoptosis vs. polyploidy upon inhibition of Aurora A. PMID:22293494

  15. Spatial Organization in Protein Kinase A Signaling Emerged at the Base of Animal Evolution.

    PubMed

    Peng, Mao; Aye, Thin Thin; Snel, Berend; van Breukelen, Bas; Scholten, Arjen; Heck, Albert J R

    2015-07-01

    In phosphorylation-directed signaling, spatial and temporal control is organized by complex interaction networks that diligently direct kinases toward distinct substrates to fine-tune specificity. How these protein networks originate and evolve into complex regulatory machineries are among the most fascinating research questions in biology. Here, spatiotemporal signaling is investigated by tracing the evolutionary dynamics of each functional domain of cAMP-dependent protein kinase (PKA) and its diverse set of A-kinase anchoring proteins (AKAPs). Homologues of the catalytic (PKA-C) and regulatory (PKA-R) domains of the (PKA-R)2-(PKA-C)2 holoenzyme were found throughout evolution. Most variation was observed in the RIIa of PKA-R, crucial for dimerization and docking to AKAPs. The RIIa domain was not observed in all PKA-R homologues. In the fungi and distinct protist lineages, the RIIa domain emerges within PKA-R, but it displays large sequence variation. These organisms do not harbor homologues of AKAPs, suggesting that efficient docking to direct spatiotemporal PKA activity evolved in multicellular eukaryotes. To test this in silico hypothesis, we experimentally screened organisms with increasing complexity by cAMP-based chemical proteomics to reveal that the occurrence of PKA-AKAP interactions indeed coincided and expanded within vertebrates, suggesting a crucial role for AKAPs in the advent of metazoan multicellularity. PMID:26066639

  16. The Effect of Anchor Test Construction on Scale Drift

    ERIC Educational Resources Information Center

    Antal, Judit; Melican, Gerald; Proctor, Thomas; Wiley, Andrew

    2010-01-01

    Presented at the Annual Meeting of National Council on Measurement in Education (NCME) in 2010. The focus of the research is to investigate the effect of applying the Sinharay & Holland (2007) midi-test idea for building anchor tests to an on-going testing program with a series of versions of the test and comparing these results to the more…

  17. Detecting Anchoring-and-Adjusting in Survey Scales

    ERIC Educational Resources Information Center

    McIntyre, Joe

    2014-01-01

    Proper survey design is essential to obtain reliable, replicable data from research subjects. One threat to inferences drawn from surveys is anchoring-and-adjusting. Tversky and Kahnemann (1974) observed that participants' responses to questions depended systematically on irrelevant information they received prior to answering. It is important for…

  18. Culturally-Anchored Values and University Education Experience Perception

    ERIC Educational Resources Information Center

    Mitsis, Ann; Foley, Patrick

    2009-01-01

    Purpose: The purpose of this paper is to examine whether business students' gender, age and culturally-anchored values affect their perceptions of their university course experience. Design/methodology/approach: Culturally diverse business students (n 1/4 548) studying at an Australian university were surveyed using previously established scales.…

  19. 20. DETAIL, ANCHOR BLOCK AND BUTTRESS AT SOUTH END OF ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    20. DETAIL, ANCHOR BLOCK AND BUTTRESS AT SOUTH END OF DAM, SHOWING PROMINENT GROOVE WHERE THE NEXT ARCH COULD HAVE BEEN JOINED TO THE NORTH-SOUTH TRENDING DAM. - Rock Creek Dam, East end of Rock Creek Road, Auburn, Placer County, CA

  20. 48 CFR 1812.7000 - Anchor tenancy contracts.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 6 2013-10-01 2013-10-01 false Anchor tenancy contracts. 1812.7000 Section 1812.7000 Federal Acquisition Regulations System NATIONAL AERONAUTICS AND SPACE ADMINISTRATION COMPETITION AND ACQUISITION PLANNING ACQUISITION OF COMMERCIAL ITEMS Commercial Space Hardware...

  1. 48 CFR 1812.7000 - Anchor tenancy contracts.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 6 2014-10-01 2014-10-01 false Anchor tenancy contracts. 1812.7000 Section 1812.7000 Federal Acquisition Regulations System NATIONAL AERONAUTICS AND SPACE ADMINISTRATION COMPETITION AND ACQUISITION PLANNING ACQUISITION OF COMMERCIAL ITEMS Commercial Space Hardware...

  2. Effective Leadership for Supervisors: Behaviorally Anchored Supervision System.

    ERIC Educational Resources Information Center

    Kukic, Stevan J.; Fister, Susan

    The paper describes the Behaviorally Anchored Supervision System (BASS), an approach to supervision in special education based on the definition of effective instruction developed by the Council for Exceptional Children in 1986. A comprehensive model for excellence in special education is based upon the following four elements: values,…

  3. Early active mobilization following UCL repair With Mitek bone anchor.

    PubMed

    Crowley, Timothy P; Stevenson, Susan; Taghizadeh, Reika; Addison, Patrick; Milner, Richard H

    2013-09-01

    Ulnar collateral ligament (UCL) injuries of the thumb are common. Surgical repair is accepted as the treatment of choice for complete rupture of the ligament. Biomechanical studies have suggested that Mitek bone anchor repairs are potentially safe and strong enough to allow early controlled active mobilization. To date, there have been no studies to compare early active mobilization following Mitek bone anchor repair to standard postoperative rehabilitation involving thumb spica immobilization for the first 4 to 6 weeks. We performed a small pilot randomized control trial to assess the outcome of this new rehabilitation technique to that of standard immobilization following UCL repair with Mitek bone anchor. Our results show that on average early active mobilization leads to an earlier return to full hand function (6 vs. 8 wk) and an earlier return to work (7 vs. 11 wk). There is no difference in the final range of motion achieved. We suggest that Mitek bone anchor repairs for UCL ruptures are robust enough to allow early active mobilization and that a larger trial is warranted to assess whether early active mobilization is superior to standard rehabilitation. PMID:23970193

  4. Isolating Suture Slippage During Cadaveric Testing of Knotless Anchors.

    PubMed

    Klinge, Stephen A; Vopat, Bryan G; Paller, David; Avery, Anthony L; Koruprolu, Sarath; Fadale, Paul D

    2015-07-01

    We evaluated a testing method designed to isolate and analyze the effectiveness of different suture-retention mechanisms in knotless suture anchors used for rotator cuff repairs. Six knotless PushLock implants (Arthrex) with a suture-retention mechanism dependent on a press-fit of suture between the anchor's outer diameter and surrounding bone were compared with 6 ReelX STT devices (Stryker) reliant on an intrinsic suture-locking mechanism. Suture slippage beyond minimal clinical failure thresholds, as well as ultimate failure load, were determined with a novel testing fixture that isolated suture slippage. Suture slippage was isolated from anchor-bone disengagement. Each PushLock exhibited suture slippage of more than 3 mm, and each ReelX exhibited slippage of less than 3 mm. The PushLock implants also exhibited significantly (P < .05) more interval and maximum slippage; 5 of these 6 implants failed via complete suture slippage before dynamic testing could be completed. All ReelX devices survived dynamic testing and ultimately failed via suture breakage. This novel axial load biomechanical testing technique isolated suture slippage in 2 uniquely designed knotless anchors. The press-fit PushLock implant was prone to slippage failure, whereas the ReelX device with its internal suture-locking mechanism exhibited minimal slippage. PMID:26161766

  5. Self Mountable and Extractable Ultrasonic/Sonic Anchor

    NASA Technical Reports Server (NTRS)

    Bar-Cohen, Yoseph (Inventor); Sherrit, Stewart (Inventor)

    2007-01-01

    Self drilling anchors and related methods and apparatus. In one embodiment an apparatus comprises a drill bit, a hammer mechanism for hammering the drill bit in a first direction and in a second direction, and a selection mechanism for controlling whether, at a given point in time, the drill bit is hammered in the first or second direction.

  6. When Does Scale Anchoring Work? A Case Study

    ERIC Educational Resources Information Center

    Sinharay, Sandip; Haberman, Shelby J.; Lee, Yi-Hsuan

    2011-01-01

    Providing information to test takers and test score users about the abilities of test takers at different score levels has been a persistent problem in educational and psychological measurement. Scale anchoring, a technique which describes what students at different points on a score scale know and can do, is a tool to provide such information.…

  7. 33 CFR 401.14 - Anchor marking buoys.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 3 2010-07-01 2010-07-01 false Anchor marking buoys. 401.14 Section 401.14 Navigation and Navigable Waters SAINT LAWRENCE SEAWAY DEVELOPMENT CORPORATION, DEPARTMENT OF TRANSPORTATION SEAWAY REGULATIONS AND RULES Regulations Condition of Vessels § 401.14...

  8. The Effect of Anchor Test Construction on Scale Drift

    ERIC Educational Resources Information Center

    Antal, Judit; Proctor, Thomas P.; Melican, Gerald J.

    2014-01-01

    In common-item equating the anchor block is generally built to represent a miniature form of the total test in terms of content and statistical specifications. The statistical properties frequently reflect equal mean and spread of item difficulty. Sinharay and Holland (2007) suggested that the requirement for equal spread of difficulty may be too…

  9. Equating without an Anchor for Nonequivalent Groups of Examinees

    ERIC Educational Resources Information Center

    Longford, Nicholas T.

    2015-01-01

    An equating procedure for a testing program with evolving distribution of examinee profiles is developed. No anchor is available because the original scoring scheme was based on expert judgment of the item difficulties. Pairs of examinees from two administrations are formed by matching on coarsened propensity scores derived from a set of…

  10. Ten Anchor Points for Teaching Principles of Marketing

    ERIC Educational Resources Information Center

    Tomkovick, Chuck

    2004-01-01

    Effective marketing instructors commonly share a love for their students, an affinity for the subject matter, and a devotion to continuous quality improvement. The purpose of this article is to highlight 10 anchor points for teaching Principles of Marketing, which are designed to better engage students in the learning process. These anchor…

  11. 24 CFR 3285.402 - Ground anchor installations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... HOUSING AND URBAN DEVELOPMENT MODEL MANUFACTURED HOME INSTALLATION STANDARDS Anchorage Against Wind § 3285... anchoring. Manufactured homes must also be stabilized against wind in the longitudinal direction in all Wind Zones. Manufactured homes located in Wind Zones II and III must have longitudinal ground...

  12. 24 CFR 3285.402 - Ground anchor installations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... HOUSING AND URBAN DEVELOPMENT MODEL MANUFACTURED HOME INSTALLATION STANDARDS Anchorage Against Wind § 3285... anchoring. Manufactured homes must also be stabilized against wind in the longitudinal direction in all Wind Zones. Manufactured homes located in Wind Zones II and III must have longitudinal ground...

  13. Aerothermal Anchoring of CBAERO Using High Fidelity CFD

    NASA Technical Reports Server (NTRS)

    Kinney, David J.

    2007-01-01

    The Configuration Based Aerodynamics (CBAERO) software package is used to predict the convective and radiative heating environments for the Crew Exploration Vehicle (CEV). A limited number of high fidelity CFD solutions are used to "anchor" the engineering level estimates obtained using CBAERO.

  14. 30 CFR 57.9311 - Anchoring stationary sizing devices.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Anchoring stationary sizing devices. 57.9311 Section 57.9311 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES...

  15. 30 CFR 57.9311 - Anchoring stationary sizing devices.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Anchoring stationary sizing devices. 57.9311 Section 57.9311 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES...

  16. 30 CFR 57.9311 - Anchoring stationary sizing devices.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Anchoring stationary sizing devices. 57.9311 Section 57.9311 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES...

  17. 30 CFR 57.9311 - Anchoring stationary sizing devices.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Anchoring stationary sizing devices. 57.9311 Section 57.9311 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES...

  18. 30 CFR 57.9311 - Anchoring stationary sizing devices.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Anchoring stationary sizing devices. 57.9311 Section 57.9311 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES...

  19. 24. STARBOARD PROFILE OF ALABAMA (ALABAMIAN); VESSEL AT ANCHOR ON ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    24. STARBOARD PROFILE OF ALABAMA (ALABAMIAN); VESSEL AT ANCHOR ON STATION IN GULF OF MEXICO WITH MOTOR BOAT TIED AT STERN Original 4-3/4'x6-3/4' photograph taken c. 1930? - Pilot Schooner "Alabama", Moored in harbor at Vineyard Haven, Vineyard Haven, Dukes County, MA

  20. 16. STRUCTURAL DETAILS: CHANNEL, BIT & CLEAT, ANCHOR BOLTS & ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    16. STRUCTURAL DETAILS: CHANNEL, BIT & CLEAT, ANCHOR BOLTS & PLATES FOR PIERS 4, 5, AND 6, DWG. NO. 97, 1-1/2" = 1', MADE BY A.F., JUNE 13, 1908 - Baltimore Inner Harbor, Pier 5, South of Pratt Street between Market Place & Concord Street, Baltimore, Independent City, MD

  1. Finding the Optimal Guidance for Enhancing Anchored Instruction

    ERIC Educational Resources Information Center

    Zydney, Janet Mannheimer; Bathke, Arne; Hasselbring, Ted S.

    2014-01-01

    This study investigated the effect of different methods of guidance with anchored instruction on students' mathematical problem-solving performance. The purpose of this research was to iteratively design a learning environment to find the optimal level of guidance. Two iterations of the software were compared. The first iteration used…

  2. Poor Anchoring Limits Dyslexics' Perceptual, Memory, and Reading Skills

    ERIC Educational Resources Information Center

    Oganian, Yulia; Ahissar, Merav

    2012-01-01

    The basic deficits underlying the severe and persistent reading difficulties in dyslexia are still highly debated. One of the major topics of debate is whether these deficits are language specific, or affect both verbal and non-verbal stimuli. Recently, Ahissar and colleagues proposed the "anchoring-deficit hypothesis" (Ahissar, Lubin,…

  3. Implementing Anchored Instruction: Guiding Principles for Curriculum Development.

    ERIC Educational Resources Information Center

    McLarty, Kim; And Others

    A curriculum based on "anchored instruction" was developed to enhance students' literacy development and acquisition of knowledge. The curriculum was designed to create a rich, shared environment that generates interest and enables students to identify and define problems while they explore the content from many perspectives. Based on what is…

  4. Development of Behaviorally-Anchored Rating Scales for Pharmacy Practice.

    ERIC Educational Resources Information Center

    Grussing, Paul G.; And Others

    1979-01-01

    Seventeen behaviorally-anchored rating scales were developed for a comprehensive measure of performance in pharmacy practice, including pharmacist selection, performance appraisal, and promotion activities. The scales (which are included) were also used to evaluate extern performance, and to serve as a criterion measure in studies of concurrent…

  5. Corrected High-Frame Rate Anchored Ultrasound with Software Alignment

    ERIC Educational Resources Information Center

    Miller, Amanda L.; Finch, Kenneth B.

    2011-01-01

    Purpose: To improve lingual ultrasound imaging with the Corrected High Frame Rate Anchored Ultrasound with Software Alignment (CHAUSA; Miller, 2008) method. Method: A production study of the IsiXhosa alveolar click is presented. Articulatory-to-acoustic alignment is demonstrated using a Tri-Modal 3-ms pulse generator. Images from 2 simultaneous…

  6. Impact of Enhanced Anchored Instruction in Inclusive Math Classrooms

    ERIC Educational Resources Information Center

    Bottge, Brian A.; Toland, Michael D.; Gassaway, Linda; Butler, Mark; Choo, Sam; Griffen, Ann Katherine; Ma, Xin

    2015-01-01

    The Common Core State Standards for Mathematics will place more pressure on special education and math teachers to raise the skill levels of all students, especially those with disabilities in math (MD). The purpose of this study was to assess the effects of enhanced anchored instruction (EAI) on students with and without MD in co-taught general…

  7. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  8. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  9. The membrane anchor of the transcriptional activator SREBP is characterized by intrinsic conformational flexibility

    PubMed Central

    Linser, Rasmus; Salvi, Nicola; Briones, Rodolfo; Rovó, Petra; de Groot, Bert L.; Wagner, Gerhard

    2015-01-01

    Regulated intramembrane proteolysis (RIP) is a conserved mechanism crucial for numerous cellular processes, including signaling, transcriptional regulation, axon guidance, cell adhesion, cellular stress responses, and transmembrane protein fragment degradation. Importantly, it is relevant in various diseases including Alzheimer’s disease, cardiovascular diseases, and cancers. Even though a number of structures of different intramembrane proteases have been solved recently, fundamental questions concerning mechanistic underpinnings of RIP and therapeutic interventions remain. In particular, this includes substrate recognition, what properties render a given substrate amenable for RIP, and how the lipid environment affects the substrate cleavage. Members of the sterol regulatory element-binding protein (SREBP) family of transcription factors are critical regulators of genes involved in cholesterol/lipid homeostasis. After site-1 protease cleavage of the inactive SREBP transmembrane precursor protein, RIP of the anchor intermediate by site-2 protease generates the mature transcription factor. In this work, we have investigated the labile anchor intermediate of SREBP-1 using NMR spectroscopy. Surprisingly, NMR chemical shifts, site-resolved solvent exposure, and relaxation studies show that the cleavage site of the lipid-signaling protein intermediate bears rigid α-helical topology. An evolutionary conserved motif, by contrast, interrupts the secondary structure ∼9–10 residues C-terminal of the scissile bond and acts as an inducer of conformational flexibility within the carboxyl-terminal transmembrane region. These results are consistent with molecular dynamics simulations. Topology, stability, and site-resolved dynamics data suggest that the cleavage of the α-helical substrate in the case of RIP may be associated with a hinge motion triggered by the molecular environment. PMID:26392539

  10. 46 CFR 32.15-15 - Anchors, Chains, and Hawsers-TB/ALL

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... will evaluate the suitability of the equipment. (e) Barges Equipped with Anchors to Comply with 33 CFR 155.230(b)(1). Each barge equipped with an anchor, to comply with 33 CFR 155.230(b)(1), must be...

  11. 46 CFR 32.15-15 - Anchors, Chains, and Hawsers-TB/ALL

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... will evaluate the suitability of the equipment. (e) Barges Equipped with Anchors to Comply with 33 CFR 155.230(b)(1). Each barge equipped with an anchor, to comply with 33 CFR 155.230(b)(1), must be...

  12. Expression of an enzymically active glycosylphosphatidylinositol-anchored form of neutral endopeptidase (EC 3.4.24.11) in Cos-1 cells.

    PubMed Central

    Howell, S; Lanctôt, C; Boileau, G; Crine, P

    1994-01-01

    Neutral endopeptidase (EC 3.4.24.11, NEP) is a type-II integral membrane protein found in a wide variety of cell types. We previously produced a secreted form of the enzyme by deletion of the cytoplasmic and transmembrane domains and in-frame fusion of the cleavable signal peptide of pro-opiomelanocortin [Lemay, Waksman, Roques, Crine and Boileau (1989) J. Biol. Chem. 264, 15620-15623]. Here we have used this secreted form of NEP and fused to it the glycosylphosphatidylinositol (GPI)-anchor attachment signal of decay-accelerating factor to produce a GPI-anchored form. Expression of this chimeric form in Cos-1 cells resulted in cell-surface activity. This activity could be released from the cell surface by phosphatidylinositol-specific phospholipase C and radiolabelling studies showed that the protein could incorporate [3H]ethanolamine, indicating that the enzyme was GPI-anchored. The Km value, using [D-Ala2,Leu5]enkephalin as substrate, of GPI-anchored NEP (62 +/- 5 microM) was comparable with that of wild-type NEP (70 +/- 4 microM), as were the sensitivities to the inhibitors phosphoramidon and thiorphan. However, pulse-chase studies showed that the biosynthesis and cell-surface delivery of GPI-anchored NEP was delayed compared with that of the wild-type transmembrane form of NEP. These results suggest a lower rate of biosynthesis and/or cellular transport for GPI-anchored NEP compared with its transmembrane counterpart. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:8166636

  13. A Nup133-dependent NPC-anchored network tethers centrosomes to the nuclear envelope in prophase

    PubMed Central

    Bolhy, Stéphanie; Bouhlel, Imène; Dultz, Elisa; Nayak, Tania; Zuccolo, Michela; Gatti, Xavier; Vallee, Richard; Ellenberg, Jan

    2011-01-01

    Centrosomes are closely associated with the nuclear envelope (NE) throughout the cell cycle and this association is maintained in prophase when they separate to establish the future mitotic spindle. At this stage, the kinetochore constituents CENP-F, NudE, NudEL, dynein, and dynactin accumulate at the NE. We demonstrate here that the N-terminal domain of the nuclear pore complex (NPC) protein Nup133, although largely dispensable for NPC assembly, is required for efficient anchoring of the dynein/dynactin complex to the NE in prophase. Nup133 exerts this function through an interaction network via CENP-F and NudE/EL. We show that this molecular chain is critical for maintaining centrosome association with the NE at mitotic entry and contributes to this process without interfering with the previously described RanBP2–BICD2-dependent pathway of centrosome anchoring. Finally, our study reveals that tethering of centrosomes to the nuclear surface at the G2/M transition contributes, along with other cellular mechanisms, to early stages of bipolar spindle assembly. PMID:21383080

  14. Biomimetic anchors applied to the host-guest antifouling functionalization of titanium substrates.

    PubMed

    Cai, Xiao Yan; Li, Ning Ning; Chen, Jiu Cun; Kang, En-Tang; Xu, Li Qun

    2016-08-01

    A biomimetic strategy was developed for the construction of antifouling titanium oxide (Ti(oxide)) surfaces based on host-guest interactions. Two catecholic derivatives, dopamine 4-(phenylazo)benzamide (AZODopa) and dopamine 1-adamantanecarboxamide (AdaDopa) were synthesized and immobilized onto the Ti(oxide) surfaces. The guest molecules-anchored Ti(oxide) surfaces were further functionalized with zwitterionic heptakis[6-deoxy-6-(N-3-sulfopropyl-N,N-dimethylammonium ethyl sulfanyl)]-β-cyclodextrin (SBCD) and hydrophilic β-CD polymer (CDP). The surface elemental compositions and hydrophobic/hydrophilic properties of the Ti(oxide) surfaces before and after modification were characterized by X-ray photoelectron spectroscopy (XPS) and static water contact angle measurements, respectively. The antifouling properties of the modified Ti(oxide) surfaces were evaluated by the protein adsorption and bacterial adhesion assays. The zwitterionic SBCD- and hydrophilic CDP-functionalized Ti(oxide) surfaces can reduce the adsorption of bovine plasma fibrinogen and adhesion of Escherichia coli, as compared to the pristine and guest molecules-anchored Ti(oxide) surfaces. PMID:27135943

  15. Raft-based interactions of gangliosides with a GPI-anchored receptor.

    PubMed

    Komura, Naoko; Suzuki, Kenichi G N; Ando, Hiromune; Konishi, Miku; Koikeda, Machi; Imamura, Akihiro; Chadda, Rahul; Fujiwara, Takahiro K; Tsuboi, Hisae; Sheng, Ren; Cho, Wonhwa; Furukawa, Koichi; Furukawa, Keiko; Yamauchi, Yoshio; Ishida, Hideharu; Kusumi, Akihiro; Kiso, Makoto

    2016-06-01

    Gangliosides, glycosphingolipids containing one or more sialic acid(s) in the glyco-chain, are involved in various important physiological and pathological processes in the plasma membrane. However, their exact functions are poorly understood, primarily because of the scarcity of suitable fluorescent ganglioside analogs. Here, we developed methods for systematically synthesizing analogs that behave like their native counterparts in regard to partitioning into raft-related membrane domains or preparations. Single-fluorescent-molecule imaging in the live-cell plasma membrane revealed the clear but transient colocalization and codiffusion of fluorescent ganglioside analogs with a fluorescently labeled glycosylphosphatidylinisotol (GPI)-anchored protein, human CD59, with lifetimes of 12 ms for CD59 monomers, 40 ms for CD59's transient homodimer rafts in quiescent cells, and 48 ms for engaged-CD59-cluster rafts, in cholesterol- and GPI-anchoring-dependent manners. The ganglioside molecules were always mobile in quiescent cells. These results show that gangliosides continually and dynamically exchange between raft domains and the bulk domain, indicating that raft domains are dynamic entities. PMID:27043189

  16. Cell wall anchor structure of BcpA pili in Bacillus anthracis.

    PubMed

    Budzik, Jonathan M; Oh, So-Young; Schneewind, Olaf

    2008-12-26

    Assembly of pili in Gram-positive bacteria and their attachment to the cell wall envelope are mediated by sortases. In Bacillus cereus and its close relative Bacillus anthracis, the major pilin protein BcpA is cleaved between the threonine and the glycine of its C-terminal LPXTG motif sorting signal by the pilin-specific sortase D. The resulting acyl enzyme intermediate is relieved by the nucleophilic attack of the side-chain amino group of lysine within the YPKN motif of another BcpA subunit. Cell wall anchoring of assembled BcpA pili requires sortase A, which also cleaves the LPXTG sorting signal of BcpA between its threonine and glycine residues. We show here that sortases A and D require only the C-terminal sorting signal of BcpA for substrate cleavage. Unlike sortase D, which accepts the YPKN motif as a nucleophile, sortase A forms an amide bond between the BcpA C-terminal carboxyl group of threonine and the side-chain amino group of diaminopimelic acid within the cell wall peptidoglycan of bacilli. These results represent the first demonstration of a cell wall anchor structure for pili, which are deposited by sortase A into the envelope of many different microbes. PMID:18940793

  17. Multivalent anchored and crosslinked hyperbranched polyglycerol monolayers as antifouling coating for titanium oxide surfaces.

    PubMed

    Wei, Qiang; Krysiak, Stefanie; Achazi, Katharina; Becherer, Tobias; Noeske, Paul-Ludwig Michael; Paulus, Florian; Liebe, Hendrik; Grunwald, Ingo; Dernedde, Jens; Hartwig, Andreas; Hugel, Thorsten; Haag, Rainer

    2014-10-01

    A set of new catecholic monolayer coatings was developed to improve the antifouling performance of TiO2 surfaces. To solve the problem of the weak charge-transfer interaction between a single catechol anchor and TiO2, multiple catechol groups were combined with hyperbranched polyglycerol (hPG) which is a distinct dendritic scaffold that exposes its multivalent anchor groups on the surface. Thus, multivalent catecholic hPGs can be easily prepared for surface modification. The immobilization of the compounds was monitored by quartz crystal microbalance with dissipation monitoring. Surface properties of the coatings were analyzed by water contact angle, X-ray photoelectron spectroscopy, and atomic force microscopy. The antifouling ability and stability were investigated by protein adsorption and cell adhesion. By increasing the number of catechol groups on the hPG scaffold, the stability and surface coverage could be significantly enhanced. Moreover, the inner-layer crosslinking of the coatings by grafting and initiating vinyl groups clearly improved their long-term stability. As a result, hPG with a catecholic functional degree of 10% (hPG-Cat10) and hPG with both catecholic and vinylic functional degree of 5% (hPG-Cat5-V5) were identified as the best catecholic hPGs to prepare bioinert and stable monolayer coatings on TiO2. PMID:25189471

  18. Inhibition of Bak Activation by VDAC2 Is Dependent on the Bak Transmembrane Anchor*

    PubMed Central

    Lazarou, Michael; Stojanovski, Diana; Frazier, Ann E.; Kotevski, Aneta; Dewson, Grant; Craigen, William J.; Kluck, Ruth M.; Vaux, David L.; Ryan, Michael T.

    2010-01-01

    Bax and Bak are pro-apoptotic factors that are required for cell death by the mitochondrial or intrinsic pathway. Bax is found in an inactive state in the cytosol and upon activation is targeted to the mitochondrial outer membrane where it releases cytochrome c and other factors that cause caspase activation. Although Bak functions in the same way as Bax, it is constitutively localized to the mitochondrial outer membrane. In the membrane, Bak activation is inhibited by the voltage-dependent anion channel isoform 2 (VDAC2) by an unknown mechanism. Using blue native gel electrophoresis, we show that in healthy cells endogenous inactive Bak exists in a 400-kDa complex that is dependent on the presence of VDAC2. Activation of Bak is concomitant with its release from the 400-kDa complex and the formation of lower molecular weight species. Furthermore, substitution of the Bak transmembrane anchor with that of the mitochondrial outer membrane tail-anchored protein hFis1 prevents association of Bak with the VDAC2 complex and increases the sensitivity of cells to an apoptotic stimulus. Our results suggest that VDAC2 interacts with the hydrophobic tail of Bak to sequester it in an inactive state in the mitochondrial outer membrane, thereby raising the stimulation threshold necessary for permeabilization of the mitochondrial outer membrane and cell death. PMID:20851889

  19. Inhibition of Bak activation by VDAC2 is dependent on the Bak transmembrane anchor.

    PubMed

    Lazarou, Michael; Stojanovski, Diana; Frazier, Ann E; Kotevski, Aneta; Dewson, Grant; Craigen, William J; Kluck, Ruth M; Vaux, David L; Ryan, Michael T

    2010-11-19

    Bax and Bak are pro-apoptotic factors that are required for cell death by the mitochondrial or intrinsic pathway. Bax is found in an inactive state in the cytosol and upon activation is targeted to the mitochondrial outer membrane where it releases cytochrome c and other factors that cause caspase activation. Although Bak functions in the same way as Bax, it is constitutively localized to the mitochondrial outer membrane. In the membrane, Bak activation is inhibited by the voltage-dependent anion channel isoform 2 (VDAC2) by an unknown mechanism. Using blue native gel electrophoresis, we show that in healthy cells endogenous inactive Bak exists in a 400-kDa complex that is dependent on the presence of VDAC2. Activation of Bak is concomitant with its release from the 400-kDa complex and the formation of lower molecular weight species. Furthermore, substitution of the Bak transmembrane anchor with that of the mitochondrial outer membrane tail-anchored protein hFis1 prevents association of Bak with the VDAC2 complex and increases the sensitivity of cells to an apoptotic stimulus. Our results suggest that VDAC2 interacts with the hydrophobic tail of Bak to sequester it in an inactive state in the mitochondrial outer membrane, thereby raising the stimulation threshold necessary for permeabilization of the mitochondrial outer membrane and cell death. PMID:20851889

  20. A protein targeting signal that functions in polarized epithelial cells in vivo.

    PubMed Central

    Ali, S; Hall, J; Hazlewood, G P; Hirst, B H; Gilbert, H J

    1996-01-01

    Eukaryotic membrane-associated polypeptides often contain a glycosylphosphatidylinositol (GPI) anchor that signals the attachment of GPI lipids to these proteins. The GPI anchor can function as a basolateral or apical targeting signal in mammalian cells cultured in vitro, although the function of the GPI anchor in vivo remains to be elucidated. In this study we have evaluated the effect of fusing a GPI anchor sequence to a prokaryotic reporter protein on the cellular location of the polypeptide in polarized epithelial cells of transgenic mice. The bacterial enzyme, when fused to a eukaryotic signal peptide, was secreted through the basolateral membrane of small-intestinal enterocytes; however, when the enzyme was lined to the GPI anchor sequence the polypeptide was redirected to the apical surface of the epithelial cells. These data provide the first direct evidence that the GPI anchor functions as an apical membrane protein sorting signal in polarized epithelial cells in vivo. PMID:8645168

  1. Corrosion of rock anchors in US coal mines

    NASA Astrophysics Data System (ADS)

    Bylapudi, Gopi

    The mining industry is a major consumer of rock bolts in the United States. Due to the high humidity in the underground mining environment, the rock bolts corrode and loose their load bearing capacity which in turn reduces the life expectancy of the ground support and, thus, creates operational difficulties and number of safety concerns[1]. Research on rock anchor corrosion has not been adequately extensive in the past and the effects of several factors in the mine atmosphere and waters are not clearly understood. One of the probable reasons for this lack of research may be attributed to the time required for gathering meaningful data that makes the study of corrosion quite challenging. In this particular work underground water samples from different mines in the Illinois coal basin were collected and the major chemical content was analyzed and used for the laboratory testing. The corrosion performance of the different commercial rock anchors was investigated by techniques such as laboratory immersion tests in five different corrosion chambers, and potentiodynamic polarization tests in simulated ground waters based on the Illinois coal basin. The experiments were conducted with simulate underground mining conditions (corrosive). The tensile strengths were measured for the selected rock anchors taken every 3 months from the salt spray corrosion chambers maintained at different pH values and temperatures. The corrosion potential (Ecorr ), corrosion current (Icorr) and the corresponding corrosion rates (CR) of the selected commercial rock bolts: #5, #6, #6 epoxy coated and #7 forged head rebar steels, #6 and #7 threaded head rebar steels were measured at the solution pH values of 5 and 8 at room temperature. The open circuit potential (OCP) values of the different rock anchors were recorded in 3 selected underground coal mines (A, B & C) in the Illinois coal basin and the data compared with the laboratory electrochemical tests for analyzing the life of the rock

  2. Lipid Bilayers Covalently Anchored to Carbon Nanotubes

    PubMed Central

    Dayani, Yasaman; Malmstadt, Noah

    2012-01-01

    The unique physical and electrical properties of carbon nanotubes make them an exciting material for applications in various fields such as bioelectronics and biosensing. Due to the poor water solubility of carbon nanotubes, functionalization for such applications has been a challenge. Of particular need are functionalization methods for integrating carbon nanotubes with biomolecules and constructing novel hybrid nanostructures for bionanoelectronic applications. We present a novel method for the fabrication of dispersible, biocompatible carbon nanotube-based materials. Multi-walled carbon nanotubes (MWCNTs) are covalently modified with primary amine-bearing phospholipids in a carbodiimide-activated reaction. These modified carbon nanotubes have good dispersibility in nonpolar solvents. Fourier transform infrared (FTIR) spectroscopy shows peaks attributable to the formation of amide bonds between lipids and the nanotube surface. Simple sonication of lipid-modified nanotubes with other lipid molecules leads to the formation of a uniform lipid bilayer coating the nanotubes. These bilayer-coated nanotubes are highly dispersible and stable in aqueous solution. Confocal fluorescence microscopy shows labeled lipids on the surface of bilayer-modified nanotubes. Transmission electron microscopy (TEM) shows the morphology of dispersed bilayer-coated MWCNTs. Fluorescence quenching of lipid-coated MWCNTs confirms the bilayer configuration of the lipids on the nanotube surface and fluorescence anisotropy measurements show that the bilayer is fluid above the gel-to-liquid transition temperature. The membrane protein α-hemolysin spontaneously inserts into the MWCNT-supported bilayer, confirming the biomimetic membrane structure. These biomimetic nanostructures are a promising platform for the integration of carbon nanotube-based materials with biomolecules. PMID:22568448

  3. Equilibrium Configuration in a Nematic Liquid Crystal Droplet with Homeotropic Anchoring of Finite Strength

    NASA Astrophysics Data System (ADS)

    Kanke, Masaki; Sasaki, Kazuo

    2013-09-01

    Equilibrium configuration of order parameter in a nematic liquid crystal droplet with homeotropic anchoring of finite strength at the surface is studied numerically by using the Landau--de Gennes approach. It is found that a hedgehog-like configuration with a disclination loop of a small radius is stable for strong anchoring while an axial configuration without defect is stable for weak anchoring. A first-order phase transition from one configuration to the other occurs as the strength of the anchoring is varied. The critical anchoring strength turns out to increase almost linearly with the inverse of the droplet radius.

  4. Simulation study of liquid crystal anchoring at a polymer surface

    NASA Astrophysics Data System (ADS)

    Hamaneh, Mehdi

    2005-03-01

    The process of rubbing a polymer substrate to induce planar anchoring has two effects. It aligns the polymer chains and also creates grooves in the surface. We have investigated which one of these effects is more important by conducting a series of simulations of molecules of 5CB in contact with a poly(vinyl alcohol) surface. The polymer surface was constructed from a set of parallel straight chains. It was then distorted to mimic the effect of grooves in a direction perpendicular to the chain direction, thus causing two opposing anchoring effects. It was found that the 5CB molecules ordered preferentially along the chain direction when the depth of the grooves was less than 20 percent of the distance between grooves. For grooves whose walls were more steeply pitched, the nematic ordering aligned with the grooves.

  5. Arthroscopic Suture Anchor Tenodesis: Loop-Suture Technique

    PubMed Central

    Shon, Min Soo; Koh, Kyoung Hwan; Lim, Tae Kang; Lee, Seung Won; Park, Young Eun; Yoo, Jae Chul

    2013-01-01

    With advancements in arthroscopic surgery, arthroscopic biceps tenodesis with suture anchor recently has been reported to be a reasonable option for the treatment of biceps pathologies, especially for those that are symptomatic or accompanied by a rotator cuff tear. We introduce our technique of arthroscopic biceps tenodesis with suture anchor that we call the loop-suture technique, which is constructed with 1 loop strand and another sutured strand. This technique can help to improve biceps grip and simultaneously minimize longitudinal splitting of the tendon. In addition, it is relatively simple and can be performed with the use of conventional devices and arthroscopic portals used for rotator cuff repair, without the formation of additional portals or a separate incision for the tenodesis. PMID:23875133

  6. Effective anchoring energy in dipolar organic film on metals surfaces

    NASA Astrophysics Data System (ADS)

    Iwamoto, Mitsumasa; Zakharov, A. V.

    2007-04-01

    The influence of electron injection from the metal electrode into organic liquid crystal dipolar film on the effective anchoring energy (EAE) of the polar organic film is discussed from the energy point of view. It is shown that the accounting for the injected carrier in organic film results in a polynomial function for the EAE expanded up to the fourth order in cos θs, where θs is the polar angle of the director nˆ at the film/metal interface. It is also shown that in a certain range of the location of centroid of the injected carrier z¯ the destabilizing surface polarization mechanism may lead to destruction of the linear anchoring strength coefficient w1. The strong influence of z¯ on the quadratic term w2 also has been demonstrated.

  7. Graphene Nanoribbons Anchored to a SiC Substrate

    NASA Astrophysics Data System (ADS)

    Woods, Lilia; Le, Nam

    Due to their exceptional fundamental characteristics graphene nanoribbons play a major role in the development of future nano-technological applications. The high chemical reactivity of the graphene nanoribbon edges can be utilized to create modified materials. Using first principles simulations we explore this possibility to construct patterned systems composed of anchored ribbons of zigzag edges covalently bonded to a SiC substrate. The hybrid edge states are found to possess interesting electronic and magnetic properties, which alter the overall behavior of the entire system as compared to the behavior of the individual components. It is found that the van der Waals interactions are important for the overall stability and structure of the anchored ribbons. Also, spin-polarization effects play a profound role in the electronic structure and associated density of states. The hybrid graphene/SiC zigzag edges are analyzed in terms of their transport characteristics as well.

  8. Anchoring transition in confined discotic columnar liquid crystal films

    NASA Astrophysics Data System (ADS)

    Brunet, Thomas; Thiebaut, Olivier; Charlet, Émilie; Bock, Harald; Kelber, Julien; Grelet, Éric

    2011-01-01

    We report the achievement of ultrathin films (down to 25 nm thick) of thermotropic columnar liquid crystals in homeotropic alignment (columns normal to the interface) confined between a glass slide and a thin metallic electrode (about 150 nm thick). The face-on orientation of the discotic compound is obtained by anchoring transition of a columnar liquid crystalline phase from a degenerate planar orientation to the homeotropic alignment without any phase transition to the isotropic liquid phase. The kinetic dependence on temperature of such anchoring transition is investigated revealing various diffusive growth regimes of the homeotropic domains. Finally, confining effects are also considered by varying the thickness of the columnar liquid crystal film to reach the typical value required in organic solar cells thus demonstrating the reliability of such alignment process in a photovoltaic context.

  9. Graphene nanoribbons anchored to SiC substrates.

    PubMed

    Le, Nam B; Woods, Lilia M

    2016-09-14

    Graphene nanoribbons are quasi-one-dimensional planar graphene allotropes with diverse properties dependent on their width and types of edges. Graphene nanoribbons anchored to substrates is a hybrid system, which offers novel opportunities for property modifications as well as experimental control. Here we present electronic structure calculations of zigzag graphene nanoribbons chemically attached via the edges to the Si or C terminated surfaces of a SiC substrate. The results show that the edge characteristics are rather robust and the properties are essentially determined by the individual nanoribbon. While the localized spin polarization of the graphene nanoribbon edge atoms is not significantly affected by the substrate, secondary energy gaps in the highest conduction and lowest valence region may emerge in the anchored structures. The van der Waals interaction together with the electrostatic interactions due to the polarity of the surface bonds are found to be important for the structure parameters and energy stability. PMID:27392014

  10. Graphene nanoribbons anchored to SiC substrates

    NASA Astrophysics Data System (ADS)

    Le, Nam B.; Woods, Lilia M.

    2016-09-01

    Graphene nanoribbons are quasi-one-dimensional planar graphene allotropes with diverse properties dependent on their width and types of edges. Graphene nanoribbons anchored to substrates is a hybrid system, which offers novel opportunities for property modifications as well as experimental control. Here we present electronic structure calculations of zigzag graphene nanoribbons chemically attached via the edges to the Si or C terminated surfaces of a SiC substrate. The results show that the edge characteristics are rather robust and the properties are essentially determined by the individual nanoribbon. While the localized spin polarization of the graphene nanoribbon edge atoms is not significantly affected by the substrate, secondary energy gaps in the highest conduction and lowest valence region may emerge in the anchored structures. The van der Waals interaction together with the electrostatic interactions due to the polarity of the surface bonds are found to be important for the structure parameters and energy stability.

  11. Arthroscopic suture anchor tenodesis: loop-suture technique.

    PubMed

    Shon, Min Soo; Koh, Kyoung Hwan; Lim, Tae Kang; Lee, Seung Won; Park, Young Eun; Yoo, Jae Chul

    2013-05-01

    With advancements in arthroscopic surgery, arthroscopic biceps tenodesis with suture anchor recently has been reported to be a reasonable option for the treatment of biceps pathologies, especially for those that are symptomatic or accompanied by a rotator cuff tear. We introduce our technique of arthroscopic biceps tenodesis with suture anchor that we call the loop-suture technique, which is constructed with 1 loop strand and another sutured strand. This technique can help to improve biceps grip and simultaneously minimize longitudinal splitting of the tendon. In addition, it is relatively simple and can be performed with the use of conventional devices and arthroscopic portals used for rotator cuff repair, without the formation of additional portals or a separate incision for the tenodesis. PMID:23875133

  12. Recycling Suture Limbs from Knotless Suture Anchors for Arthroscopic Shoulder Stabilization

    PubMed Central

    Johnson, Timothy S.; DiPompeo, Christine M.; Ismaeli, Zahra C.; Porter, Polly A.; Nicholson, Shannon L.; Johnson, David C.

    2014-01-01

    Recurrent shoulder instability often leads to labral abnormality that requires surgical intervention that may require fixation with suture anchors. The proposed surgical technique allows the surgeon to achieve 2 points of fixation around the labrum and/or capsule with a single suture secured to the glenoid with a knotless anchor. Instead of cutting and discarding the residual suture limbs after anchor insertion, this technique uses the residual suture limbs of the knotless anchor for a second suture pass. This technique (1) creates a more cost- and time-efficient surgical procedure than using multiple single-loaded anchors or double-loaded anchors, (2) decreases the known risk of glenoid fracture from the stress riser at the implant tips of multi-anchor repairs by reducing the number of anchors required for stabilization, (3) decreases the surgical time compared with the use of double-loaded anchors through simpler suture management and less knot tying, (4) allows for the secure reapproximation of the labrum to the glenoid while offering a convenient option for capsulorrhaphy without the need to insert another anchor, and (5) yields more points of soft-tissue fixation with fewer anchors drilled into the glenoid. PMID:25126504

  13. Taking the high (or low) road: a quantifier priming perspective on basic anchoring effects.

    PubMed

    Sleeth-Keppler, David

    2013-01-01

    Current explanations of basic anchoring effects, defined as the influence of an arbitrary number standard on an uncertain judgment, confound numerical values with vague quantifiers. I show that the consideration of numerical anchors may bias subsequent judgments primarily through the priming of quantifiers, rather than the numbers themselves. Study 1 varied the target of a numerical comparison judgment in a between--participants design, while holding the numerical anchor value constant. This design yielded an anchoring effect consistent with a quantifier priming hypothesis. Study 2 included a direct manipulation of vague quantifiers in the traditional anchoring paradigm. Finally, Study 3 examined the notion that specific associations between quantifiers, reflecting values on separate judgmental dimensions (i.e., the price and height of a target) can affect the direction of anchoring effects. Discussion focuses on the nature of vague quantifier priming in numerically anchored judgments. PMID:23951950

  14. Increased Diversity of the HLA-B40 Ligandome by the Presentation of Peptides Phosphorylated at Their Main Anchor Residue*

    PubMed Central

    Marcilla, Miguel; Alpízar, Adán; Lombardía, Manuel; Ramos-Fernandez, Antonio; Ramos, Manuel; Albar, Juan Pablo

    2014-01-01

    Human leukocyte antigen (HLA) class I molecules bind peptides derived from the intracellular degradation of endogenous proteins and present them to cytotoxic T lymphocytes, allowing the immune system to detect transformed or virally infected cells. It is known that HLA class I–associated peptides may harbor posttranslational modifications. In particular, phosphorylated ligands have raised much interest as potential targets for cancer immunotherapy. By combining affinity purification with high-resolution mass spectrometry, we identified more than 2000 unique ligands bound to HLA-B40. Sequence analysis revealed two major anchor motifs: aspartic or glutamic acid at peptide position 2 (P2) and methionine, phenylalanine, or aliphatic residues at the C terminus. The use of immobilized metal ion and TiO2 affinity chromatography allowed the characterization of 85 phosphorylated ligands. We further confirmed every sequence belonging to this subset by comparing its experimental MS2 spectrum with that obtained upon fragmentation of the corresponding synthetic peptide. Remarkably, three phospholigands lacked a canonical anchor residue at P2, containing phosphoserine instead. Binding assays showed that these peptides bound to HLA-B40 with high affinity. Together, our data demonstrate that the peptidome of a given HLA allotype can be broadened by the presentation of peptides with posttranslational modifications at major anchor positions. We suggest that ligands with phosphorylated residues at P2 might be optimal targets for T-cell-based cancer immunotherapy. PMID:24366607

  15. N-acetylglucosamine deacetylases modulate the anchoring of the gamma-glutamyl capsule to the cell wall of Bacillus anthracis.

    PubMed

    Candela, Thomas; Balomenou, Stavroula; Aucher, Willy; Bouriotis, Vassilis; Simore, Jean-Pierre; Fouet, Agnes; Boneca, Ivo G

    2014-06-01

    Bacillus anthracis has a complex cell wall structure composed of a peptidoglycan (PG) layer to which major structures are anchored such as a neutral polysaccharide, an S-layer, and a poly-γ-D-glutamate (PDGA) capsule. Many of these structures have central roles in the biology of B. anthracis, particularly, in virulence. However, little attention has been devoted to structurally study the PG and how it is modified in the presence of these secondary cell wall components. We present here the fine structure of the PG of the encapsulated RPG1 strain harboring both pXO1 and pXO2 virulence plasmids. We show that B. anthracis has a high degree of cross-linking and its GlcNAc residues are highly modified by N-deacetylation. The PG composition is not dependent on the presence of either LPXTG proteins or the capsule. Using NMR analysis of the PG-PDGA complex, we provide evidence for the anchoring of the PDGA to the glucosamine residues. We show that anchoring of the PDGA capsule is impaired in two PG N-deacetylase mutants, Ba1961 and Ba3679. Thus, these multiple N-deactylase activities would constitute excellent drug targets in B. anthracis by simultaneously affecting its resistance to lysozyme and to phagocytosis impairing B. anthracis survival in the host. PMID:24833281

  16. Centriolar satellite– and hMsd1/SSX2IP-dependent microtubule anchoring is critical for centriole assembly

    PubMed Central

    Hori, Akiko; Peddie, Christopher J.; Collinson, Lucy M.; Toda, Takashi

    2015-01-01

    Centriolar satellites are numerous electron-dense granules dispersed around the centrosome. Mutations in their components are linked to various human diseases, but their molecular roles remain elusive. In particular, the significance of spatial communication between centriolar satellites and the centrosome is unknown. hMsd1/SSX2IP localizes to both the centrosome and centriolar satellites and is required for tethering microtubules to the centrosome. Here we show that hMsd1/SSX2IP-mediated microtubule anchoring is essential for proper centriole assembly and duplication. On hMsd1/SSX2IP knockdown, the centriolar satellites become stuck at the microtubule minus end near the centrosome. Intriguingly, these satellites contain many proteins that normally localize to the centrosome. Of importance, microtubule structures, albeit not being anchored properly, are still required for the emergence of abnormal satellites, as complete microtubule depolymerization results in the disappearance of these aggregates from the vicinity of the centrosome. We highlighted, using superresolution and electron microscopy, that under these conditions, centriole structures are faulty. Remarkably, these cells are insensitive to Plk4 overproduction–induced ectopic centriole formation, yet they accelerate centrosome reduplication upon hydroxyurea arrest. Finally, the appearance of satellite aggregates is cancer cell specific. Together our findings provide novel insights into the mechanism of centriole assembly and microtubule anchoring. PMID:25833712

  17. Centriolar satellite- and hMsd1/SSX2IP-dependent microtubule anchoring is critical for centriole assembly.

    PubMed

    Hori, Akiko; Peddie, Christopher J; Collinson, Lucy M; Toda, Takashi

    2015-06-01

    Centriolar satellites are numerous electron-dense granules dispersed around the centrosome. Mutations in their components are linked to various human diseases, but their molecular roles remain elusive. In particular, the significance of spatial communication between centriolar satellites and the centrosome is unknown. hMsd1/SSX2IP localizes to both the centrosome and centriolar satellites and is required for tethering microtubules to the centrosome. Here we show that hMsd1/SSX2IP-mediated microtubule anchoring is essential for proper centriole assembly and duplication. On hMsd1/SSX2IP knockdown, the centriolar satellites become stuck at the microtubule minus end near the centrosome. Intriguingly, these satellites contain many proteins that normally localize to the centrosome. Of importance, microtubule structures, albeit not being anchored properly, are still required for the emergence of abnormal satellites, as complete microtubule depolymerization results in the disappearance of these aggregates from the vicinity of the centrosome. We highlighted, using superresolution and electron microscopy, that under these conditions, centriole structures are faulty. Remarkably, these cells are insensitive to Plk4 overproduction-induced ectopic centriole formation, yet they accelerate centrosome reduplication upon hydroxyurea arrest. Finally, the appearance of satellite aggregates is cancer cell specific. Together our findings provide novel insights into the mechanism of centriole assembly and microtubule anchoring. PMID:25833712

  18. N-Acetylglucosamine Deacetylases Modulate the Anchoring of the Gamma-Glutamyl Capsule to the Cell Wall of Bacillus anthracis

    PubMed Central

    Candela, Thomas; Balomenou, Stavroula; Aucher, Willy; Bouriotis, Vassilis; Simore, Jean-Pierre; Fouet, Agnes

    2014-01-01

    Bacillus anthracis has a complex cell wall structure composed of a peptidoglycan (PG) layer to which major structures are anchored such as a neutral polysaccharide, an S-layer, and a poly-γ-D-glutamate (PDGA) capsule. Many of these structures have central roles in the biology of B. anthracis, particularly, in virulence. However, little attention has been devoted to structurally study the PG and how it is modified in the presence of these secondary cell wall components. We present here the fine structure of the PG of the encapsulated RPG1 strain harboring both pXO1 and pXO2 virulence plasmids. We show that B. anthracis has a high degree of cross-linking and its GlcNAc residues are highly modified by N-deacetylation. The PG composition is not dependent on the presence of either LPXTG proteins or the capsule. Using NMR analysis of the PG-PDGA complex, we provide evidence for the anchoring of the PDGA to the glucosamine residues. We show that anchoring of the PDGA capsule is impaired in two PG N-deacetylase mutants, Ba1961 and Ba3679. Thus, these multiple N-deactylase activities would constitute excellent drug targets in B. anthracis by simultaneously affecting its resistance to lysozyme and to phagocytosis impairing B. anthracis survival in the host. PMID:24833281

  19. Genome Assembly Anchored QTL Map of Bovine Chromosome 14

    PubMed Central

    Wibowo, Tito A.; Gaskins, Charles T.; Newberry, Ruth C.; Thorgaard, Gary H.; Michal, Jennifer J.; Jiang, Zhihua

    2008-01-01

    Bovine chromosome 14 (BTA14) has been widely explored for quantitative trait loci (QTL) and genes related to economically important traits in both dairy and beef cattle. We reviewed more than 40 investigations and anchored 126 QTL to the current genome assembly (Btau 4_0). Using this anchored QTL map, we observed that, in dairy cattle, the region spanning 0 – 10 Mb on BTA14 has the highest density QTL map with a total of 56 QTL, mainly for milk production traits. It is very likely that both somatic cell score (SCS) and clinical mastitis share some common QTL in two regions: 61.48 Mb - 73.84 Mb and 7.86 Mb – 39.55 Mb, respectively. As well, both ovulation rate and twinning rate might share a common QTL region from 34.16 Mb to 65.38 Mb. However, there are no common QTL locations in three pregnancy related phenotypes: non-return rate, pregnancy rate and daughter pregnancy rate. In beef cattle, the majority of QTL are located in a broad region of 15 Mb – 45 Mb on the chromosome. Functional genes, such as CRH, CYP11B1, DGAT1, FABP4 and TG, as potential candidates for some of these QTL, were also reviewed. Therefore, our review provides a standardized QTL map anchored within the current genome assembly, which would enhance the process of selecting positional and physiological candidate genes for many important traits in cattle. PMID:19043607

  20. A MYRISTOYL/PHOSPHOSERINE SWITCH CONTROLS cAMP-DEPENDENT PROTEIN KINASE ASSOCIATION TO MEMBRANES

    PubMed Central

    Gaffarogullari, Ece C.; Masterson, Larry R.; Metcalfe, Emily E.; Traaseth, Nathaniel J.; Balatri, Erica; Musa, Musa M.; Mullen, Daniel; Distefano, Mark D.; Veglia, Gianluigi

    2012-01-01

    The cAMP-dependent protein kinase (PKA) mediates a myriad of cellular signaling events and its activity is tightly regulated both in space and time. Among these regulatory mechanisms is N-myristoylation, whose biological role has been elusive. Using a combination of thermodynamics, kinetics, and spectroscopic methods, we analyzed the effects of N-myristoylation and phosphorylation at Ser10 on the interactions of PKA with model membranes. We found that in the absence of lipids, the myristoyl group is tucked into the hydrophobic binding pocket of the enzyme (myr-in state). Upon association with lipid bilayers, the myristoyl group is extruded and inserts into the hydrocarbon region of the lipid bilayer (myr-out state). NMR data indicate that the enzyme undergoes conformational equilibrium between myr-in and myr-out states, which can be shifted either by interaction with membranes and/or phosphorylation at Ser10. Our results provide evidence that the membrane binding motif of myristoylated PKA-C steers the enzyme towards lipids independent of its regulatory subunit or an A-kinase anchoring protein (AKAP), providing an additional mechanism to localize the enzyme near membrane-bound substrates. PMID:21740913

  1. GPI-anchored single chain Fv - an effective way to capture transiently-exposed neutralization epitopes on HIV-1 envelope spike

    PubMed Central

    2010-01-01

    Background Identification of broad neutralization epitopes in HIV-1 envelope spikes is paramount for HIV-1 vaccine development. A few broad neutralization epitopes identified so far are present on the surface of native HIV-1 envelope spikes whose recognition by antibodies does not depend on conformational changes of the envelope spikes. However, HIV-1 envelope spikes also contain transiently-exposed neutralization epitopes, which are more difficult to identify. Results In this study, we constructed single chain Fvs (scFvs) derived from seven human monoclonal antibodies and genetically linked them with or without a glycosyl-phosphatidylinositol (GPI) attachment signal. We show that with a GPI attachment signal the scFvs are targeted to lipid rafts of plasma membranes. In addition, we demonstrate that four of the GPI-anchored scFvs, but not their secreted counterparts, neutralize HIV-1 with various degrees of breadth and potency. Among them, GPI-anchored scFv (X5) exhibits extremely potent and broad neutralization activity against multiple clades of HIV-1 strains tested. Moreover, we show that GPI-anchored scFv (4E10) also exhibited more potent neutralization activity than its secretory counterpart. Finally, we demonstrate that expression of GPI-anchored scFv (X5) in the lipid raft of plasma membrane of human CD4+ T cells confers long-term resistance to HIV-1 infection, HIV-1 envelope-mediated cell-cell fusion, and the infection of HIV-1 captured and transferred by human DCs. Conclusions Thus GPI-anchored scFv could be used as a general and effective way to identify antibodies that react with transiently-exposed neutralization epitopes in envelope proteins of HIV-1 and other enveloped viruses. The GPI-anchored scFv (X5), because of its breadth and potency, should have a great potential to be developed into anti-viral agent for HIV-1 prevention and therapy. PMID:20923574

  2. A Novel Glycosylphosphatidylinositol-Anchored Glycoside Hydrolase from Ustilago esculenta Functions in β-1,3-Glucan Degradation

    PubMed Central

    Nakajima, Masahiro; Yamashita, Tetsuro; Takahashi, Machiko; Nakano, Yuki

    2012-01-01

    A glycoside hydrolase responsible for laminarin degradation was partially purified to homogeneity from a Ustilago esculenta culture filtrate by weak-cation-exchange, strong-cation-exchange, and size-exclusion chromatography. Three proteins in enzymatically active fractions were digested with chymotrypsin followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis, resulting in the identification of three peptide sequences that shared significant similarity to a putative β-1,3-glucanase, a member of glucoside hydrolase family 16 (GH16) from Sporisorium reilianum SRZ2. A gene encoding a laminarin-degrading enzyme from U. esculenta, lam16A, was isolated by PCR using degenerate primers designed based on the S. reilianum SRZ2 β-1,3-glucanase gene. Lam16A possesses a GH16 catalytic domain with an N-terminal signal peptide and a C-terminal glycosylphosphatidylinositol (GPI) anchor peptide. Recombinant Lam16A fused to an N-terminal FLAG peptide (Lam16A-FLAG) overexpressed in Aspergillus oryzae exhibited hydrolytic activity toward β-1,3-glucan specifically and was localized both in the extracellular and in the membrane fractions but not in the cell wall fraction. Lam16A without a GPI anchor signal peptide was secreted extracellularly and was not detected in the membrane fraction. Membrane-anchored Lam16A-FLAG was released completely by treatment with phosphatidylinositol-specific phospholipase C. These results suggest that Lam16A is anchored in the plasma membrane in order to modify β-1,3-glucan associated with the inner cell wall and that Lam16A is also used for the catabolism of β-1,3-glucan after its release in the extracellular medium. PMID:22685137

  3. Membrane anchoring of diacylglycerol-lactones substituted with rigid hydrophobic acyl domains correlates with biological activities

    PubMed Central

    Raifman, Or; Kolusheva, Sofiya; Comin, Maria J.; Kedei, Noemi; Lewin, Nancy E.; Blumberg, Peter M.; Marquez, Victor E.; Jelinek, Raz

    2009-01-01

    Summary Synthetic diacylglycerol lactones (DAG-lactones) are effective modulators of critical cellular signaling pathways, downstream of the lipophilic second messenger diacylglycerol, that activate a host of protein kinase C (PKC) isozymes as well as other non-kinase proteins that share with PKC similar C1 membrane-targeting domains. A fundamental determinant of the biological activity of these amphiphilic molecules is the nature of their interactions with cellular membranes. This study characterizes the membrane interactions and bilayer anchoring of a series of DAG-lactones in which the hydrophobic moiety is a “molecular rod”, namely a rigid 4-[2-(R-phenyl)ethynyl]benzoate moiety in the acyl position. Application of assays employing chromatic biomimetic vesicles and biophysical techniques reveals that the mode of membrane anchoring of the DAG-lactone derivatives was markedly affected by the presence of the hydrophobic diphenyl rod and by the size of the functional unit displayed at the terminus of the rod. Two primary mechanisms of interaction were observed: surface binding of the DAG-lactones at the lipid/water interface and deep insertion of the ligands into the alkyl core of the lipid bilayer. These membrane-insertion properties could explain the different patterns of PKC translocation from cytosol to membranes induced by the molecular-rod DAG-lactones. This investigation emphasizes that the side-residues of DAG-lactones, rather than simply conferring hydrophobicity, profoundly influence membrane interactions and in that fashion may further contribute to the diversity of biological actions of these synthetic biomimetic ligands. PMID:19961537

  4. The Effects of Different Types of Anchor Tests on Observed Score Equating. Research Report. ETS RR-09-41

    ERIC Educational Resources Information Center

    Liu, Jinghua; Sinharay, Sandip; Holland, Paul W.; Feigenbaum, Miriam; Curley, Edward

    2009-01-01

    This study explores the use of a different type of anchor, a "midi anchor", that has a smaller spread of item difficulties than the tests to be equated, and then contrasts its use with the use of a "mini anchor". The impact of different anchors on observed score equating were evaluated and compared with respect to systematic error (bias), random…

  5. Covalent binding and anchoring of cytochrome c to mitochondrial mimetic membranes promoted by cholesterol carboxyaldehyde.

    PubMed

    Genaro-Mattos, Thiago C; Appolinário, Patricia P; Mugnol, Katia C U; Bloch, Carlos; Nantes, Iseli L; Di Mascio, Paolo; Miyamoto, Sayuri

    2013-10-21

    Mitochondrial cholesterol has been reported to be increased under specific pathological conditions associated with enhanced oxidative stress parameters. In this scenario, cholesterol oxidation would be increased, leading to the production of reactive aldehydes, including cholesterol carboxyaldehyde (ChAld). By using SDS micelles as a mitochondrial mimetic model, we have demonstrated that ChAld covalently modifies cytochrome c (cytc), a protein known to participate in electron transport and apoptosis signaling. This mimetic model induces changes in cytc structure in the same way as mitochondrial membranes do. Tryptic digestion of the cytc-ChAld adduct followed by MALDI-TOF/TOF analyses revealed that modifications occur at Lys residues (K22) localized at cytc site L, a site involved in protein-protein and protein-membrane interactions. Interestingly, ChAld ligation prevented cytc detachment from liposomes even under high ionic strength conditions. Overall, it can be concluded that ChAld ligation to Lys residues at site L creates a hydrophobic tail at cytc, which promotes cytc anchoring to the membrane. Although not investigated in detail in this study, cytc adduction to cholesterol derived aldehydes could have implications in cytc release from mitochondria under apoptotic stimuli. PMID:24059586

  6. Liberation of GPI-anchored prion from phospholipids accelerates amyloidogenic conversion.

    PubMed

    Lin, Shen-Jie; Yu, Kun-Hua; Wu, Jhih-Ru; Lee, Chin-Fa; Jheng, Cheng-Ping; Chen, Hau-Ren; Lee, Cheng-I

    2013-01-01

    Prion diseases or transmissible spongiform encephalopathies are a rare group of fatal neurodegenerative illnesses in humans and animals caused by misfolding of prion protein (PrP). Prion protein is a cell-surface glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed mostly in the central and peripheral nervous system, and this membrane-bound protein can be cleaved from the cell membranes by phosphoinositide phospholipase C. Numerous studies have investigated GPI-free recombinant PrP, but the role of GPI on misfolding of PrP is not well known. In this study, we synthesized a GPI analog that was covalently linking to a PrP S230C mutant, resulting in S230C-GPI. The structural changes in S230C-GPI upon binding to lipid vesicles composed of mixtures of the zwitterionic lipid (POPC) and the anionic lipid (POPG) were analyzed by circular dichroism spectroscopy, and the amyloid aggregation of S230C-GPI in the liberation from phospholipid vesicles was monitored by proteinase K-digestion assay. Our results indicate that S230C-GPI in the liberation of lipid vesicles has high tendency to misfold into amyloid fibrils, while the membrane-bound S230C-GPI proteins are highly stable and rarely convert into amyloid forms. In addition, the role of cholesterol in S230C-GPI was studied. The effect of GPI, cholesterol and phospholipid vesicles on misfolding of PrP is further discussed. PMID:24005859

  7. The cystic fibrosis transmembrane recruiter the alter ego of CFTR as a multi-kinase anchor

    PubMed Central

    2007-01-01

    This review focuses on a newly discovered interaction between protein kinases involved in cellular energetics, a process that may be disturbed in cystic fibrosis for unknown reasons. I propose a new model where kinase-mediated cellular transmission of energy provides mechanistic insight to a latent role of the cystic fibrosis transmembrane conductance regulator (CFTR). I suggest that CFTR acts as a multi-kinase recruiter to the apical epithelial membrane. My group finds that, in the cytosol, two protein kinases involved in cell energy homeostasis, nucleoside diphosphate kinase (NDPK) and AMP-activated kinase (AMPK), bind one another. Preliminary data suggest that both can also bind CFTR (function unclear). The disrupted role of this CFTR-kinase complex as ‘membrane transmitter to the cell’ is proposed as an alternative paradigm to the conventional ion transport mediated and CFTR/chloride-centric view of cystic fibrosis pathogenesis. Chloride remains important, but instead, chloride-induced control of the phosphohistidine content of one kinase component (NDPK, via a multi-kinase complex that also includes a third kinase, CK2; formerly casein kinase 2). I suggest that this complex provides the necessary near-equilibrium conditions needed for efficient transmission of phosphate energy to proteins controlling cellular energetics. Crucially, a new role for CFTR as a kinase controller is proposed with ionic concentration acting as a signal. The model posits a regulatory control relay for energy sensing involving a cascade of protein kinases bound to CFTR. PMID:17805562

  8. Not all nutrition claims are perceived equal: anchoring effects and moderating mechanisms in food advertising.

    PubMed

    Paek, Hye-Jin; Yoon, Hye Jin; Hove, Thomas

    2011-03-01

    Despite the increased use of health claims in food advertising, few studies have investigated how specific nutrition claims have differential effects depending on how they are presented. In this context, the current study tests the anchoring hypothesis. Anchoring refers to a common human tendency to evaluate information differently depending on the presence or absence of a numerical "anchor" or reference point. Two (pilot and main) experimental studies explore anchoring effects on audience response to food advertising both directly and moderated by cognitive, motivational, and message factors. The pilot study finds that food product ads employing nutrition claims with an anchor rather than without an anchor generate two results: First, participants perceive the product to have lower fat/lower calorie contents (anchoring hypothesis); second, they prefer the messages with an anchor over those without an anchor. The main study reports that when anchoring is successfully evoked, it produces favorable attitudes toward the ad, favorable attitudes toward the brand, and purchase intention-but only when moderated by health orientation, claim believability, and nutrition knowledge. Practical implications are provided with respect to regulatory guidelines and effective communication strategies for promoting low-fat and low-calorie products in food advertising. PMID:21308579

  9. Study of anchoring behavior of nematic fluids at the interface of polymer-dispersed liquid crystals

    NASA Astrophysics Data System (ADS)

    Zhou, Jian

    A liquid crystal (LC) at its boundary surface adopts a preferential alignment, which is referred to as anchoring. The direction of this alignment (i.e., anchoring direction) may be perpendicular, parallel or tilted with respect to the surface. Transitions from one anchoring condition to another may occur when the parameters (e.g., temperature) charactering the surface change, as referred to as anchoring transitions. In the LC-polymer composite systems under our study, the anchoring and temperature-driven anchoring transitions of nematic fluids is very sensitive to the structure of the side chain of poly (alkyl acrylate) matrixes that encapsulate the LC. We have shown that the anchoring transition temperature of these systems can be tuned far below the nematic-to-isotropic transition temperature, by varying either the length, branching structure of the side chains of homopolymers, or the composition of copolymer of two dissimilar monomers. Both sharp and broad anchoring transitions with respect to the temperature range over which a transition occurs were observed. It is postulated that microscopic interactions between the polymer side chains and LC molecules play an important role in determining the anchoring. In particular, the conformation of the polymer side chain is proposed to have important control over the anchoring. Anchoring strength and tilt angle as a function of temperature during the anchoring transitions were also experimentally investigated, which contribute to understanding of the microscopic mechanism for such transitions. Based on the LC-polymer composites with controlled anchoring, a LC display with reverse switching mode and a novel electrically switchable diffraction grating have been demonstrated. The advantages of these devices are ease of manufacturing, low operation voltage, and mechanical stability offered by polymer matrix. Moreover, a detailed study of the director configuration of wall defects found in these composite films was carried

  10. Displaying Lipase B from Candida antarctica in Pichia pastoris Using the Yeast Surface Display Approach: Prospection of a New Anchor and Characterization of the Whole Cell Biocatalyst

    PubMed Central

    Moura, Marcelo Victor Holanda; da Silva, Giulia Pontes; Machado, Antônio Carlos de Oliveira; Torres, Fernando Araripe Gonçalves; Freire, Denise Maria Guimarães; Almeida, Rodrigo Volcan

    2015-01-01

    Yeast Surface Display (YSD) is a strategy to anchor proteins on the yeast cell wall which has been employed to increase enzyme stability thus decreasing production costs. Lipase B from Candida antarctica (LipB) is one of the most studied enzymes in the context of industrial biotechnology. This study aimed to assess the biochemical features of this important biocatalyst when immobilized on the cell surface of the methylotrophic yeast Pichia pastoris using the YSD approach. For that purpose, two anchors were tested. The first (Flo9) was identified after a prospection of the P. pastoris genome being related to the family of flocculins similar to Flo1 but significantly smaller. The second is the Protein with Internal Repeats (Pir1) from P. pastoris. An immunolocalization assay showed that both anchor proteins were able to display the reporter protein EGFP in the yeast outer cell wall. LipB was expressed in P. pastoris fused either to Flo9 (FLOLIPB) or Pir1 (PIRLIPB). Both constructions showed hydrolytic activity towards tributyrin (>100 U/mgdcw and >80 U/mgdcw, respectively), optimal hydrolytic activity around 45°C and pH 7.0, higher thermostability at 45°C and stability in organic solvents when compared to a free lipase. PMID:26510006

  11. A novel role of Rab11 in trafficking GPI-anchored trans-sialidase to the plasma membrane of Trypanosoma cruzi

    PubMed Central

    Niyogi, Sayantanee; Docampo, Roberto

    2015-01-01

    Trypanosoma cruzi, the causative agent of Chagas disease, is a unicellular parasite that possesses a contractile vacuole complex (CVC). This organelle is usually present in free-living protists and is mainly involved in osmoregulation. However, in some organisms, like for example Dictyostelium discoideum, other roles include calcium homeostasis and transference of proteins to the plasma membrane. T. cruzi plasma membrane is very rich in glycosylphosphatidylinositol anchored proteins (GPI-AP) and a very important group of GPI-AP is that of the trans-sialidases. These enzymes catalyze the transfer of sialic acid from host glycoconjugates to mucins present in the surface of the parasite and are important for host cell invasion among other functions. We recently reported that a pathway dependent on the Rab GTPase Rab11 is involved in the traffic of trans-sialidases to the plasma membrane through the CVC of the infective stages of the parasite and that preventing this traffic results in considerable reduction in the ability of T. cruzi to infect host cells. We also found that traffic of other GPI-anchored proteins is also through the CVC but uses a Rab11-independent pathway. These represent unconventional pathways of GPI-anchored protein traffic to the plasma membrane. PMID:25862161

  12. Aromatic Anchor at an Invariant Hormone-Receptor Interface

    PubMed Central

    Pandyarajan, Vijay; Smith, Brian J.; Phillips, Nelson B.; Whittaker, Linda; Cox, Gabriella P.; Wickramasinghe, Nalinda; Menting, John G.; Wan, Zhu-li; Whittaker, Jonathan; Ismail-Beigi, Faramarz; Lawrence, Michael C.; Weiss, Michael A.

    2014-01-01

    Crystallographic studies of insulin bound to fragments of the insulin receptor have recently defined the topography of the primary hormone-receptor interface. Here, we have investigated the role of PheB24, an invariant aromatic anchor at this interface and site of a human mutation causing diabetes mellitus. An extensive set of B24 substitutions has been constructed and tested for effects on receptor binding. Although aromaticity has long been considered a key requirement at this position, MetB24 was found to confer essentially native affinity and bioactivity. Molecular modeling suggests that this linear side chain can serve as an alternative hydrophobic anchor at the hormone-receptor interface. These findings motivated further substitution of PheB24 by cyclohexanylalanine (Cha), which contains a nonplanar aliphatic ring. Contrary to expectations, [ChaB24]insulin likewise exhibited high activity. Furthermore, its resistance to fibrillation and the rapid rate of hexamer disassembly, properties of potential therapeutic advantage, were enhanced. The crystal structure of the ChaB24 analog, determined as an R6 zinc-stabilized hexamer at a resolution of 1.5 Å, closely resembles that of wild-type insulin. The nonplanar aliphatic ring exhibits two chair conformations with partial occupancies, each recapitulating the role of PheB24 at the dimer interface. Together, these studies have defined structural requirements of an anchor residue within the B24-binding pocket of the insulin receptor; similar molecular principles are likely to pertain to insulin-related growth factors. Our results highlight in particular the utility of nonaromatic side chains as probes of the B24 pocket and suggest that the nonstandard Cha side chain may have therapeutic utility. PMID:25305014

  13. Influence of anchoring on miscarriage risk perception associated with amniocentesis.

    PubMed

    Nuccio, Regina; Hashmi, S Shahrukh; Mastrobattista, Joan; Noblin, Sarah Jane; Refuerzo, Jerrie; Smith, Janice L; Singletary, Claire N

    2015-04-01

    One factor women consider when deciding whether to pursue amniocentesis is the risk of miscarriage. People use mechanisms like anchoring, or the prior belief regarding the magnitude of risk, as a frame of reference for new information. This study aimed to determine a woman's perception of miscarriage risk associated with amniocentesis before and after genetic counseling and to determine what factors anchor a woman's perception of miscarriage risk. One hundred thirteen women being seen for prenatal genetic counseling and possible amniocentesis at six Houston clinics participated in the two-part anonymous survey. While most women (56.7 %) perceived the risk as low or average pre-counseling and indicated the numeric risk of amniocentesis as <1 %, significantly more patients (73 %) correctly identified the numeric risk as <1 % post-counseling (p < 0.0001). However, the majority of patients' qualitative risk perception did not change after the genetic counseling session (60 %). Those who changed their feeling about the risk after counseling showed a decreased perception of the risk (p < 0.0001). Participants who elected amniocentesis had a significantly lower perception of the risk (p = 0.017) whereas those who declined amniocentesis were more likely to view the risk as high (p = 0.004). The only two anchoring factors that had an effect were having a friend or relative with a personal or family history of a genetic disorder (p = 0.001) and having a child already (p = 0.038); both were associated with a lower risk perception. The lack of significant factors may reflect the uniqueness of each patient's risk assessment framework and reinforces the importance of genetic counseling to elucidate individual concerns, particularly as non-invasive prenatal testing becomes more widely available and further complicates the prenatal testing landscape. PMID:25224777

  14. Control of anchoring of nematic fluids at polymer surfaces created by in situ photopolymerization.

    PubMed

    Zhou, Jian; Collard, David M; Park, Jung O; Srinivasarao, Mohan

    2005-05-12

    In situ photopolymerization of alkyl acrylate monomers in the presence of a nematic fluid provides a cellular matrix of liquid crystalline droplets in which the chemical structure of the encapsulating polymer exerts control over the alignment (anchoring) of the liquid crystalline molecules. Control is obtained by variation of the alkyl side chains and through copolymerization of two dissimilar monofunctional acrylates. For example, among a series of poly(methylheptyl acrylate)s, the 1-methylheptyl analogue prefers planar anchoring of a nematic (TL205) over the temperature range studied. However, the polymers of other methylheptyl side chains display a homeotropic-to-planar anchoring thermal transition temperature similar to that of the n-heptyl analogue. Copolymerization of two monofunctional acrylates with opposing tendencies of aligning liquid crystal leads to tunability of anchoring behavior over a wide temperature range. The broad anchoring transitions we observed provide a way of achieving highly tilted anchoring. PMID:16852050

  15. Doubly anchored nematic polymer brushes: Shear, field effects, and quasipiezoelectricity

    NASA Astrophysics Data System (ADS)

    Halperin, A.; Williams, D. R. M.

    1994-02-01

    The shear behavior of a doubly anchored brush of liquid-crystalline polymers immersed in a nematic solvent is investigated. In such a brush the grafted polymers bridge two plates. Remarkably, the Franck nematic elasticity in this system can dominate the shear modulus. In the presence of a field the system undergoes a Fréedericksz transition modified by the bridging polymers. In particular: (i) the polymeric elasticity affects the critical field Ec; (ii) the shear modulus vanishes as the field approaches Ec; and (iii) the nematic distortion couples to a shear strain. The system exhibits quasipiezoelectricity, which is nonlinear and is not associated with any inverse effect.

  16. Molecular rectifiers: a new design based on asymmetric anchoring moieties.

    PubMed

    Van Dyck, Colin; Ratner, Mark A

    2015-03-11

    The quest for a molecular rectifier is among the major challenges of molecular electronics. We introduce three simple rules to design an efficient rectifying molecule and demonstrate its functioning at the theoretical level, relying on the NEGF-DFT technique. The design rules notably require both the introduction of asymmetric anchoring moieties and a decoupling bridge. They lead to a new rectification mechanism based on the compression and control of the HOMO/LUMO gap by the electrode Fermi levels, arising from a pinning effect. Significant rectification ratios up to 2 orders of magnitude are theoretically predicted as the mechanism opposes resonant to nonresonant tunneling. PMID:25706442

  17. Photolithography of Dithiocarbamate-Anchored Monolayers and Polymers on Gold

    PubMed Central

    Leonov, Alexei P.; Wei, Alexander

    2011-01-01

    Dithiocarbamate (DTC)-anchored monolayers and polymers were investigated as positive resists for UV photolithography on planar and roughened Au surfaces. DTCs were formed in situ by the condensation of CS2 with monovalent or polyvalent amines such as linear polyethyleneimine (PEI) under mildly basic aqueous conditions, just prior to surface passivation. The robust adsorption of the polyvalent PEI-DTC to Au surfaces supported high levels of resistance to photoablation, providing opportunities to generate thin films with gradient functionality. Treatment of photopatterned substrates with alkanethiols produced binary coatings, enabling a direct visual comparison of DTC- and thiol-passivated surfaces against chemically induced corrosion using confocal microscopy. PMID:21894240

  18. Simulation of weak anchoring effects on nematic liquid crystal hemispheres

    NASA Astrophysics Data System (ADS)

    Gillen, Sean; Somers, David A. T.; Munday, Jeremy N.

    The free energy of a nematic liquid crystal droplet depends on an interplay between elastic and surface interactions. When the two contributions are of similar magnitude, there exists a transition of the nematic structure of the droplet. Because the two contributions scale differently with length scales, this transition is visible as a function of the size of the droplet. We carry out numerical simulations to explore the use of this transition in measuring surface anchoring energies. This technique could help elucidate alignment forces on liquid crystals, such as those caused by rubbed surfaces, electric fields, or even the Casimir torque. Electrical and Computer Engineering.

  19. 18. VIEW OF ANCHOR BRIDGE NUMBER 310 LOOKING WEST ALONG ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    18. VIEW OF ANCHOR BRIDGE NUMBER 310 LOOKING WEST ALONG THE MAIN TRACK LOCATED TO THE NORTH OF THE COS COB POWER PLANT. THE LOAD DISPATCHER'S TOWER IS SHOWN IN THE LEFT FOREGROUND. AT THIS STATION THE DISPATCHER CONTROLLED POWER OUTPUT TO VARIOUS PARTS OF THE SYSTEM. THE STRUCTURE SERVES THE SAME PURPOSE IN 1993 AND CAN BE OPERATED LOCALLY OR REMOTELY FROM METRO-NORTH'S HEADQUARTERS IN MANHATTAN. THE STEEL STRUCTURE AND STACK IN THE BACKGROUND ARE PART OF THE BOILER 902-903 INSTALLATION. - New York, New Haven & Hartford Railroad, Cos Cob Power Plant, Sound Shore Drive, Greenwich, Fairfield County, CT

  20. Direct imaging of rotating molecules anchored on graphene

    NASA Astrophysics Data System (ADS)

    Choe, Jeongheon; Lee, Yangjin; Fang, Lei; Lee, Gun-Do; Bao, Zhenan; Kim, Kwanpyo

    2016-07-01

    There has been significant research interest in controlling and imaging molecular dynamics, such as translational and rotational motions, especially at a single molecular level. Here we applied aberration-corrected transmission electron microscopy (ACTEM) to actuate and directly image the rotational motions of molecules anchored on a single-layer-graphene sheet. Nanometer-sized carbonaceous molecules anchored on graphene provide ideal systems for monitoring rotational motions via ACTEM. We observed the preferential registry of longer molecular axis along graphene zigzag or armchair lattice directions due to the stacking-dependent molecule-graphene energy landscape. The calculated cross section from elastic scattering theory was used to experimentally estimate the rotational energy barriers of molecules on graphene. The observed energy barrier was within the range of 1.5-12 meV per atom, which is in good agreement with previous calculation results. We also performed molecular dynamics simulations, which revealed that the edge atoms of the molecule form stably bonds to graphene defects and can serve as a pivot point for rotational dynamics. Our study demonstrates the versatility of ACTEM for the investigation of molecular dynamics and configuration-dependent energetics at a single molecular level.There has been significant research interest in controlling and imaging molecular dynamics, such as translational and rotational motions, especially at a single molecular level. Here we applied aberration-corrected transmission electron microscopy (ACTEM) to actuate and directly image the rotational motions of molecules anchored on a single-layer-graphene sheet. Nanometer-sized carbonaceous molecules anchored on graphene provide ideal systems for monitoring rotational motions via ACTEM. We observed the preferential registry of longer molecular axis along graphene zigzag or armchair lattice directions due to the stacking-dependent molecule-graphene energy landscape. The

  1. Dephosphorylated synapsin I anchors synaptic vesicles to actin cytoskeleton: an analysis by videomicroscopy.

    PubMed

    Ceccaldi, P E; Grohovaz, F; Benfenati, F; Chieregatti, E; Greengard, P; Valtorta, F

    1995-03-01

    Synapsin I is a synaptic vesicle-associated protein which inhibits neurotransmitter release, an effect which is abolished upon its phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Based on indirect evidence, it was suggested that this effect on neurotransmitter release may be achieved by the reversible anchoring of synaptic vesicles to the actin cytoskeleton of the nerve terminal. Using video-enhanced microscopy, we have now obtained experimental evidence in support of this model: the presence of dephosphorylated synapsin I is necessary for synaptic vesicles to bind actin; synapsin I is able to promote actin polymerization and bundling of actin filaments in the presence of synaptic vesicles; the ability to cross-link synaptic vesicles and actin is specific for synapsin I and is not shared by other basic proteins; the cross-linking between synaptic vesicles and actin is specific for the membrane of synaptic vesicles and does not reflect either a non-specific binding of membranes to the highly surface active synapsin I molecule or trapping of vesicles within the thick bundles of actin filaments; the formation of the ternary complex is virtually abolished when synapsin I is phosphorylated by CaM kinase II. The data indicate that synapsin I markedly affects synaptic vesicle traffic and cytoskeleton assembly in the nerve terminal and provide a molecular basis for the ability of synapsin I to regulate the availability of synaptic vesicles for exocytosis and thereby the efficiency of neurotransmitter release. PMID:7876313

  2. The HP1 homolog Rhino anchors a nuclear complex that suppresses piRNA precursor splicing

    PubMed Central

    Zhang, Zhao; Wang, Jie; Schultz, Nadine; Zhang, Fan; Parhad, Swapnil S.; Tu, Shikui; Vreven, Thom; Zamore, Phillip D.; Weng, Zhiping; Theurkauf, William E.

    2014-01-01

    Summary piRNAs guide an adaptive genome defense system that silences transposons during germline development. The Drosophila HP1 homolog Rhino is required for germline piRNA production. We show that Rhino binds specifically to the heterochromatic clusters that produce piRNA precursors, and that binding directly correlates with piRNA production. Rhino co-localizes to germline nuclear foci with Rai1/DXO related protein Cuff and the DEAD box protein UAP56, which are also required for germline piRNA production. RNA sequencing indicates that most cluster transcripts are not spliced, and that rhino, cuff and uap56 mutations increase expression of spliced cluster transcripts over 100 fold. LacI∷Rhino fusion protein binding suppresses splicing of a reporter transgene, and is sufficient to trigger piRNA production from a trans combination of sense and antisense reporters. We therefore propose that Rhino anchors a nuclear complex that suppresses cluster transcript splicing, and speculate that stalled splicing differentiates piRNA precursors from mRNAs. PMID:24906152

  3. Inhibition of the Aurora A kinase augments the anti-tumor efficacy of oncolytic measles virotherapy.

    PubMed

    Iankov, I D; Kurokawa, C B; D'Assoro, A B; Ingle, J N; Domingo-Musibay, E; Allen, C; Crosby, C M; Nair, A A; Liu, M C; Aderca, I; Federspiel, M J; Galanis, E

    2015-09-01

    Oncolytic measles virus (MV) strains have demonstrated broad spectrum preclinical anti-tumor efficacy, including breast cancer. Aurora A kinase controls mitotic spindle formation and has a critical role in malignant transformation. We hypothesized that the Aurora A kinase inhibitor MLN8237 (alisertib) can increase MV oncolytic effect and efficacy by causing mitotic arrest. Alisertib enhanced MV oncolysis in vitro and significantly improved outcome in vivo against breast cancer xenografts. In a disseminated MDA-231-lu-P4 lung metastatic model, the MV/alisertib combination treatment markedly increased median survival to 82.5 days with 20% of the animals being long-term survivors versus 48 days median survival for the control animals. Similarly, in a pleural effusion model of advanced breast cancer, the MV/alisertib combination significantly improved outcome with a 74.5 day median survival versus the single agent groups (57 and 40 days, respectively). Increased viral gene expression and IL-24 upregulation were demonstrated, representing possible mechanisms for the observed increase in anti-tumor effect. Inhibiting Aurora A kinase with alisertib represents a novel approach to enhance MV-mediated oncolysis and antitumor effect. Both oncolytic MV strains and alisertib are currently tested in clinical trials, this study therefore provides the basis for translational applications of this combinatorial strategy in the treatment of patients with advanced breast cancer. PMID:26272026

  4. Percutaneous retrieval of a radiolucent anchoring sleeve embolized in pulmonary artery during pacemaker implantation.

    PubMed

    Tokuda, Michifumi; Yamane, Teiichi; Sadaoka, Shunichi; Tokutake, Kenichi; Yokoyama, Kenichi; Hioki, Mika; Narui, Ryohsuke; Tanigawa, Shinichi; Inada, Keiichi; Matsuo, Seiichiro; Yoshimura, Michihiro

    2016-08-01

    An 85-year-old female presented to our institution with symptomatic sick sinus syndrome. During pacemaker implantation, an anchoring sleeve in the right ventricular lead was embolized in the left pulmonary artery. Although the anchoring sleeve was radiolucent, digital subtraction angiography revealed an angiographic filling defect in the lower branch of the left pulmonary artery, and a snare catheter enabled the anchoring sleeve to be grasped and extracted. PMID:26391679

  5. Effects of anchoring and adjustment in the evaluation of product pricing.

    PubMed

    Elaad, Eitan; Sayag, Neta; Ezer, Aliya

    2010-08-01

    Anchoring and adjustment comprise a heuristic that creates expectations. Two types of anchors were applied on participants' evaluation of products: the price reference of the product (maximum, minimum, or no price reference) and the context in which the products were evaluated (the prestige of the shopping center). Results showed that both factors anchored evaluations of products' value. Context effects were explained by the different expectations of visitors in prestigious (looking for quality) and less prestigious (seeking a bargain) centers. PMID:20923048

  6. An Improved Anchor Shot Detection Method Using Fitness of Face Location and Dissimilarity of Icon Region

    NASA Astrophysics Data System (ADS)

    Keum, Ji-Soo; Lee, Hyon-Soo; Hagiwara, Masafumi

    In this letter, we propose an improved anchor shot detection (ASD) method in order to effectively retrieve anchor shots from news video. The face location and dissimilarity of icon region are used to reduce false alarms in the proposed method. According to the results of the experiment on several types of news video, the proposed method obtained high anchor detection results compared with previous methods.

  7. Knotless single-row rotator cuff repair: a comparative biomechanical study of 2 knotless suture anchors.

    PubMed

    Efird, Chad; Traub, Shaun; Baldini, Todd; Rioux-Forker, Dana; Spalazzi, Jeffrey P; Davisson, Twana; Hawkins, Monica; McCarty, Eric

    2013-08-01

    The purpose of this study was to compare the gap formation during cyclic loading, maximum repair strength, and failure mode of single-row full-thickness supraspinatus repairs performed using 2 knotless suture anchors with differing internal suture-retention mechanisms in a human cadaver model. Nine matched pairs of cadaver shoulders were used. Full-thickness tears were induced by detaching the supraspinatus tendon from the greater tuberosity. Single-row repairs were performed with either type I (Opus Magnum PI; ArthroCare, Austin, Texas) or type II (ReelX STT; Stryker, Mahwah, New Jersey) knotless suture anchors. The repaired tendon was cycled from 10 to 90 N for 500 cycles, followed by load to failure. Gap formation was measured at 5, 100, 200, 300, 400, and 500 cycles with a video digitizing system. Anchor type or location (anterior or posterior) had no effect on gap formation during cyclic loading regardless of position (anterior, P=.385; posterior, P=.389). Maximum load to failure was significantly greater (P=.018) for repairs performed with type II anchors (288±62 N) compared with type I anchors (179±39 N). Primary failure modes were anchor pullout and tendon tearing for type II anchors and suture slippage through the anchor for type I anchors. The internal ratcheting suture-retention mechanism of type II anchors may have helped this anchor outperform the suture-cinching mechanism of type I anchors by supporting significantly higher loads before failure and minimizing suture slippage, potentially leading to stronger repairs clinically. PMID:23937749

  8. Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts

    SciTech Connect

    Du, Yijun; Pattnaik, Asit K.; Song, Cheng; Yoo, Dongwan; Li, Gang

    2012-03-01

    The porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 4 (GP4) resembles a typical type I membrane protein in its structure but lacks a hydrophilic tail at the C-terminus, suggesting that GP4 may be a lipid-anchored membrane protein. Using the human decay-accelerating factor (DAF; CD55), a known glycosyl-phosphatidylinositol (GPI) lipid-anchored protein, chimeric constructs were made to substitute the GPI-anchor domain of DAF with the putative lipid-anchor domain of GP4, and their membrane association and lipase cleavage were determined in cells. The DAF-GP4 fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC), indicating that the C-terminal domain of GP4 functions as a GPI anchor. Mutational studies for residues adjacent to the GPI modification site and characterization of respective mutant viruses generated from infectious cDNA clones show that the ability of GP4 for membrane association corresponded to virus viability and growth characteristics. The residues T158 ({omega} - 2, where {omega} is the GPI moiety at E160), P159 ({omega} - 1), and M162 ({omega} + 2) of GP4 were determined to be important for virus replication, with M162 being of particular importance for virus infectivity. The complete removal of the peptide-anchor domain in GP4 resulted in a complete loss of virus infectivity. The depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in PRRSV infection. Remarkably, GP4 was found to co-localize with CD163 in the lipid rafts on the plasma membrane. Since CD163 has been reported as a cellular receptor for PRRSV and GP4 has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus.

  9. Sialic Acid on the Glycosylphosphatidylinositol Anchor Regulates PrP-mediated Cell Signaling and Prion Formation.

    PubMed

    Bate, Clive; Nolan, William; Williams, Alun

    2016-01-01

    The prion diseases occur following the conversion of the cellular prion protein (PrP(C)) into disease-related isoforms (PrP(Sc)). In this study, the role of the glycosylphosphatidylinositol (GPI) anchor attached to PrP(C) in prion formation was examined using a cell painting technique. PrP(Sc) formation in two prion-infected neuronal cell lines (ScGT1 and ScN2a cells) and in scrapie-infected primary cortical neurons was increased following the introduction of PrP(C). In contrast, PrP(C) containing a GPI anchor from which the sialic acid had been removed (desialylated PrP(C)) was not converted to PrP(Sc). Furthermore, the presence of desialylated PrP(C) inhibited the production of PrP(Sc) within prion-infected cortical neurons and ScGT1 and ScN2a cells. The membrane rafts surrounding desialylated PrP(C) contained greater amounts of sialylated gangliosides and cholesterol than membrane rafts surrounding PrP(C). Desialylated PrP(C) was less sensitive to cholesterol depletion than PrP(C) and was not released from cells by treatment with glimepiride. The presence of desialylated PrP(C) in neurons caused the dissociation of cytoplasmic phospholipase A2 from PrP-containing membrane rafts and reduced the activation of cytoplasmic phospholipase A2. These findings show that the sialic acid moiety of the GPI attached to PrP(C) modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP(Sc) formation. These results suggest that pharmacological modification of GPI glycosylation might constitute a novel therapeutic approach to prion diseases. PMID:26553874

  10. Detecting disulfide-bound complexes and the oxidative regulation of cyclic nucleotide-dependent protein kinases by H2O2.

    PubMed

    Burgoyne, Joseph R; Eaton, Philip

    2013-01-01

    Hydrogen peroxide regulates intracellular signaling by oxidatively converting susceptible cysteine thiols to a modified state, which includes the formation of intermolecular disulfides. This type of oxidative modification can occur within the cAMP- and cGMP-dependent protein kinases often referred to as PKA and PKG, which have important roles in regulating cardiac contractility and systemic blood pressure. Both kinases are stimulated through conical pathways that elevate their respective cyclic nucleotides leading to direct kinase stimulation. However, PKA and PKG can also be functionally modulated independently of cyclic nucleotide stimulation through direct cysteine thiol oxidation leading to intermolecular disulfide formation. In the case of PKG, the formation of an intermolecular disulfide between two parallel dimeric subunits leads to enhanced kinase affinity for substrate. For PKA, the formation of two intermolecular disulfides between antiparallel dimeric regulatory RI subunits increases the affinity of this kinase for its binding partners, the A-kinase anchoring proteins, leading to increased PKA localization to its substrates. In this chapter, we describe the methods for detecting intermolecular disulfide-bound proteins and monitoring PKA and PKG oxidation within biological samples. PMID:23849862

  11. Effects of Director Angle Anchoring Conditions on the Dynamic Moduli of Heterogeneous Nematic Polymers

    NASA Astrophysics Data System (ADS)

    Choate, Eric P.; Forest, M. Gregory; Cui, Zhenlu; Ju, Lili

    2008-07-01

    We examine the linear viscoelastic response of heterogeneous nematic polymers to small amplitude oscillatory shear flow, paying special attention to the macroscopic influence of strong plate anchoring conditions. To predict the dynamic moduli, we model the system with Stokes hydrodynamic equations with viscous and nematic stresses coupled with orientational dynamics driven by the flow, an excluded volume potential, and an elasticity potential. First, we show that for special cases of normal and tangential anchoring, we recover explicitly solvable Leslie-Ericksen-Frank behavior. In this case we observe significant differences between the moduli for normal and tangential anchoring, including a two-to-three order of magnitude enhancement of the storage modulus for normal over tangential anchoring. Then, we turn to a numerical study of oblique anchoring conditions, which are more complicated due to the appearance of order parameter gradients at leading order. When the anchoring angle is near 45 degrees, we observe significantly different scaling behavior in the storage modulus for high frequencies compared to the behavior for normal or tangential anchoring. Furthermore, we find that for low frequencies, normal anchoring gives the largest storage modulus and tangential the smallest; however, we see a brief window of higher frequencies where the maximum storage modulus occurs at an oblique anchoring angle.

  12. Glucosamine-Anchored Graphene Oxide Nanosheets: Fabrication, Ultraviolet Irradiation, and Electrochemical Properties.

    PubMed

    Veerapandian, Murugan; Lévaray, Nicolas; Lee, Min-Ho; Giasson, Suzanne; Zhu, X X

    2015-07-15

    A biofunctionalized graphene oxide (GO) nanosheet with improved physicochemical properties is useful for electrocatalysis and sensor development. Herein, a new class of functionalized GO with a chemically anchored biomolecule glucosamine is developed. Structural and chemical analyses confirm the glucosamine anchoring. Ultraviolet irradiation transforms the surface chemistry of GO. Glucosamine-anchored GO nanosheets exhibit improved cyclic voltammetric and amperometric sensing activity toward the model redox probe, ruthenium(II) and N-acetylneuraminic acid, respectively. The biomolecular anchoring and ultraviolet irradiation helped to tune and enhance the properties of GO, which may find multiple applications in optimizing sensor platforms. PMID:26120929

  13. SNP discovery and chromosome anchoring provide the first physically-anchored hexaploid oat map and reveal synteny with model species.

    PubMed

    Oliver, Rebekah E; Tinker, Nicholas A; Lazo, Gerard R; Chao, Shiaoman; Jellen, Eric N; Carson, Martin L; Rines, Howard W; Obert, Donald E; Lutz, Joseph D; Shackelford, Irene; Korol, Abraham B; Wight, Charlene P; Gardner, Kyle M; Hattori, Jiro; Beattie, Aaron D; Bjørnstad, Åsmund; Bonman, J Michael; Jannink, Jean-Luc; Sorrells, Mark E; Brown-Guedira, Gina L; Mitchell Fetch, Jennifer W; Harrison, Stephen A; Howarth, Catherine J; Ibrahim, Amir; Kolb, Frederic L; McMullen, Michael S; Murphy, J Paul; Ohm, Herbert W; Rossnagel, Brian G; Yan, Weikai; Miclaus, Kelci J; Hiller, Jordan; Maughan, Peter J; Redman Hulse, Rachel R; Anderson, Joseph M; Islamovic, Emir; Jackson, Eric W

    2013-01-01

    A physically anchored consensus map is foundational to modern genomics research; however, construction of such a map in oat (Avena sativa L., 2n = 6x = 42) has been hindered by the size and complexity of the genome, the scarcity of robust molecular markers, and the lack of aneuploid stocks. Resources developed in this study include a modified SNP discovery method for complex genomes, a diverse set of oat SNP markers, and a novel chromosome-deficient SNP anchoring strategy. These resources were applied to build the first complete, physically-anchored consensus map of hexaploid oat. Approximately 11,000 high-confidence in silico SNPs were discovered based on nine million inter-varietal sequence reads of genomic and cDNA origin. GoldenGate genotyping of 3,072 SNP assays yielded 1,311 robust markers, of which 985 were mapped in 390 recombinant-inbred lines from six bi-parental mapping populations ranging in size from 49 to 97 progeny. The consensus map included 985 SNPs and 68 previously-published markers, resolving 21 linkage groups with a total map distance of 1,838.8 cM. Consensus linkage groups were assigned to 21 chromosomes using SNP deletion analysis of chromosome-deficient monosomic hybrid stocks. Alignments with sequenced genomes of rice and Brachypodium provide evidence for extensive conservation of genomic regions, and renewed encouragement for orthology-based genomic discovery in this important hexaploid species. These results also provide a framework for high-resolution genetic analysis in oat, and a model for marker development and map construction in other species with complex genomes and limited resources. PMID:23533580

  14. Decisions Under Distress Stress Profiles Influence Anchoring and Adjustment

    PubMed Central

    Kassam, Karim S.; Koslov, Katrina; Mendes, Wendy Berry

    2009-01-01

    People frequently make decisions under stress. Understanding how stress affects decision making is complicated by the fact that not all stress responses are created equal. Challenge states, for example, occur when individuals appraise a stressful situation as demanding, but believe they have the personal resources to cope, and are characterized by efficient cardiovascular reactivity and approach motivation. Threat states, in contrast, occur when situational demands are perceived to outweigh resources and are characterized by less efficient cardiovascular reactivity and withdrawal motivation. We randomly assigned participants to social-feedback conditions (i.e., positive or negative feedback) designed to engender challenge or threat, or a no-stress condition. Participants then completed an anchoring-and-adjustment questionnaire. Those assigned to the challenge condition adjusted more from self-generated anchors than those assigned to the threat condition. Cardiovascular responses mediated the relationship between condition and adjustment. This study demonstrates the importance of considering profiles of cardiovascular reactivity when examining the influence of stress on decision making. PMID:19843261

  15. Lightness perception in simple images: Testing the anchoring rules

    PubMed Central

    Radonjić, Ana; Gilchrist, Alan L.

    2014-01-01

    One approach toward understanding how vision computes surface lightness is to first determine what principles govern lightness in simple stimuli and then test whether these hold for more complex stimuli. Gilchrist (2006) proposed that in the simplest images that produce the experience of a surface (two surfaces differing in luminance that fill the entire visual field) lightness can be predicted based on two anchoring rules: the highest luminance rule and the area rule, plus a scale normalization. To test whether these anchoring rules hold when critical features of the stimuli are varied, we probed lightness in simple stimuli, painted onto the inside of hemispheric domes viewed under diffuse lighting. We find that although the highest luminance surface appears nearly white across a large variation in illumination (as predicted by the highest luminance rule), its lightness tends to increase as its luminance increases. This effect is small relative to the size of the overall luminance change. Further, we find that when the darker region fills more than half of the visual field, it appears to lighten with further increases in area but only if it is a single surface. Splitting the dark region into smaller sectors that cover an equal cumulative area diminishes or eliminates the area effect. PMID:25424860

  16. Reliable anchoring groups for single-molecule junctions

    NASA Astrophysics Data System (ADS)

    González, M. Teresa; Leary, Edmund; Evangeli, Charalambos; Arroyo, Carlos; Rubio-Bollinger, Gabino; Agraïit, Nicolás

    2012-02-01

    In the field of molecular electronics, thiols have been extensively used as the most common anchoring groups to bind molecules to gold electrodes. However, other anchoring groups as amines can provide interesting advantages. Recently, C-60 has been also proposed as a possible very efficient binding group. In this talk, I will present our studies on molecular junctions formed by thiol-, amine-, and C-60-terminated molecules. We use a STM (scanning tunneling microscope) break-junction technique to create and characterized single-molecule junctions both in ambient and liquid environment. We compare thiols and amines on the alkane family and an oligo(phenylene ethynylene). Our study of the molecular-junction stretching length allows us to conclude that thiols affect atomic rearrangement at the electrodes significantly more than amines. Using C-60-terminated molecules, we have recently introduced a new technique for controllably wiring one molecule at a time. We first get STM images to located isolated molecules on a gold substrate, which are then specifically targeted and contacted using a STM gold tip. This technique offers a significant improvement over other techniques, as it guaranties that one and only one molecule is contacted at a time between the electrodes.

  17. Cognitive Advantages of Blending with Material Anchors in Energy Instruction

    NASA Astrophysics Data System (ADS)

    Close, Hunter; Close, Eleanor; Scherr, Rachel; McKagan, Sarah

    2012-03-01

    Conceptual blending theory [1] explains how the human imagination creates unreal situations that help us think about reality. In these imaginary blended situations, we establish new correspondences, interactions, and dynamics, and the outcomes of the dynamics lend insight to the nature of various real situations that were used to compose the blend. Blends are not just in the head, however; in some cases, a material system participates in the blend by lending its material structure as conceptual structure [2]. In the instructional activity Energy Theater [3], people represent units of energy and move around in order to solve puzzles of energy transfer and transformation. We use the ideas of blending and material anchors to understand how learners are able to use the representation to their cognitive advantage. [4pt] [1] Fauconnier, G. & Turner, M. (2002). The Way We Think: Conceptual Blending and the Mind's Hidden Complexities. New York: Basic Books.[0pt] [2] Hutchins, E. (2005) Material anchors for conceptual blends. Journal of Pragmatics 37, 1555-1577.[0pt] [3] Scherr, R. E., Close, H. G., McKagan, S. B., & Close, E. W. (2010) ``Energy Theater'': Using the body symbolically to understand energy. In C. Singh, M. Sabella, & S. Rebello (Eds.) 2010 PERC Proceedings. Melville, NY: AIP Press.

  18. Computations of flow in an anchored Solar Vortex

    NASA Astrophysics Data System (ADS)

    Min, Dahhea; Fischer, Paul F.; Pearlstein, Arne J.

    2015-11-01

    In regions with high solar insolation, there is a potential to extract mechanical energy from the gravitationally unstable ground-heated air layer, using the substantial axial and azimuthal momentum of an anchored buoyancy-induced columnar vortex to drive a vertical-axis turbine. The seasonal and diurnal availability (which extends well into the late afternoon and even past sunset, due to utilization of the thermal capacity of the ground to heat the air, rather than direct use of photons) is well-matched to air-conditioning loads in the southwestern US. Critical issues in the design of such systems are the geometry of the enclosure that serves to anchor the dust devil-like vortex and prevent it from being blown away by ambient wind, as well as the geometry of the stationary vanes used both to enhance entrainment of ground-heated air into the vortex from a collection area much larger than that of the enclosure, and to utilize any ambient wind to enhance the vortex. Here, we report computations (using the spectral-element code Nek5000) of heated and unheated flows in several geometries of interest. The results are discussed in the context of field experiments. Supported by ARPA-E award DE-AR0000296.

  19. Direct imaging of rotating molecules anchored on graphene.

    PubMed

    Choe, Jeongheon; Lee, Yangjin; Fang, Lei; Lee, Gun-Do; Bao, Zhenan; Kim, Kwanpyo

    2016-07-21

    There has been significant research interest in controlling and imaging molecular dynamics, such as translational and rotational motions, especially at a single molecular level. Here we applied aberration-corrected transmission electron microscopy (ACTEM) to actuate and directly image the rotational motions of molecules anchored on a single-layer-graphene sheet. Nanometer-sized carbonaceous molecules anchored on graphene provide ideal systems for monitoring rotational motions via ACTEM. We observed the preferential registry of longer molecular axis along graphene zigzag or armchair lattice directions due to the stacking-dependent molecule-graphene energy landscape. The calculated cross section from elastic scattering theory was used to experimentally estimate the rotational energy barriers of molecules on graphene. The observed energy barrier was within the range of 1.5-12 meV per atom, which is in good agreement with previous calculation results. We also performed molecular dynamics simulations, which revealed that the edge atoms of the molecule form stably bonds to graphene defects and can serve as a pivot point for rotational dynamics. Our study demonstrates the versatility of ACTEM for the investigation of molecular dynamics and configuration-dependent energetics at a single molecular level. PMID:27333828

  20. Dithiocarbamate anchoring in molecular wire junctions: a first principles study.

    PubMed

    Li, Zhenyu; Kosov, Daniel S

    2006-05-25

    Recent experimental realization [J. Am. Chem. Soc., 127 (2005) 7328] of various dithiocarbamate self-assembly on gold surface opens the possibility for use of dithiocarbamate linkers to anchor molecular wires to gold electrodes. In this paper, we explore this hypothesis computationally. We computed the electron transport properties of 4,4'-bipyridine (BP), 4,4'-bipyridinium-1,1'-bis(carbodithioate) (BPBC), 4-(4'-pyridyl)-peridium-1-carbodithioate (BPC) molecule junctions based on the density functional theory and nonequilibrium Green's functions. We demonstrated that the stronger molecule-electrode coupling associated with the conjugated dithiocarbamate linker broadens transmission resonances near the Fermi energy. The broadening effect along with the extension of the pi conjugation from the molecule to the gold electrodes lead to enhanced electrical conductance for BPBC molecule. The conductance enhancement factor is as large as 25 at applied voltage bias 1.0 V. Rectification behavior is predicted for BPC molecular wire junction, which has the asymmetric anchoring groups. PMID:16706444