Science.gov

Sample records for a-kinase anchoring proteins

  1. Organizing signal transduction through A-kinase anchoring proteins (AKAPs).

    PubMed

    Logue, Jeremy S; Scott, John D

    2010-11-01

    A fundamental role for protein-protein interactions in the organization of signal transduction pathways is evident. Anchoring, scaffolding and adapter proteins function to enhance the precision and directionality of these signaling events by bringing enzymes together. The cAMP signaling pathway is organized by A-kinase anchoring proteins. This family of proteins assembles enzyme complexes containing the cAMP-dependent protein kinase, phosphoprotein phosphatases, phosphodiesterases and other signaling effectors to optimize cellular responses to cAMP and other second messengers. Selected A-kinase anchoring protein signaling complexes are highlighted in this minireview. © 2010 The Authors Journal compilation © 2010 FEBS.

  2. A-Kinase Anchoring Protein-Lbc: A Molecular Scaffold Involved in Cardiac Protection.

    PubMed

    Diviani, Dario; Osman, Halima; Reggi, Erica

    2018-02-08

    Heart failure is a lethal disease that can develop after myocardial infarction, hypertension, or anticancer therapy. In the damaged heart, loss of function is mainly due to cardiomyocyte death and associated cardiac remodeling and fibrosis. In this context, A-kinase anchoring proteins (AKAPs) constitute a family of scaffolding proteins that facilitate the spatiotemporal activation of the cyclic adenosine monophosphate (AMP)-dependent protein kinase (PKA) and other transduction enzymes involved in cardiac remodeling. AKAP-Lbc, a cardiac enriched anchoring protein, has been shown to act as a key coordinator of the activity of signaling pathways involved in cardiac protection and remodeling. This review will summarize and discuss recent advances highlighting the role of the AKAP-Lbc signalosome in orchestrating adaptive responses in the stressed heart.

  3. Roles of A-Kinase Anchoring Proteins and Phosphodiesterases in the Cardiovascular System

    PubMed Central

    Ercu, Maria; Klussmann, Enno

    2018-01-01

    A-kinase anchoring proteins (AKAPs) and cyclic nucleotide phosphodiesterases (PDEs) are essential enzymes in the cyclic adenosine 3′-5′ monophosphate (cAMP) signaling cascade. They establish local cAMP pools by controlling the intensity, duration and compartmentalization of cyclic nucleotide-dependent signaling. Various members of the AKAP and PDE families are expressed in the cardiovascular system and direct important processes maintaining homeostatic functioning of the heart and vasculature, e.g., the endothelial barrier function and excitation-contraction coupling. Dysregulation of AKAP and PDE function is associated with pathophysiological conditions in the cardiovascular system including heart failure, hypertension and atherosclerosis. A number of diseases, including autosomal dominant hypertension with brachydactyly (HTNB) and type I long-QT syndrome (LQT1), result from mutations in genes encoding for distinct members of the two classes of enzymes. This review provides an overview over the AKAPs and PDEs relevant for cAMP compartmentalization in the heart and vasculature and discusses their pathophysiological role as well as highlights the potential benefits of targeting these proteins and their protein-protein interactions for the treatment of cardiovascular diseases. PMID:29461511

  4. Molecular evolution of a-kinase anchoring protein (AKAP)-7: implications in comparative PKA compartmentalization

    PubMed Central

    2012-01-01

    Background A-Kinase Anchoring Proteins (AKAPs) are molecular scaffolding proteins mediating the assembly of multi-protein complexes containing cAMP-dependent protein kinase A (PKA), directing the kinase in discrete subcellular locations. Splice variants from the AKAP7 gene (AKAP15/18) are vital components of neuronal and cardiac phosphatase complexes, ion channels, cardiac Ca2+ handling and renal water transport. Results Shown in evolutionary analyses, the formation of the AKAP7-RI/RII binding domain (required for AKAP/PKA-R interaction) corresponds to vertebrate-specific gene duplication events in the PKA-RI/RII subunits. Species analyses of AKAP7 splice variants shows the ancestral AKAP7 splice variant is AKAP7α, while the ancestral long form AKAP7 splice variant is AKAP7γ. Multi-species AKAP7 gene alignments, show the recent formation of AKAP7δ occurs with the loss of native AKAP7γ in rats and basal primates. AKAP7 gene alignments and two dimensional Western analyses indicate that AKAP7γ is produced from an internal translation-start site that is present in the AKAP7δ cDNA of mice and humans but absent in rats. Immunofluorescence analysis of AKAP7 protein localization in both rat and mouse heart suggests AKAP7γ replaces AKAP7δ at the cardiac sarcoplasmic reticulum in species other than rat. DNA sequencing identified Human AKAP7δ insertion-deletions (indels) that promote the production of AKAP7γ instead of AKAP7δ. Conclusions This AKAP7 molecular evolution study shows that these vital scaffolding proteins developed in ancestral vertebrates and that independent mutations in the AKAP7 genes of rodents and early primates has resulted in the recent formation of AKAP7δ, a splice variant of likely lesser importance in humans than currently described. PMID:22834419

  5. Mutation of an A-kinase-anchoring protein causes long-QT syndrome

    PubMed Central

    Chen, Lei; Marquardt, Michelle L.; Tester, David J.; Sampson, Kevin J.; Ackerman, Michael J.; Kass, Robert S.

    2007-01-01

    A-kinase anchoring proteins (AKAPs) recruit signaling molecules and present them to downstream targets to achieve efficient spatial and temporal control of their phosphorylation state. In the heart, sympathetic nervous system (SNS) regulation of cardiac action potential duration (APD), mediated by β-adrenergic receptor (βAR) activation, requires assembly of AKAP9 (Yotiao) with the IKs potassium channel α subunit (KCNQ1). KCNQ1 mutations that disrupt this complex cause type 1 long-QT syndrome (LQT1), one of the potentially lethal heritable arrhythmia syndromes. Here, we report identification of (i) regions on Yotiao critical to its binding to KCNQ1 and (ii) a single putative LQTS-causing mutation (S1570L) in AKAP9 (Yotiao) localized to the KCNQ1 binding domain in 1/50 (2%) subjects with a clinically robust phenotype for LQTS but absent in 1,320 reference alleles. The inherited S1570L mutation reduces the interaction between KCNQ1 and Yotiao, reduces the cAMP-induced phosphorylation of the channel, eliminates the functional response of the IKs channel to cAMP, and prolongs the action potential in a computational model of the ventricular cardiocyte. These reconstituted cellular consequences of the inherited S1570L-Yotiao mutation are consistent with delayed repolarization of the ventricular action potential observed in the affected siblings. Thus, we have demonstrated a link between genetic perturbations in AKAP and human disease in general and AKAP9 and LQTS in particular. PMID:18093912

  6. PKA RIα/A-kinase anchoring proteins 10 signaling pathway and the prognosis of colorectal cancer.

    PubMed

    Wang, Mojin; Li, Yuan; Wang, Rui; Wang, Ziqiang; Chen, Keling; Zhou, Bin; Zhou, Zongguang; Sun, Xiaofeng

    2015-03-01

    Previously study showed that the loss of the control of cAMP-dependent protein kinase A RIα (PKA RIα)/ A-kinase anchoring proteins 10 (AKAP10) signaling pathway initiate dysregulation of cellular healthy physiology leading to tumorigenesis. The aim of this study was to investigate the role of PKA RIα/AKAP10 signaling pathway in colorectal cancer (CRC). The AKAP10 expression at the mRNA and protein level have been analyzed in colon cancer cell lines, primary CRCs and matched normal mucosa samples, and compared in accordance with specific clinicopathological features of CRC. The correlation between expression of AKAP10 and PKA RIα were also analyzed. Compared with HCT116 and SW480 cells, the AKAP10 was significantly upregulated in the colon cell line KM12C and its metastatic counterparts, KM12SM and KM12L4A. Moreover, the KM12SM and KM12L4A having high metastatic potentials displayed the elevated levels of AKAP10 compared with KM12C having poor metastatic potential. A notably higher level of AKAP10 expression was found in CRC tissues at both mRNA and protein levels. Increased expression of AKAP10 in CRC patients was positively associated with the depth of invasion and the grade of differentiation. Univariate survival analysis showed that the increased expression of AKAP10 was related to poorer survival. Cox multivariate regression analysis confirmed that AKAP10 was an independent predictor of the overall survival of CRC patients. PKA RIα mRNA was also expressed at high levels in CRC. The correlation coefficient between mRNA expression of AKAP10 and PKA RIα in CRC was 0.417. AKAP10 mRNA overexpression was correlated significantly with PKA RIα. Our data indicated that PKA RIα/AKAP10 signaling pathway is associated with the progression and prognosis of CRC. © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

  7. Spatial Distribution of Protein Kinase A Activity during Cell Migration Is Mediated by A-kinase Anchoring Protein AKAP Lbc*

    PubMed Central

    Paulucci-Holthauzen, Adriana A.; Vergara, Leoncio A.; Bellot, Larry J.; Canton, David; Scott, John D.; O'Connor, Kathleen L.

    2009-01-01

    Protein kinase A (PKA) has been suggested to be spatially regulated in migrating cells due to its ability to control signaling events that are critical for polarized actin cytoskeletal dynamics. Here, using the fluorescence resonance energy transfer-based A-kinase activity reporter (AKAR1), we find that PKA activity gradients form with the strongest activity at the leading edge and are restricted to the basal surface in migrating cells. The existence of these gradients was confirmed using immunocytochemistry using phospho-PKA substrate antibodies. This observation holds true for carcinoma cells migrating randomly on laminin-1 or stimulated to migrate on collagen I with lysophosphatidic acid. Phosphodiesterase inhibition allows the formation of PKA activity gradients; however, these gradients are no longer polarized. PKA activity gradients are not detected when a non-phosphorylatable mutant of AKAR1 is used, if PKA activity is inhibited with H-89 or protein kinase inhibitor, or when PKA anchoring is perturbed. We further find that a specific A-kinase anchoring protein, AKAP-Lbc, is a major contributor to the formation of these gradients. In summary, our data show that PKA activity gradients are generated at the leading edge of migrating cells and provide additional insight into the mechanisms of PKA regulation of cell motility. PMID:19106088

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

    PubMed Central

    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

  9. The C-terminal region of A-kinase anchor protein 350 (AKAP350A) enables formation of microtubule-nucleation centers and interacts with pericentriolar proteins.

    PubMed

    Kolobova, Elena; Roland, Joseph T; Lapierre, Lynne A; Williams, Janice A; Mason, Twila A; Goldenring, James R

    2017-12-15

    Microtubules in animal cells assemble (nucleate) from both the centrosome and the cis-Golgi cisternae. A-kinase anchor protein 350 kDa (AKAP350A, also called AKAP450/CG-NAP/AKAP9) is a large scaffolding protein located at both the centrosome and Golgi apparatus. Previous findings have suggested that AKAP350 is important for microtubule dynamics at both locations, but how this scaffolding protein assembles microtubule nucleation machinery is unclear. Here, we found that overexpression of the C-terminal third of AKAP350A, enhanced GFP-AKAP350A(2691-3907), induces the formation of multiple microtubule-nucleation centers (MTNCs). Nevertheless, these induced MTNCs lacked "true" centriole proteins, such as Cep135. Mapping analysis with AKAP350A truncations demonstrated that AKAP350A contains discrete regions responsible for promoting or inhibiting the formation of multiple MTNCs. Moreover, GFP-AKAP350A(2691-3907) recruited several pericentriolar proteins to MTNCs, including γ-tubulin, pericentrin, Cep68, Cep170, and Cdk5RAP2. Proteomic analysis indicated that Cdk5RAP2 and Cep170 both interact with the microtubule nucleation-promoting region of AKAP350A, whereas Cep68 interacts with the distal C-terminal AKAP350A region. Yeast two-hybrid assays established a direct interaction of Cep170 with AKAP350A. Super-resolution and deconvolution microscopy analyses were performed to define the association of AKAP350A with centrosomes, and these studies disclosed that AKAP350A spans the bridge between centrioles, co-localizing with rootletin and Cep68 in the linker region. siRNA-mediated depletion of AKAP350A caused displacement of both Cep68 and Cep170 from the centrosome. These results suggest that AKAP350A acts as a scaffold for factors involved in microtubule nucleation at the centrosome and coordinates the assembly of protein complexes associating with the intercentriolar bridge.

  10. Phospholipase Cϵ Scaffolds to Muscle-specific A Kinase Anchoring Protein (mAKAPβ) and Integrates Multiple Hypertrophic Stimuli in Cardiac Myocytes*

    PubMed Central

    Zhang, Lianghui; Malik, Sundeep; Kelley, Grant G.; Kapiloff, Michael S.; Smrcka, Alan V.

    2011-01-01

    To define a role for phospholipase Cϵ (PLCϵ) signaling in cardiac myocyte hypertrophic growth, PLCϵ protein was depleted from neonatal rat ventricular myocytes (NRVMs) using siRNA. NRVMs with PLCϵ depletion were stimulated with endothelin (ET-1), norepinephrine, insulin-like growth factor-1 (IGF-1), or isoproterenol and assessed for development of hypertrophy. PLCϵ depletion dramatically reduced hypertrophic growth and gene expression induced by all agonists tested. PLCϵ catalytic activity was required for hypertrophy development, yet PLCϵ depletion did not reduce global agonist-stimulated inositol phosphate production, suggesting a requirement for localized PLC activity. PLCϵ was found to be scaffolded to a muscle-specific A kinase anchoring protein (mAKAPβ) in heart and NRVMs, and mAKAPβ localizes to the nuclear envelope in NRVMs. PLCϵ-mAKAP interaction domains were defined and overexpressed to disrupt endogenous mAKAPβ-PLCϵ complexes in NRVMs, resulting in significantly reduced ET-1-dependent NRVM hypertrophy. We propose that PLCϵ integrates multiple upstream signaling pathways to generate local signals at the nucleus that regulate hypertrophy. PMID:21550986

  11. Differential expression of the tumor suppressor A-kinase anchor protein 12 in human diffuse and pilocytic astrocytomas is regulated by promoter methylation.

    PubMed

    Goeppert, Benjamin; Schmidt, Christopher R; Geiselhart, Lea; Dutruel, Céline; Capper, David; Renner, Marcus; Vogel, Monika Nadja; Zachskorn, Cornelia; Zinke, Jenny; Campos, Benito; Schmezer, Peter; Popanda, Odilia; Wick, Wolfgang; Weller, Michael; Meyermann, Richard; Schittenhelm, Jens; Harter, Patrick Nikolaus; Simon, Perikles; Weichert, Wilko; Schirmacher, Peter; Plass, Christoph; Mittelbronn, Michel

    2013-10-01

    The scaffold protein A-kinase anchor protein 12 (AKAP12) exerts tumor suppressor activity and is downregulated in several tumor entities. We characterized AKAP12 expression and regulation in astrocytomas, including pilocytic and diffusely infiltrating astrocytomas. We examined 194 human gliomas and 23 normal brain white matter samples by immunohistochemistry or immunoblotting for AKAP12 expression. We further performed quantitative methylation analysis of the AKAP12 promoter by MassARRAY® of normal brain, World Health Organization (WHO) grade I to IV astrocytomas, and glioma cell lines. Our results show that AKAP12 is expressed in a perivascular distribution in normal CNS, strongly upregulated in tumor cells in pilocytic astrocytomas, and weakly expressed in diffuse astrocytomas of WHO grade II to IV. Methylation analyses revealed specific hypermethylation of AKAP12α promoter in WHO grade II to IV astrocytomas. Restoration experiments using 5-aza-2'-deoxycytidine in primary glioblastoma cells decreased AKAP12α promoter methylation and markedly increased AKAP12α mRNA levels. In summary, we demonstrate that AKAP12 is differentially expressed in human astrocytomas showing high expression in pilocytic but low expression in diffuse astrocytomas of all WHO-grades. Our results further indicate that epigenetic mechanisms are involved in silencing AKAP12 in diffuse astrocytomas; however, a tumor suppressive role of AKAP12 in distinct astrocytoma subtypes remains to be determined.

  12. Human muscle-specific A-kinase anchoring protein (mAKAP) polymorphisms modulate the susceptibility to cardiovascular diseases by altering cAMP/ PKA signaling.

    PubMed

    Suryavanshi, Santosh V; Jadhav, Shweta M; Anderson, Kody L; Katsonis, Panagiotis; Lichtarge, Olivier; McConnell, Bradley K

    2018-03-30

    One of the crucial cardiac signaling pathways is cAMP-mediated PKA signal transduction which is regulated by a family of scaffolding proteins, A-kinase anchoring proteins (AKAPs). Muscle-specific AKAP (mAKAP) partly regulates cardiac cAMP/PKA signaling by binding to PKA and phosphodiesterase4D3 (PDE4D3) among other proteins and plays a central role in modulating cardiac remodeling. Moreover, genetics plays an incomparable role in modifying the risk of cardiovascular diseases (CVDs). Especially, single nucleotide polymorphisms (SNPs) in various proteins have been shown to predispose individuals to CVDs. Hence, we hypothesized that human mAKAP polymorphisms found in humans with CVDs alter cAMP/PKA pathway influencing the susceptibility of individuals to CVDs. Our computational analyses revealed two mAKAP SNPs found in cardiac disease related patients with highest predicted deleterious effects, Ser(S) 1653 Arg(R) and Glu(E) 2124 Gly(G). Co-immunoprecipitation data in HEK293T cells showed that S1653R SNP, present in the PDE4D3 binding domain of mAKAP, changed the binding of PDE4D3 to mAKAP and E2124G SNP, flanking the 3'-PKA binding domain, changed the binding of PKA before and after stimulation with isoproterenol. These SNPs significantly altered intracellular cAMP levels, global PKA activity and cytosolic PDE activity when compared with the wild-type (WT) before and after isoproterenol stimulation. PKA-mediated phosphorylation of pathological markers was found to be up-regulated after cell stimulation in both mutants. In conclusion, human mAKAP polymorphisms may influence the propensity of developing CVDs by affecting cAMP/PKA signaling supporting the clinical significance of PKA-mAKAP-PDE4D3 interactions.

  13. The novel cancer-testis antigen A-kinase anchor protein 4 (AKAP4) is a potential target for immunotherapy of ovarian serous carcinoma

    PubMed Central

    Agarwal, Sumit; Saini, Shikha; Parashar, Deepak; Verma, Archana; Sinha, Abhilasha; Jagadish, Nirmala; Batra, Aruna; Suri, Sushma; Gupta, Anju; Ansari, Abdul S.; Lohiya, Nirmal Kumar; Suri, Anil

    2013-01-01

    Ovarian cancer is one of the neoplasms affecting the reproductive tract associated with high mortality rate because of limited therapeutic options and an elevated incidence of chemoresistance and recurrence. In this context, immunotherapy may constitute a promising approach to improve survival rates and clinical outcome, raising the need for specific target antigens. Cancer-testis antigens (CTAs) are considered promising candidates in this sense because they are aberrant expressed by various malignancies but not by non-transformed tissue, with the exception of testes. Here, we examined the expression and potential to promote humoral immune responses of a novel CTA, A-kinase anchor protein 4 (AKAP4), among 38 ovarian carcinoma patients. Our results reveal that AKAP4 was expressed at both the mRNA and protein levels in 89% (34/38) of ovarian carcinoma tissue specimens but not in 21 matched adjacent non-cancerous tissues. In addition, a humoral response against AKAP4 was detected in 58% (22/38) of ovarian carcinoma patients by ELISA. In particular, 65% (22/34) patients bearing an AKAP4-expressing tumor exhibited circulating anti-AKAP4 antibodies. Interestingly, the majority of specimens were categorized as ovarian serous adenocarcinoma and serous papillary carcinoma, of which 93% (28/30) and 100% (6/6), respectively, expressed AKAP4. A humoral response against AKAP4 was detected in 79% (19/24) and 67% (4/6) of ovarian serous adenocarcinoma and serous papillary carcinoma patients, respectively. The presence of circulating anti-AKAP4 antibodies suggests the AKAP4 is highly immunogenic in ovarian serous carcinoma patients. Our study lays the foundations for exploring AKAP4 as a potential target for the immunotherapy of ovarian cancer. PMID:23762804

  14. Detection of glycophospholipid anchors on proteins.

    PubMed

    Doering, T L; Englund, P T; Hart, G W

    2001-05-01

    Many eukaryotic proteins are tethered to the plasma membrane by glycosyl phosphatidylinositol (GPI) membrane anchors. This unit provides a general approach for detecting GPI-anchored proteins. First, the detergent-partitioning behavior of a protein of interest is examined for characteristics of GPI-linked species. The protein may also be subjected to specific enzymatic or chemical cleavages to release the protein from its GPI anchor. Protocols for phospholipase cleavage and chemical cleavage with nitrous acid are provided for this purpose. If GPI-anchored proteins are radiolabeled with fatty acids, it facilitates the detection of the GPI protein products following the cleavage reactions. Separation of lipid moieties and base hydrolysis of proteins are detailed herein.

  15. Anchor residues in protein–protein interactions

    PubMed Central

    Rajamani, Deepa; Thiel, Spencer; Vajda, Sandor; Camacho, Carlos J.

    2004-01-01

    We show that the mechanism for molecular recognition requires one of the interacting proteins, usually the smaller of the two, to anchor a specific side chain in a structurally constrained binding groove of the other protein, providing a steric constraint that helps to stabilize a native-like bound intermediate. We identify the anchor residues in 39 protein–protein complexes and verify that, even in the absence of their interacting partners, the anchor side chains are found in conformations similar to those observed in the bound complex. These ready-made recognition motifs correspond to surface side chains that bury the largest solvent-accessible surface area after forming the complex (≥100 Å2). The existence of such anchors implies that binding pathways can avoid kinetically costly structural rearrangements at the core of the binding interface, allowing for a relatively smooth recognition process. Once anchors are docked, an induced fit process further contributes to forming the final high-affinity complex. This later stage involves flexible (solvent-exposed) side chains that latch to the encounter complex in the periphery of the binding pocket. Our results suggest that the evolutionary conservation of anchor side chains applies to the actual structure that these residues assume before the encounter complex and not just to their loci. Implications for protein docking are also discussed. PMID:15269345

  16. Pseudoscaffolds and anchoring proteins: the difference is in the details

    PubMed Central

    Aggarwal-Howarth, Stacey; Scott, John D.

    2017-01-01

    Pseudokinases and pseudophosphatases possess the ability to bind substrates without catalyzing their modification, thereby providing a mechanism to recruit potential phosphotargets away from active enzymes. Since many of these pseudoenzymes possess other characteristics such as localization signals, separate catalytic sites, and protein–protein interaction domains, they have the capacity to influence signaling dynamics in local environments. In a similar manner, the targeting of signaling enzymes to subcellular locations by A-kinase-anchoring proteins (AKAPs) allows for precise and local control of second messenger signaling events. Here, we will discuss how pseudoenzymes form ‘pseudoscaffolds’ and compare and contrast this compartment-specific regulatory role with the signal organization properties of AKAPs. The mitochondria will be the focus of this review, as they are dynamic organelles that influence a broad range of cellular processes such as metabolism, ATP synthesis, and apoptosis. PMID:28408477

  17. GPI-anchor and GPI-anchored protein expression in PMM2-CDG patients

    PubMed Central

    2013-01-01

    Background Mutations in PMM2 impair phosphomannomutase-2 activity and cause the most frequent congenital disorder of glycosylation, PMM2-CDG. Mannose-1-phosphate, that is deficient in this disorder, is also implicated in the biosynthesis of glycosylphosphatidyl inositol (GPI) anchors. Objective To evaluate whether GPI-anchor and GPI-anchored proteins are defective in PMM2-CDG patients. Methods The expression of GPI-anchor and seven GPI-anchored proteins was evaluated by flow cytometry in different cell types from twelve PMM2-CDG patients. Additionally, neutrophil CD16 and plasma hepatic proteins were studied by Western blot. Transferrin glycoforms were evaluated by HPLC. Results Patients and controls had similar surface expression of GPI-anchor and most GPI-anchored proteins. Nevertheless, patients displayed a significantly diminished binding of two anti-CD16 antibodies (3G8 and KD1) to neutrophils and also of anti-CD14 (61D3) to monocytes. Interestingly, CD16 immunostaining and asialotransferrin levels significantly correlated with patients’ age. Analysis by flow cytometry of CD14 with MΦP9, and CD16 expression in neutrophils by Western blot using H-80 ruled out deficiencies of these antigens. Conclusions PMM2 mutations do not impair GPI-anchor or GPI-anchored protein expression. However, the glycosylation anomalies caused by PMM2 mutations might affect the immunoreactivity of monoclonal antibodies and lead to incorrect conclusions about the expression of different proteins, including GPI-anchored proteins. Neutrophils and monocytes are sensitive to PMM2 mutations, leading to abnormal glycosylation in immune receptors, which might potentially affect their affinity to their ligands, and contribute to infection. This study also confirms less severe hypoglycosylation defects in older PMM2-CDG patients. PMID:24139637

  18. 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

  19. Ultrasensitivity in Multisite Phosphorylation of Membrane-Anchored Proteins

    PubMed Central

    Dushek, Omer; van der Merwe, P.Anton; Shahrezaei, Vahid

    2011-01-01

    Cellular signaling is initially confined to the plasma membrane, where the cytoplasmic tails of surface receptors and other membrane-anchored proteins are phosphorylated in response to ligand binding. These proteins often contain multiple phosphorylation sites that are regulated by membrane-confined enzymes. Phosphorylation of these proteins is thought to be tightly regulated, because they initiate and regulate signaling cascades leading to cellular activation, yet how their phosphorylation is regulated is poorly understood. Ultrasensitive or switchlike responses in their phosphorylation state are not expected because the modifying enzymes are in excess. Here, we describe a novel mechanism of ultrasensitivity exhibited by multisite membrane-anchored proteins, but not cytosolic proteins, even when enzymes are in excess. The mechanism underlying this concentration-independent ultrasensitivity is the local saturation of a single enzyme by multiple sites on the substrate. Local saturation is a passive process arising from slow membrane diffusion, steric hindrances, and multiple sites, and therefore may be widely applicable. Critical to this ultrasensitivity is the brief enzymatic inactivation that follows substrate modification. Computations are presented using ordinary differential equations and stochastic spatial simulations. We propose a new role, to our knowledge, for multisite membrane-anchored proteins, discuss experiments that can be used to probe the model, and relate our findings to previous theoretical work. PMID:21354391

  20. Glycosylphosphatidylinositol Anchors from Galactomannan and GPI-Anchored Protein Are Synthesized by Distinct Pathways in Aspergillus fumigatus

    PubMed Central

    Li, Jizhou; Mouyna, Isabelle; Henry, Christine; Moyrand, Frédérique; Malosse, Christian; Chamot-Rooke, Julia; Janbon, Guilhem; Latgé, Jean-Paul; Fontaine, Thierry

    2018-01-01

    Glycosylphosphatidylinositols (GPIs) are lipid anchors allowing the exposure of proteins at the outer layer of the plasma membrane. In fungi, a number of GPI-anchored proteins (GPI-APs) are involved in the remodeling of the cell wall polymers. GPIs follow a specific biosynthetic pathway in the endoplasmic reticulum. After the transfer of the protein onto the GPI-anchor, a lipid remodeling occurs to substitute the diacylglycerol moiety by a ceramide. In addition to GPI-APs, A. fumigatus produces a GPI-anchored polysaccharide, the galactomannan (GM), that remains unique in the fungal kingdom. To investigate the role of the GPI pathway in the biosynthesis of the GM and cell wall organization, the deletion of PER1—coding for a phospholipase required for the first step of the GPI lipid remodeling—was undertaken. Biochemical characterization of the GPI-anchor isolated from GPI-APs showed that the PER1 deficient mutant produced a lipid anchor with a diacylglycerol. The absence of a ceramide on GPI-anchors in the Δper1 mutant led to a mislocation of GPI-APs and to an alteration of the composition of the cell wall alkali-insoluble fraction. On the other hand, the GM isolated from the Δper1 mutant membranes possesses a ceramide moiety as the parental strain, showing that GPI anchor of the GM follow a distinct unknown biosynthetic pathway. PMID:29393895

  1. Development of a prediction system for tail-anchored proteins.

    PubMed

    Shigemitsu, Shunsuke; Cao, Wei; Terada, Tohru; Shimizu, Kentaro

    2016-09-15

    "Tail-anchored (TA) proteins" is a collective term for transmembrane proteins with a C-terminal transmembrane domain (TMD) and without an N-terminal signal sequence. TA proteins account for approximately 3-5 % of all transmembrane proteins that mediate membrane fusion, regulation of apoptosis, and vesicular transport. The combined use of TMD and signal sequence prediction tools is typically required to predict TA proteins. Here we developed a prediction system named TAPPM that predicted TA proteins solely from target amino acid sequences according to the knowledge of the sequence features of TMDs and the peripheral regions of TA proteins. Manually curated TA proteins were collected from published literature. We constructed hidden markov models of TA proteins as well as three different types of transmembrane proteins with similar structures and compared their likelihoods as TA proteins. Using the HMM models, we achieved high prediction accuracy; area under the receiver operator curve values reaching 0.963. A command line tool written in Python is available at https://github.com/davecao/tappm_cli .

  2. Tail-anchored membrane proteins: exploring the complex diversity of tail-anchored-protein targeting in plant cells.

    PubMed

    Abell, Ben M; Mullen, Robert T

    2011-02-01

    Tail-anchored (TA) proteins are special class of integral membrane proteins that in recent years have received a considerable amount of attention due to their diverse cellular functions and unique targeting and insertion mechanisms. Defined by the presence of a single, hydrophobic membrane-spanning domain at or near their C terminus, TA proteins must be inserted into membranes post-translationally and are orientated such that their larger N-terminal domain (most often the functional domain) faces the cytosol, while their shorter C-terminal domain faces the interior of the organelle. The C-terminal domain of TA proteins also usually contains the information responsible for their selective targeting to the proper subcellular membrane, a process that, based primarily on studies with yeasts and mammals, appears to be highly complex due to the presence of multiple pathways. Within this context, we discuss here the biogenesis of plant TA proteins and the potential for hundreds of new TA proteins identified via bioinformatics screens to contribute to the already remarkable number of roles that this class of membrane proteins participates in throughout plant growth and development.

  3. Trafficking of glycosylphosphatidylinositol anchored proteins from the endoplasmic reticulum to the cell surface

    PubMed Central

    Muñiz, Manuel; Riezman, Howard

    2016-01-01

    In eukaryotes, many cell surface proteins are attached to the plasma membrane via a glycolipid glycosylphosphatidylinositol (GPI) anchor. GPI-anchored proteins (GPI-APs) receive the GPI anchor as a conserved posttranslational modification in the lumen of the endoplasmic reticulum (ER). After anchor attachment, the GPI anchor is structurally remodeled to function as a transport signal that actively triggers the delivery of GPI-APs from the ER to the plasma membrane, via the Golgi apparatus. The structure and composition of the GPI anchor confer a special mode of interaction with membranes of GPI-APs within the lumen of secretory organelles that lead them to be differentially trafficked from other secretory membrane proteins. In this review, we examine the mechanisms by which GPI-APs are selectively transported through the secretory pathway, with special focus on the recent progress made in their actively regulated export from the ER and the trans-Golgi network. PMID:26450970

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

    USDA-ARS?s Scientific Manuscript database

    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...

  5. Cellular aspects of M protein and SfbI anchoring to Streptococcus pyogenes wall

    PubMed Central

    Raz, Assaf; Talay, Susanne; Fischetti, Vincent

    2012-01-01

    Summary Wall-anchored surface proteins are critical for the in vivo survival of Streptococcus pyogenes. Cues in the signal sequence direct the membrane translocation of surface proteins: M protein to the septum, and SfbI to the poles. Both proteins are subsequently anchored to the wall by the membrane bound enzyme sortase A. However, the cellular features of these pathways are not fully understood. Here we show that M protein and SfbI are anchored simultaneously throughout the cell cycle. M protein is rapidly anchored at the septum, and in part of the cell cycle, is anchored simultaneously at the mother and daughter septa. Conversely, SfbI accumulates gradually on peripheral peptidoglycan, resulting in a polar distribution. Sortase is not required for translocation of M protein or SfbI at their respective locations. Methicillin-induced unbalanced peptidoglycan synthesis diminishes surface M protein but not SfbI. Furthermore, overexpression of the division regulator DivIVA also diminishes surface M protein but increases SfbI. These results demonstrate a close connection between the regulation of cell division and protein anchoring. Better understanding of the spatial regulation of surface anchoring may lead to the identification of novel targets for the development of anti-infective agents, given the importance of surface molecules for pathogenesis. PMID:22512736

  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 anchoredmore » 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.« less

  7. Requirements for the membrane insertion of signal-anchor type proteins

    PubMed Central

    1991-01-01

    Proteins which are inserted and anchored in the membrane of the ER by an uncleaved signal-anchor sequence can assume two final orientations. Type I signal-anchor proteins translocate the NH2 terminus across the membrane while type II signal-anchor proteins translocate the COOH terminus. We investigated the requirements for cytosolic protein components and nucleotides for the membrane targeting and insertion of single-spanning type I signal-anchor proteins. Besides the ribosome, signal recognition particle (SRP), GTP, and rough microsomes (RMs) no other components were found to be required. The GTP analogue GMPPNP could substitute for GTP in supporting the membrane insertion of IMC- CAT. By using a photocrosslinking assay we show that for secreted, type I and type II signal-anchor proteins the presence of both GTP and RMs is required for the release of the nascent chain from the 54-kD subunit of SRP. For two of the proteins studied the release of the nascent chain from SRP54 was accompanied by a new interaction with components of the ER. We conclude that the GTP-dependent release of the nascent chain from SRP54 occurs in an identical manner for each of the proteins studied. PMID:1848865

  8. GPI anchor transamidase of Trypanosoma brucei: in vitro assay of the recombinant protein and VSG anchor exchange.

    PubMed

    Kang, Xuedong; Szallies, Alexander; Rawer, Marc; Echner, Hartmut; Duszenko, Michael

    2002-06-15

    GPI8 from Trypanosoma brucei was cloned and expressed in Escherichia coli. TbGPI8 encodes a 37 kDa protein (35 kDa after removal of the putative signal sequence) with a pI of 5.5. It contains one potential N-glycosylation site near the N-terminus but no C-terminal hydrophobic region. Enzyme activity assays using trypanosomal lysates or recombinant TbGpi8 exhibited cleavage of the synthetic peptide acetyl-S-V-L-N-aminomethyl-coumarine, indicating that TbGpi8 is indeed directly involved in the proteolytic processing of the GPI anchoring signal. Intracellular localization of TbGpi8 within tubular structures, such as the endoplasmic reticulum, was observed by using specific anti-TbGpi8 antibodies. The transamidase mechanism of GPI anchoring was studied in bloodstream forms of Trypanosoma brucei using media containing hydrazine or biotinylated hydrazine. In the presence of the latter nucleophile, part of the newly formed VSG was linked to this instead of the GPI anchor and was not transferred to the cell surface. VSG-hydrazine-biotin was detected by streptavidin in western blots and intracellularly in Golgi-like compartments.

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  11. Aurora-A kinase phosphorylation of Aurora-A kinase interacting protein (AIP) and stabilization of the enzyme-substrate complex.

    PubMed

    Katayama, Hiroshi; Sasai, Kaori; Czerniak, Bogdan A; Carter, Jennifer L; Sen, Subrata

    2007-12-01

    Aurora-A is an oncogenic kinase that plays essential roles in mitosis as well as cell survival. Aurora-A interacting protein (AIP) was identified as a negative regulator of Aurora-A with its ectopic over expression inducing destabilization of Aurora-A protein. Here we present evidence that in human cells, contrary to the earlier report, AIP functions in stabilizing rather than destabilizing Aurora-A. Furthermore, AIP is phosphorylated on Serine 70 by Aurora-A but not Aurora-B and expression of phosphorylation mimic mutant of AIP results in prolonged protein stability compared to unphosphorylatable mutant. We observed that when co-expressed with AIP, protein levels of both Aurora-A and Aurora-B are markedly elevated regardless of their kinase activities and phosphorylation state of AIP. Interaction of Aurora kinases with AIP is necessary for this elevated stability. This phenomenon is commonly detected in several human cancer cell lines used in this study. Depletion of AIP by RNA interference decreased Aurora-A but not Aurora-B in two of the three cell lines analyzed, indicating that under physiological condition, AIP functions in stabilization of Aurora-A but not Aurora-B, though this regulation may be dependent on additional factors as well. Further, AIP siRNA induced cell cycle arrest at G2/M, which is consistent with anticipated loss of function of Aurora-A in these cells. Thus, our study provides the first evidence of a role for AIP in G2/M cell cycle progression by cooperatively regulating protein stabilization of its up-stream regulator, Aurora-A kinase through protein-protein interaction as well as protein phosphorylation. (c) 2007 Wiley-Liss, Inc.

  12. Tritium labelling of a cholesterol amphiphile designed for cell membrane anchoring of proteins.

    PubMed

    Schäfer, Balázs; Orbán, Erika; Kele, Zoltán; Tömböly, Csaba

    2015-01-01

    Cell membrane association of proteins can be achieved by the addition of lipid moieties to the polypeptide chain, and such lipid-modified proteins have important biological functions. A class of cell surface proteins contains a complex glycosylphosphatidylinositol (GPI) glycolipid at the C-terminus, and they are accumulated in cholesterol-rich membrane microdomains, that is, lipid rafts. Semisynthetic lipoproteins prepared from recombinant proteins and designed lipids are valuable probes and model systems of the membrane-associated proteins. Because GPI-anchored proteins can be reinserted into the cell membrane with the retention of the biological function, they are appropriate candidates for preparing models via reduction of the structural complexity. A synthetic headgroup was added to the 3β-hydroxyl group of cholesterol, an essential lipid component of rafts, and the resulting cholesterol derivative was used as a simplified GPI mimetic. In order to quantitate the membrane integrated GPI mimetic after the exogenous addition to live cells, a tritium labelled cholesterol anchor was prepared. The radioactive label was introduced into the headgroup, and the radiolabelled GPI mimetic anchor was obtained with a specific activity of 1.37 TBq/mmol. The headgroup labelled cholesterol derivative was applied to demonstrate the sensitive detection of the cell membrane association of the anchor under in vivo conditions. Copyright © 2015 John Wiley & Sons, Ltd.

  13. Protein-Anchoring Therapy of Biglycan for Mdx Mouse Model of Duchenne Muscular Dystrophy.

    PubMed

    Ito, Mikako; Ehara, Yuka; Li, Jin; Inada, Kosuke; Ohno, Kinji

    2017-05-01

    Duchenne muscular dystrophy (DMD) is a devastating muscle disease caused by loss-of-function mutations in DMD encoding dystrophin. No rational therapy is currently available. Utrophin is a paralog of dystrophin and is highly expressed at the neuromuscular junction. In mdx mice, utrophin is naturally upregulated throughout the muscle fibers, which mitigates muscular dystrophy. Protein-anchoring therapy was previously reported, in which a recombinant extracellular matrix (ECM) protein is delivered to and anchored to a specific target using its proprietary binding domains. Being prompted by a report that intramuscular and intraperitoneal injection of an ECM protein, biglycan, upregulates expression of utrophin and ameliorates muscle pathology in mdx mice, protein-anchoring therapy was applied to mdx mice. Recombinant adeno-associated virus serotype 8 (rAAV8) carrying hBGN encoding human biglycan was intravenously injected into 5-week-old mdx mice. The rAAV8-hBGN treatment improved motor deficits and decreased plasma creatine kinase activities. In muscle sections of treated mice, the number of central myonuclei and the distribution of myofiber sizes were improved. The treated mice increased gene expressions of utrophin and β1-syntrophin, as well as protein expressions of biglycan, utrophin, γ-sarcoglycan, dystrobrevin, and α1-syntrophin. The expression of hBGN in the skeletal muscle of the treated mice was 1.34-fold higher than that of the native mouse Bgn (mBgn). The low transduction efficiency and improved motor functions suggest that biglycan expressed in a small number of muscle fibers was likely to have been secreted and anchored to the cell surface throughout the whole muscular fibers. It is proposed that the protein-anchoring strategy can be applied not only to deficiency of an ECM protein as previously reported, but also to augmentation of a naturally induced ECM protein.

  14. Msp1 Is a Membrane Protein Dislocase for Tail-Anchored Proteins.

    PubMed

    Wohlever, Matthew L; Mateja, Agnieszka; McGilvray, Philip T; Day, Kasey J; Keenan, Robert J

    2017-07-20

    Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Biosynthesis of GPI-anchored proteins: special emphasis on GPI lipid remodeling

    PubMed Central

    Kinoshita, Taroh; Fujita, Morihisa

    2016-01-01

    Glycosylphosphatidylinositols (GPIs) act as membrane anchors of many eukaryotic cell surface proteins. GPIs in various organisms have a common backbone consisting of ethanolamine phosphate (EtNP), three mannoses (Mans), one non-N-acetylated glucosamine, and inositol phospholipid, whose structure is EtNP-6Manα-2Manα-6Manα-4GlNα-6myoinositol-P-lipid. The lipid part is either phosphatidylinositol of diacyl or 1-alkyl-2-acyl form, or inositol phosphoceramide. GPIs are attached to proteins via an amide bond between the C-terminal carboxyl group and an amino group of EtNP. Fatty chains of inositol phospholipids are inserted into the outer leaflet of the plasma membrane. More than 150 different human proteins are GPI anchored, whose functions include enzymes, adhesion molecules, receptors, protease inhibitors, transcytotic transporters, and complement regulators. GPI modification imparts proteins with unique characteristics, such as association with membrane microdomains or rafts, transient homodimerization, release from the membrane by cleavage in the GPI moiety, and apical sorting in polarized cells. GPI anchoring is essential for mammalian embryogenesis, development, neurogenesis, fertilization, and immune system. Mutations in genes involved in remodeling of the GPI lipid moiety cause human diseases characterized by neurological abnormalities. Yeast Saccharomyces cerevisiae has >60 GPI-anchored proteins (GPI-APs). GPI is essential for growth of yeast. In this review, we discuss biosynthesis of GPI-APs in mammalian cells and yeast with emphasis on the lipid moiety. PMID:26563290

  16. Current perspective on protein S-acylation in plants: more than just a fatty anchor?

    PubMed

    Hurst, Charlotte H; Hemsley, Piers A

    2015-03-01

    Membranes are an important signalling platform in plants. The plasma membrane is the point where information about the external environment must be converted into intracellular signals, while endomembranes are important sites of protein trafficking, organization, compartmentalization, and intracellular signalling. This requires co-ordinating the spatial distribution of proteins, their activation state, and their interacting partners. This regulation frequently occurs through post-translational modification of proteins. Proteins that associate with the cell membrane do so through transmembrane domains, protein-protein interactions, lipid binding motifs/domains or use the post-translational addition of lipid groups as prosthetic membrane anchors. S-acylation is one such lipid modification capable of anchoring proteins to the membrane. Our current knowledge of S-acylation function in plants is fairly limited compared with other post-translational modifications and S-acylation in other organisms. However, it is becoming increasingly clear that S-acylation can act as more than just a simple membrane anchor: it can also act as a regulatory mechanism in signalling pathways in plants. S-acylation is, therefore, an ideal mechanism for regulating protein function at membranes. This review discusses our current knowledge of S-acylated proteins in plants, the interaction of different lipid modifications, and the general effects of S-acylation on cellular function. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  17. 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 themore » 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.« less

  18. Computationally-guided optimization of small-molecule inhibitors of the Aurora A kinase-TPX2 protein-protein interaction.

    PubMed

    Cole, Daniel J; Janecek, Matej; Stokes, Jamie E; Rossmann, Maxim; Faver, John C; McKenzie, Grahame J; Venkitaraman, Ashok R; Hyvönen, Marko; Spring, David R; Huggins, David J; Jorgensen, William L

    2017-08-17

    Free energy perturbation theory, in combination with enhanced sampling of protein-ligand binding modes, is evaluated in the context of fragment-based drug design, and used to design two new small-molecule inhibitors of the Aurora A kinase-TPX2 protein-protein interaction.

  19. 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

  20. 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? © 2016 Authors; published by Portland Press Limited.

  1. Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46

    SciTech Connect

    Balachandran, Manasi; Giannone, Richard J.; Bemis, David A.

    2017-08-31

    Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins withmore » an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.« less

  2. Yeast telomerase and the SUN domain protein Mps3 anchor telomeres and repress subtelomeric recombination.

    PubMed

    Schober, Heiko; Ferreira, Helder; Kalck, Véronique; Gehlen, Lutz R; Gasser, Susan M

    2009-04-15

    Telomeres form the ends of linear chromosomes and protect these ends from being recognized as DNA double-strand breaks. Telomeric sequences are maintained in most cells by telomerase, a reverse transcriptase that adds TG-rich repeats to chromosome ends. In budding yeast, telomeres are organized in clusters at the nuclear periphery by interactions that depend on components of silent chromatin and the telomerase-binding factor yeast Ku (yKu). In this study, we examined whether the subnuclear localization of telomeres affects end maintenance. A telomere anchoring pathway involving the catalytic yeast telomerase subunits Est2, Est1, and Tlc1 is shown to be necessary for the perinuclear anchoring activity of Yku80 during S phase. Additionally, we identify the conserved Sad1-UNC-84 (SUN) domain protein Mps3 as the principal membrane anchor for this pathway. Impaired interference with Mps3 anchoring through overexpression of the Mps3 N terminus in a tel1 deletion background led to a senescence phenotype and to deleterious levels of subtelomeric Y' recombination. This suggests that telomere binding to the nuclear envelope helps protect telomeric repeats from recombination. Our results provide an example of a specialized structure that requires proper spatiotemporal localization to fulfill its biological role, and identifies a novel pathway of telomere protection.

  3. Yeast telomerase and the SUN domain protein Mps3 anchor telomeres and repress subtelomeric recombination

    PubMed Central

    Schober, Heiko; Ferreira, Helder; Kalck, Véronique; Gehlen, Lutz R.; Gasser, Susan M.

    2009-01-01

    Telomeres form the ends of linear chromosomes and protect these ends from being recognized as DNA double-strand breaks. Telomeric sequences are maintained in most cells by telomerase, a reverse transcriptase that adds TG-rich repeats to chromosome ends. In budding yeast, telomeres are organized in clusters at the nuclear periphery by interactions that depend on components of silent chromatin and the telomerase-binding factor yeast Ku (yKu). In this study, we examined whether the subnuclear localization of telomeres affects end maintenance. A telomere anchoring pathway involving the catalytic yeast telomerase subunits Est2, Est1, and Tlc1 is shown to be necessary for the perinuclear anchoring activity of Yku80 during S phase. Additionally, we identify the conserved Sad1–UNC-84 (SUN) domain protein Mps3 as the principal membrane anchor for this pathway. Impaired interference with Mps3 anchoring through overexpression of the Mps3 N terminus in a tel1 deletion background led to a senescence phenotype and to deleterious levels of subtelomeric Y′ recombination. This suggests that telomere binding to the nuclear envelope helps protect telomeric repeats from recombination. Our results provide an example of a specialized structure that requires proper spatiotemporal localization to fulfill its biological role, and identifies a novel pathway of telomere protection. PMID:19390087

  4. Determination of the non-ionic detergent insolubility and phosphoprotein associations of glycosylphosphatidylinositol-anchored proteins expressed on T cells.

    PubMed Central

    Solomon, K R; Mallory, M A; Finberg, R W

    1998-01-01

    Glycosylphosphatidylinositol (GPI)-anchored proteins are poorly solublized in non-ionic detergents such as Triton X-100 and Nonidet P40, but are easily solublized by detergents with high critical micelle concentrations such as octylglucoside. This solubility profile has been suggested to be due to the localization of GPI-anchored proteins to lipid microdomains rich in cholesterol and sphingolipids. Additionally, GPI-anchored proteins expressed on haemopoietic cells have been shown to associate with src-family tyrosine kinases and heterotrimeric G proteins. Despite these observations, the non-ionic detergent insolubility of GPI-anchored proteins on haemopoietic cells has not been quantified nor has a relationship between the non-ionic detergent insolubility of these proteins and their association with signal-transduction molecules been identified. Here we show that GPI-anchored proteins found on T-cell tumours and activated T cells, although significantly more insoluble then transmembrane proteins, are not uniform in their detergent insolubility. Whereas CD59 was between 4% and 13% soluble, CD48 was between 13% and 25% soluble, CD55 was between 20% and 30% soluble, and CD109 was between 34% and 75% soluble. The ability of these GPI-anchored proteins to associate with phosphoproteins was correlated with their detergent insolubility: the more detergent-insoluble that a GPI-anchored protein was, the greater the level of phosphoprotein associations. These experiments reveal a relationship between non-ionic detergent insolubility and association with signal-transduction molecules and suggest a cause-and-effect relationship between these two properties. In total, these experiments support the hypothesis that the association of GPI-anchored proteins with signalling molecules is due to their sorting to lipid microdomains. PMID:9716490

  5. Determination of the non-ionic detergent insolubility and phosphoprotein associations of glycosylphosphatidylinositol-anchored proteins expressed on T cells.

    PubMed

    Solomon, K R; Mallory, M A; Finberg, R W

    1998-09-01

    Glycosylphosphatidylinositol (GPI)-anchored proteins are poorly solublized in non-ionic detergents such as Triton X-100 and Nonidet P40, but are easily solublized by detergents with high critical micelle concentrations such as octylglucoside. This solubility profile has been suggested to be due to the localization of GPI-anchored proteins to lipid microdomains rich in cholesterol and sphingolipids. Additionally, GPI-anchored proteins expressed on haemopoietic cells have been shown to associate with src-family tyrosine kinases and heterotrimeric G proteins. Despite these observations, the non-ionic detergent insolubility of GPI-anchored proteins on haemopoietic cells has not been quantified nor has a relationship between the non-ionic detergent insolubility of these proteins and their association with signal-transduction molecules been identified. Here we show that GPI-anchored proteins found on T-cell tumours and activated T cells, although significantly more insoluble then transmembrane proteins, are not uniform in their detergent insolubility. Whereas CD59 was between 4% and 13% soluble, CD48 was between 13% and 25% soluble, CD55 was between 20% and 30% soluble, and CD109 was between 34% and 75% soluble. The ability of these GPI-anchored proteins to associate with phosphoproteins was correlated with their detergent insolubility: the more detergent-insoluble that a GPI-anchored protein was, the greater the level of phosphoprotein associations. These experiments reveal a relationship between non-ionic detergent insolubility and association with signal-transduction molecules and suggest a cause-and-effect relationship between these two properties. In total, these experiments support the hypothesis that the association of GPI-anchored proteins with signalling molecules is due to their sorting to lipid microdomains.

  6. Plasmid DNA immunization against Japanese encephalitis virus: immunogenicity of membrane-anchored and secretory envelope protein.

    PubMed

    Kaur, Rupinderjeet; Sachdeva, Geetanjali; Vrati, Sudhanshu

    2002-01-01

    Plasmid DNA synthesizing membrane-anchored or secretory Japanese encephalitis virus (JEV) envelope (E) protein and premembrane protein was delivered to mice by intramuscular injection or gene gun. Intramuscular plasmid immunization induced anti-E antibody responses similar to those associated with commercial JEV vaccine. The gene gun induced less antibody response. The 2 forms of the E protein induced similar antibody titers when administered by the same delivery mode. Both plasmids generated high titers of JEV-neutralizing antibodies, although the titers were lower than those induced by the vaccine. Intramuscular DNA immunization induced T helper 1 (Th1) immune responses, and the gene gun induced Th2 responses. Compared with secretory E protein, the membrane-anchored protein heavily skewed the immune response toward either Th1 or Th2, depending on the route of immunization. In an intracerebral JEV challenge model, plasmid-immunized mice had approximately 60% protection; this was not affected by the form of the E protein or by immunization route.

  7. Mechanism for the Posttranslational Membrane Insertion of the Tail-Anchored Proteins by Get Complex

    NASA Astrophysics Data System (ADS)

    Yamagata, Atsushi

    Tail anchored proteins, defined by a single transmembrane domain at C-terminus, are post-translationally inserted into the membrane by Get3 ATPase. Here we report the crystal structure of Get3 in ADP-bound and nucleotide free forms. Get3 forms an open dimer conformation, in which two subunits are linked by a Zn2+ ion. Together with the biochemical studies, we propose the ATP independent TA protein binding, and the membrane insertion by conformational change of Get3 coupled with ATP hydrolysis.

  8. A protean clamp guides membrane targeting of tail-anchored proteins

    PubMed Central

    Chio, Un Seng; Chung, SangYoon; Weiss, Shimon

    2017-01-01

    Proper localization of proteins to target membranes is a fundamental cellular process. How the nature and dynamics of the targeting complex help guide substrate proteins to the target membrane is not understood for most pathways. Here, we address this question for the conserved ATPase guided entry of tail-anchored protein 3 (Get3), which targets the essential class of tail-anchored proteins (TAs) to the endoplasmic reticulum (ER). Single-molecule fluorescence spectroscopy showed that, contrary to previous models of a static closed Get3•TA complex, Get3 samples open conformations on the submillisecond timescale upon TA binding, generating a fluctuating “protean clamp” that stably traps the substrate. Point mutations at the ATPase site bias Get3 toward closed conformations, uncouple TA binding from induced Get3•Get4/5 disassembly, and inhibit the ER targeting of the Get3•TA complex. These results demonstrate an essential role of substrate-induced Get3 dynamics in driving TA targeting to the membrane, and reveal a tightly coupled channel of communication between the TA-binding site, ATPase site, and effector interaction surfaces of Get3. Our results provide a precedent for large-scale dynamics in a substrate-bound chaperone, which provides an effective mechanism to retain substrate proteins with high affinity while also generating functional switches to drive vectorial cellular processes. PMID:28973888

  9. Yeast arming systems: pros and cons of different protein anchors and other elements required for display.

    PubMed

    Andreu, Cecilia; Del Olmo, Marcel Lí

    2018-03-01

    Yeast display is a powerful strategy that consists in exposing peptides or proteins of interest on the cell surface of this microorganism. Ever since initial experiments with this methodology were carried out, its scope has extended and many applications have been successfully developed in different science and technology fields. Several yeast display systems have been designed, which all involve introducting into yeast cells the gene fusions that contain the coding regions of a signal peptide, an anchor protein, to properly attach the target to the cell surface, and the protein of interest to be exposed, all of which are controlled by a strong promoter. In this work, we report the description of such elements for the alternative systems introduced by focusing particularly on anchor proteins. The comparisons made between them are included whenever possible, and the main advantages and inconveniences of each one are discussed. Despite the huge number of publications on yeast surface display and the revisions published to date, this topic has not yet been widely considered. Finally, given the growing interest in developing systems for non-Saccharomyces yeasts, the main strategies reported for some are also summarized.

  10. MARCH6 and TRC8 facilitate the quality control of cytosolic and tail-anchored proteins.

    PubMed

    Stefanovic-Barrett, Sandra; Dickson, Anna S; Burr, Stephen P; Williamson, James C; Lobb, Ian T; van den Boomen, Dick Jh; Lehner, Paul J; Nathan, James A

    2018-03-08

    Misfolded or damaged proteins are typically targeted for destruction by proteasome-mediated degradation, but the mammalian ubiquitin machinery involved is incompletely understood. Here, using forward genetic screens in human cells, we find that the proteasome-mediated degradation of the soluble misfolded reporter, mCherry-CL1, involves two ER-resident E3 ligases, MARCH6 and TRC8. mCherry-CL1 degradation is routed via the ER membrane and dependent on the hydrophobicity of the substrate, with complete stabilisation only observed in double knockout MARCH6/TRC8 cells. To identify a more physiological correlate, we used quantitative mass spectrometry and found that TRC8 and MARCH6 depletion altered the turnover of the tail-anchored protein heme oxygenase-1 (HO-1). These E3 ligases associate with the intramembrane cleaving signal peptide peptidase (SPP) and facilitate the degradation of HO-1 following intramembrane proteolysis. Our results highlight how ER-resident ligases may target the same substrates, but work independently of each other, to optimise the protein quality control of selected soluble and tail-anchored proteins. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

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

    USDA-ARS?s Scientific Manuscript database

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

    USDA-ARS?s Scientific Manuscript database

    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...

  13. Implications of lipid moiety in oligomerization and immunoreactivities of GPI-anchored proteins

    PubMed Central

    Seong, Jihyoun; Wang, Yetao; Kinoshita, Taroh; Maeda, Yusuke

    2013-01-01

    Glycosylphosphatidylinositol (GPI) enriches GPI-anchored proteins (GPI-AP) in lipid rafts by intimate interaction of its lipid moiety with sphingolipids and cholesterol. In addition to such lipid-lipid interactions, it has been reported that GPI may interact with protein moiety linked to GPI and affect protein conformations because GPI delipidation reduced immunoreactivities of protein. Here, we report that GPI-APs that have not undergone fatty acid remodeling exhibit reduced immunoreactivities in Western blotting, similar to delipidated proteins, compared with normal remodeled GPI-APs. In contrast, immunostaining in flow cytometry and immunoprecipitation did not show significant differences between remodeled and unremodeled GPI-APs. Moreover, detection with premixed primary/secondary antibody complexes or Fab fragments eliminated this difference in Western blotting. These results indicate that normally remodeled GPI enhanced oligomerization of GPI-APs and that inefficient oligomerization of unremodeled GPI-APs was responsible for reduced immunoreactivities. Moreover, the reduction in immunoreactivities of delipidated GPI-APs was most likely caused by the same effect. Finally, by chemical cross-linking of surface proteins in living cells and cell killing assay using a pore-forming bacterial toxin, we showed that enhanced oligomerization by GPI-remodeling occurs under a physiological membrane environment. Thus, this study clarifies the significance of GPI fatty acid remodeling in oligomerization of GPI-APs and provides useful information for technical studies of these cell components. PMID:23378600

  14. 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

  15. Ground anchors and anchored systems

    DOT National Transportation Integrated Search

    1999-06-01

    This document presents state-of-the-practice information on the design and installation of cement-grouted ground anchors and anchored systems for highway applications. The anchored systems discussed include flexible anchored walls, slopes supported u...

  16. Structural insights into tail-anchored protein binding and membrane insertion by Get3

    PubMed Central

    Bozkurt, Gunes; Stjepanovic, Goran; Vilardi, Fabio; Amlacher, Stefan; Wild, Klemens; Bange, Gert; Favaloro, Vincenzo; Rippe, Karsten; Hurt, Ed; Dobberstein, Bernhard; Sinning, Irmgard

    2009-01-01

    Tail-anchored (TA) membrane proteins are involved in a variety of important cellular functions, including membrane fusion, protein translocation, and apoptosis. The ATPase Get3 (Asna1, TRC40) was identified recently as the endoplasmic reticulum targeting factor of TA proteins. Get3 consists of an ATPase and α-helical subdomain enriched in methionine and glycine residues. We present structural and biochemical analyses of Get3 alone as well as in complex with a TA protein, ribosome-associated membrane protein 4 (Ramp4). The ATPase domains form an extensive dimer interface that encloses 2 nucleotides in a head-to-head orientation and a zinc ion. Amide proton exchange mass spectrometry shows that the α-helical subdomain of Get3 displays considerable flexibility in solution and maps the TA protein-binding site to the α-helical subdomain. The non-hydrolyzable ATP analogue AMPPNP-Mg2+- and ADP-Mg2+-bound crystal structures representing the pre- and posthydrolysis states are both in a closed form. In the absence of a TA protein cargo, ATP hydrolysis does not seem to be possible. Comparison with the ADP·AlF4−-bound structure representing the transition state (Mateja A, et al. (2009) Nature 461:361–366) indicates how the presence of a TA protein is communicated to the ATP-binding site. In vitro membrane insertion studies show that recombinant Get3 inserts Ramp4 in a nucleotide- and receptor-dependent manner. Although ATP hydrolysis is not required for Ramp4 insertion per se, it seems to be required for efficient insertion. We postulate that ATP hydrolysis is needed to release Get3 from its receptor. Taken together, our results provide mechanistic insights into posttranslational targeting of TA membrane proteins by Get3. PMID:19948960

  17. Ice-Nucleating and Antifreeze Proteins Recognize Ice through a Diversity of Anchored Clathrate and Ice-like Motifs.

    PubMed

    Hudait, Arpa; Odendahl, Nathan; Qiu, Yuqing; Paesani, Francesco; Molinero, Valeria

    2018-04-11

    Cold-adapted organisms produce antifreeze and ice-nucleating proteins to prevent and promote ice formation. The crystal structure of hyperactive bacterial antifreeze protein (AFP) MpAFP suggests that this protein binds ice through an anchored clathrate motif. It is not known whether other hyperactive AFPs and ice-nucleating proteins (INPs) use the same motif to recognize or nucleate ice. Here we use molecular simulations to elucidate the ice-binding motifs of hyperactive insect AFPs and a model INP of Pseudomonas syringae. We find that insect AFPs recognize ice through anchored clathrate motifs distinct from that of MpAFP. By performing simulations of ice nucleation by PsINP, we identify two distinct ice-binding sites on opposite sides of the β-helix. The ice-nucleating sequences identified in the simulations agree with those previously proposed for the closely related INP of Pseudomonas borealis based on the structure of the protein. The simulations indicate that these sites have comparable ice-nucleating efficiency, but distinct binding motifs, controlled by the amino acid sequence: one is an anchored clathrate and the other ice-like. We conclude that anchored clathrate and ice-like motifs can be equally effective for binding proteins to ice and promoting ice nucleation.

  18. Reprogramming of G protein-coupled receptor recycling and signaling by a kinase switch

    PubMed Central

    Vistein, Rachel; Puthenveedu, Manojkumar A.

    2013-01-01

    The postendocytic recycling of signaling receptors is subject to multiple requirements. Why this is so, considering that many other proteins can recycle without apparent requirements, is a fundamental question. Here we show that cells can leverage these requirements to switch the recycling of the beta-2 adrenergic receptor (B2AR), a prototypic signaling receptor, between sequence-dependent and bulk recycling pathways, based on extracellular signals. This switch is determined by protein kinase A-mediated phosphorylation of B2AR on the cytoplasmic tail. The phosphorylation state of B2AR dictates its partitioning into spatially and functionally distinct endosomal microdomains mediating bulk and sequence-dependent recycling, and also regulates the rate of B2AR recycling and resensitization. Our results demonstrate that G protein-coupled receptor recycling is not always restricted to the sequence-dependent pathway, but may be reprogrammed as needed by physiological signals. Such flexible reprogramming might provide a versatile method for rapidly modulating cellular responses to extracellular signaling. PMID:24003153

  19. The elusive activity of the Yersinia protein kinase A kinase domain is revealed.

    PubMed

    Laskowski-Arce, Michelle A; Orth, Kim

    2007-10-01

    Yersinia spp. pathogens use their type III secretion system to translocate effectors that manipulate host signaling pathways during infection. Although molecular targets for five of the six known Yersinia effectors are known, the target for the serine/threonine kinase domain of Yersinia protein kinase A (YpkA) has remained elusive. Recently, Navarro et al. (2007) demonstrated that YpkA phosphorylates Galphaq, and inhibits Galphaq-mediated signaling. Inhibition by YpkA could contribute to one of the most documented symptoms of Yersinia pestis infection, extensive bleeding.

  20. 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

  1. Characterization of the GPI-anchored lipid transfer proteins in the moss Physcomitrella patens.

    PubMed

    Edstam, Monika M; Laurila, Maiju; Höglund, Andrey; Raman, Amitha; Dahlström, Käthe M; Salminen, Tiina A; Edqvist, Johan; Blomqvist, Kristina

    2014-02-01

    The non-specific lipid transfer proteins (nsLTPs) are characterized by a compact structure with a central hydrophobic cavity very suitable for binding hydrophobic ligands, such as lipids. The nsLTPs are encoded by large gene families in all land plant lineages, but seem to be absent from green algae. The nsLTPs are classified to different types based on molecular weight, 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 may attach the protein to the exterior side of the plasma membrane. Here, we present the first characterization of nsLTPs from an early diverged plant, the moss Physcomitrella patens. Moss LTPGs were heterologously produced and purified from Pichia pastoris. The purified moss LTPGs were found to be extremely heat stable and showed a binding preference for unsaturated fatty acids. Structural modeling implied that high alanine content could be important for the heat stability. Lipid profiling revealed that cutin monomers, such as C16 and C18 mono- and di-hydroxylated fatty acids, could be identified in P. patens. Expression of a moss LTPG-YFP fusion revealed localization to the plasma membrane. The expressions of many of the moss LTPGs were found to be upregulated during drought and cold treatments. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

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

    PubMed

    Lecomte, Xavier; Gagnaire, Valérie; Briard-Bion, Valérie; Jardin, Julien; Lortal, Sylvie; Dary, Annie; Genay, Magali

    2014-06-05

    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. 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 and anchoring of a

  3. 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

  4. 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

  5. A novel CXCL10-based GPI-anchored fusion protein as adjuvant in NK-based tumor therapy.

    PubMed

    Muenchmeier, Niklas; Boecker, Sophia; Bankel, Lorenz; Hinz, Laura; Rieth, Nicole; Lapa, Constantin; Mendler, Anna N; Noessner, Elfriede; Mocikat, Ralph; Nelson, Peter J

    2013-01-01

    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. 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. 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 targeted recruitment of cells in various settings.

  6. Functional convergence of signalling by GPI-anchored and anchorless forms of a salamander protein implicated in limb regeneration

    PubMed Central

    Blassberg, Robert A.; Garza-Garcia, Acely; Janmohamed, Azara; Gates, Phillip B.; Brockes, Jeremy P.

    2011-01-01

    The GPI-anchor is an established determinant of molecular localisation and various functional roles have been attributed to it. The newt GPI-anchored three-finger protein (TFP) Prod1 is an important regulator of cell behaviour during limb regeneration, but it is unclear how it signals to the interior of the cell. Prod1 was expressed by transfection in cultured newt limb cells and activated transcription and expression of matrix metalloproteinase 9 (MMP9) by a pathway involving ligand-independent activation of epidermal growth factor receptor (EGFR) signalling and phosphorylation of extracellular regulated kinase 1 and 2 (ERK1/2). This was dependent on the presence of the GPI-anchor and critical residues in the α-helical region of the protein. Interestingly, Prod1 in the axolotl, a salamander species that also regenerates its limbs, was shown to activate ERK1/2 signalling and MMP9 transcription despite being anchorless, and both newt and axolotl Prod1 co-immunoprecipitated with the newt EGFR after transfection. The substitution of the axolotl helical region activated a secreted, anchorless version of the newt molecule. The activity of the newt molecule cannot therefore depend on a unique property conferred by the anchor. Prod1 is a salamander-specific TFP and its interaction with the phylogenetically conserved EGFR has implications for our view of regeneration as an evolutionary variable. PMID:21118959

  7. New Method for Measuring the Anchoring Energy of Strongly-Bound Membrane-Associated Proteins [Method for measuring the anchoring energy of strongly-bound membrane-associated proteins].

    DOE PAGES

    Kent, Michael S.; La Bauve, Elisa; Vernon, Briana C.; ...

    2016-02-01

    Here, we describe a new method to measure the activation energy required to remove a strongly-bound membrane-associated protein from a lipid membrane (anchoring energy). It is based on measuring the rate of release of a liposome-bound protein during centrifugation on a sucrose gradient as a function of time and temperature. The method was used to determine anchoring energy for the soluble dengue virus envelope protein (sE) strongly bound to 80:20 POPC:POPG liposomes at pH 5.5. We also measured the binding energy of sE at the same pH for the initial, predominantly reversible, phase of binding to a 70:30 PC:PG lipidmore » bilayer. The anchoring energy (37 +/- 1.7 kcal/mol, 20% PG) was found to be much larger than the binding energy (7.8 +/- 0.3 kcal/mol for 30% PG, or est. 7.0 kcal/mol for 20% PG). This is consistent with data showing that free sE is a monomer at pH 5.5, but assembles into trimers after associating with membranes. But, trimerization alone is insufficient to account for the observed difference in energies, and we conclude that some energy dissipation occurs during the release process. This new method to determine anchoring energy should be useful to understand the complex interactions of integral monotopic proteins and strongly-bound peripheral membrane proteins with lipid membranes.« less

  8. Coarse-grained simulations of the HIV-1 matrix protein anchoring: revisiting its assembly on membrane domains.

    PubMed

    Charlier, Landry; Louet, Maxime; Chaloin, Laurent; Fuchs, Patrick; Martinez, Jean; Muriaux, Delphine; Favard, Cyril; Floquet, Nicolas

    2014-02-04

    In the accepted model for human immunodeficiency virus preassembly in infected host cells, the anchoring to the intracellular leaflet of the membrane of the matrix domain (MA) that lies at the N-terminus of the viral Gag protein precursor appears to be one of the crucial steps for particle assembly. In this study, we simulated the membrane anchoring of human immunodeficiency virus-1 myristoylated MA protein using a coarse-grained representation of both the protein and the membrane. Our calculations first suggest that the myristoyl group could spontaneously release from its initial hydrophobic pocket before MA protein interacts with the lipid membrane. All-atom simulations confirmed this possibility with a related energy cost estimated to be ~5 kcal.mol(-1). The phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) head binds preferentially to the MA highly basic region as described in available NMR data, but interestingly without flipping of its 2' acyl chain into the MA protein. Moreover, MA was able to confine PI(4,5)P2 lipids all around its molecular surface after having found a stable orientation at the membrane surface. Our results suggest that this orientation is dependent on Myr anchoring and that this confinement induces a lateral segregation of PI(4,5)P2 in domains. This is consistent with a PI(4,5)P2 enrichment of the virus envelope as compared to the host cell membrane. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  9. Coarse-Grained Simulations of the HIV-1 Matrix Protein Anchoring: Revisiting Its Assembly on Membrane Domains

    PubMed Central

    Charlier, Landry; Louet, Maxime; Chaloin, Laurent; Fuchs, Patrick; Martinez, Jean; Muriaux, Delphine; Favard, Cyril; Floquet, Nicolas

    2014-01-01

    In the accepted model for human immunodeficiency virus preassembly in infected host cells, the anchoring to the intracellular leaflet of the membrane of the matrix domain (MA) that lies at the N-terminus of the viral Gag protein precursor appears to be one of the crucial steps for particle assembly. In this study, we simulated the membrane anchoring of human immunodeficiency virus-1 myristoylated MA protein using a coarse-grained representation of both the protein and the membrane. Our calculations first suggest that the myristoyl group could spontaneously release from its initial hydrophobic pocket before MA protein interacts with the lipid membrane. All-atom simulations confirmed this possibility with a related energy cost estimated to be ∼5 kcal.mol−1. The phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) head binds preferentially to the MA highly basic region as described in available NMR data, but interestingly without flipping of its 2′ acyl chain into the MA protein. Moreover, MA was able to confine PI(4,5)P2 lipids all around its molecular surface after having found a stable orientation at the membrane surface. Our results suggest that this orientation is dependent on Myr anchoring and that this confinement induces a lateral segregation of PI(4,5)P2 in domains. This is consistent with a PI(4,5)P2 enrichment of the virus envelope as compared to the host cell membrane. PMID:24507598

  10. Collagen XVII/BP180: a collagenous transmembrane protein and component of the dermoepidermal anchoring complex.

    PubMed

    Powell, A M; Sakuma-Oyama, Y; Oyama, N; Black, M M

    2005-11-01

    Collagen XVII, or BP180, is a collagenous transmembrane protein and a structural component of the dermoepidermal anchoring complex. Molecular studies reveal that it has a globular cytosolic amino-terminal domain and flexible-rod extracellular carboxyterminal domain. The extracellular portion of collagen XVII is constitutively shed from the cell surface by ADAMs (proteinases that contain adhesive and metalloprotease domains). Cell biological analyses suggest that collagen XVII functions as a cell-matrix adhesion molecule through stabilization of the hemidesmosome complex. This concept is supported by investigations into human diseases of the dermoepidermal junction, in which collagen XVII is either genetically defective or absent (as in some forms of nonlethal junctional epidermolysis bullosa). Autoantibodies against collagen XVII (BP180) are seen in bullous pemphigoid, pemphigoid gestationis, mucous membrane pemphigoid, linear IgA disease, lichen planus pemphigoides and pemphigoid nodularis. In vivo and in vitro studies provide evidence for a pathogenic role of these autoantibodies, and suggest that the serum level and epitope specificity of these antibodies influences disease severity and phenotype. This review summarizes the structural and biological features of collagen XVII and its role in diseases of the basement membrane zone.

  11. Regulation of ciliary motility: conserved protein kinases and phosphatases are targeted and anchored in the ciliary axoneme

    PubMed Central

    Wirschell, Maureen; Yamamoto, Ryosuke; Alford, Lea; Gokhale, Avanti; Gaillard, Anne; Sale, Winfield S.

    2011-01-01

    Recent evidence has revealed that the dynein motors and highly conserved signaling proteins are localized within the ciliary 9 + 2 axoneme. One key mechanism for regulation of motility is phosphorylation. Here, we review diverse evidence, from multiple experimental organisms, that ciliary motility is regulated by phosphorylation / dephosphorylation of the dynein arms through kinases and phosphatases that are anchored immediately adjacent to their axonemal substrates. PMID:21513695

  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 Inner Membrane Protein ElaB Increases Resistance to Stress While Reducing Persistence in Escherichia coli

    PubMed Central

    Guo, Yunxue; Liu, Xiaoxiao; Li, Baiyuan; Yao, Jianyun; Wood, Thomas K.

    2017-01-01

    ABSTRACT Host-associated bacteria, such as Escherichia coli, often encounter various host-related stresses, such as nutritional deprivation, oxidative stress, and temperature shifts. There is growing interest in searching for small endogenous proteins that mediate stress responses. Here, we characterized the small C-tail-anchored inner membrane protein ElaB in E. coli. ElaB belongs to a class of tail-anchored inner membrane proteins with a C-terminal transmembrane domain but lacking an N-terminal signal sequence for membrane targeting. Proteins from this family have been shown to play vital roles, such as in membrane trafficking and apoptosis, in eukaryotes; however, their role in prokaryotes is largely unexplored. Here, we found that the transcription of elaB is induced in the stationary phase in E. coli and stationary-phase sigma factor RpoS regulates elaB transcription by binding to the promoter of elaB. Moreover, ElaB protects cells against oxidative stress and heat shock stress. However, unlike membrane peptide toxins TisB and GhoT, ElaB does not lead to cell death, and the deletion of elaB greatly increases persister cell formation. Therefore, we demonstrate that disruption of C-tail-anchored inner membrane proteins can reduce stress resistance; it can also lead to deleterious effects, such as increased persistence, in E. coli. IMPORTANCE Escherichia coli synthesizes dozens of poorly understood small membrane proteins containing a predicted transmembrane domain. In this study, we characterized the function of the C-tail-anchored inner membrane protein ElaB in E. coli. ElaB increases resistance to oxidative stress and heat stress, while inactivation of ElaB leads to high persister cell formation. We also demonstrated that the transcription of elaB is under the direct regulation of stationary-phase sigma factor RpoS. Thus, our study reveals that small inner membrane proteins may have important cellular roles during the stress response. PMID:28242719

  14. 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

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

    PubMed Central

    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

  16. 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.

  17. Construction of Lactococcus lactis expressing secreted and anchored Eimeria tenella 3-1E protein and comparison of protective immunity against homologous challenge.

    PubMed

    Ma, Chunli; Zhang, Lili; Gao, Mingyang; Ma, Dexing

    2017-07-01

    Two novel plasmids pTX8048-SP-Δ3-1E and pTX8048-SP-NAΔ3-1E-CWA were constructed. The plasmids were respectively electrotransformed into L. lactis NZ9000 to generate strain of L. lactis/pTX8048-SP-Δ3-1E in which 3-1E protein was expressed in secretion, and L. lactis/pTX8048-SP-NAΔ3-1E-CWA on which 3-1E protein was covalently anchored to the surface of bacteria cells. The expression of target proteins were examined by Western blot. The live lactococci expressing secreted 3-1E protein, anchored 3-1E protein, and cytoplasmic 3-1E protein was administered orally to chickens respectively, and the protective immunity and efficacy were compared by animal experiment. The results showed oral immunization to chickens with recombinant lactococci expressing anchored 3-1E protein elicited high 3-1E-specific serum IgG, increased high proportion of CD4 + and CD8α + cells in spleen, alleviated average lesion score in cecum, decreased the oocyst output per chicken compared to lactococci expressing cytoplasmic or secreted 3-1E protein. Taken together, these findings indicated the surface anchored Eimeria protein displayed by L. lacits can induce protective immunity and partial protection against homologous infection. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. A ternary membrane protein complex anchors the spindle pole body in the nuclear envelope in budding yeast.

    PubMed

    Kupke, Thomas; Malsam, Jörg; Schiebel, Elmar

    2017-05-19

    In budding yeast ( Saccharomyces cerevisia e) the multilayered spindle pole body (SPB) is embedded in the nuclear envelope (NE) at fusion sites of the inner and outer nuclear membrane. The SPB is built from 18 different proteins, including the three integral membrane proteins Mps3, Ndc1, and Mps2. These membrane proteins play an essential role in the insertion of the new SPB into the NE. How the huge core structure of the SPB is anchored in the NE has not been investigated thoroughly until now. The present model suggests that the NE protein Mps2 interacts via Bbp1 with Spc29, one of the coiled-coil proteins forming the central plaque of the SPB. To test this model, we purified and reconstituted the Mps2-Bbp1 complex from yeast and incorporated the complex into liposomes. We also demonstrated that Mps2-Bbp1 directly interacts with Mps3 and Ndc1. We then purified Spc29 and reconstituted the ternary Mps2-Bbp1-Spc29 complex, proving that Bbp1 can simultaneously interact with Mps2 and Spc29 and in this way link the central plaque of the SPB to the nuclear envelope. Interestingly, Bbp1 induced oligomerization of Spc29, which may represent an early step in SPB duplication. Together, this analysis provides important insights into the interaction network that inserts the new SPB into the NE and indicates that the Mps2-Bbp1 complex is the central unit of the SPB membrane anchor. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. 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

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

    SciTech Connect

    Hu, Junbin; Li, Jingzhi; Qian, Xinguo

    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. Amore » 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.« less

  1. An efficient bacterial surface display system based on a novel outer membrane anchoring element from the Escherichia coli protein YiaT.

    PubMed

    Han, Mee-Jung; Lee, Seung Hwan

    2015-01-01

    In a bacterial surface display system, the display of a successful recombinant protein is highly dependent on the choice of anchoring motif. In this study, we developed an efficient Escherichia coli display system using novel anchoring motifs derived from the protein YiaT. To determine the best surface-anchoring motif, full-length YiaT and two of its C-terminal truncated forms, cut at the R181 and R232 sites, were evaluated. Two industrial enzymes, a lipase from Pseudomonas fluorescens SIK W1 and an α-amylase from Bacillus subtilis, were used as the target proteins for display. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, immunofluorescence microscopy and whole-cell enzyme activity measurements confirmed the expression of the fusion proteins on the E. coli surface. Using YiaTR181 or YiaTR232 as the anchoring motif, the fusion proteins showed very high enzyme activities and did not exert any adverse effects on either cell growth or the outer membrane integrity. Additionally, these fusion proteins were suitable for displaying proteins of large molecular size in an active form. Compared with the previous anchoring motifs FadL and OprF, YiaTR181 and YiaTR232 had approximately 10-fold and 20-fold higher enzyme activities, respectively. These results suggest that YiaT can be used as an E. coli anchoring motif to efficiently display various enzymes; hence, this system could be employed in a variety of biotechnological and industrial applications. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. The major surface protein of Leishmania promastigotes is anchored in the membrane by a myristic acid-labeled phospholipid.

    PubMed Central

    Etges, R; Bouvier, J; Bordier, C

    1986-01-01

    Promastigotes of the protozoan parasite Leishmania major were biosynthetically labeled with myristic acid. Solubilization and phase separation in the non-ionic detergent Triton X-114 shows that the label is not incorporated into soluble hydrophilic proteins, but is incorporated into a few insoluble proteins. The bulk of the incorporated fatty acid is associated with a heterogeneous phosphorylated glycolipid and a few amphiphilic integral membrane proteins. Among these, the major surface protein of Leishmania promastigotes, p63, is predominantly labeled. Upon digestion with Bacillus cereus phospholipase C, amphiphilic p63 is shown to lose its myristic acid label and to acquire concomitantly the characteristic electrophoretic mobility and solubility behavior of hydrophilic p63. These data show that the amphiphilic character of the major surface protein of Leishmania promastigotes is due to a covalently attached phospholipid. We propose that this phospholipid provides the sole hydrophobic moiety anchoring the protein to the pellicular membrane of the protozoan parasite. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. Fig. 6. PMID:3709520

  3. Clumping factor A, von Willebrand factor-binding protein and von Willebrand factor anchor Staphylococcus aureus to the vessel wall.

    PubMed

    Claes, J; Liesenborghs, L; Peetermans, M; Veloso, T R; Missiakas, D; Schneewind, O; Mancini, S; Entenza, J M; Hoylaerts, M F; Heying, R; Verhamme, P; Vanassche, T

    2017-05-01

    Essentials Staphylococcus aureus (S. aureus) binds to endothelium via von Willebrand factor (VWF). Secreted VWF-binding protein (vWbp) mediates S. aureus adhesion to VWF under shear stress. vWbp interacts with VWF and the Sortase A-dependent surface protein Clumping factor A (ClfA). VWF-vWbp-ClfA anchor S. aureus to vascular endothelium under shear stress. Objective When establishing endovascular infections, Staphylococcus aureus (S. aureus) overcomes shear forces of flowing blood by binding to von Willebrand factor (VWF). Staphylococcal VWF-binding protein (vWbp) interacts with VWF, but it is unknown how this secreted protein binds to the bacterial cell wall. We hypothesized that vWbp interacts with a staphylococcal surface protein, mediating the adhesion of S. aureus to VWF and vascular endothelium under shear stress. Methods We studied the binding of S. aureus to vWbp, VWF and endothelial cells in a micro-parallel flow chamber using various mutants deficient in Sortase A (SrtA) and SrtA-dependent surface proteins, and Lactococcus lactis expressing single staphylococcal surface proteins. In vivo adhesion of bacteria was evaluated in the murine mesenteric circulation using real-time intravital vascular microscopy. Results vWbp bridges the bacterial cell wall and VWF, allowing shear-resistant binding of S. aureus to inflamed or damaged endothelium. Absence of SrtA and Clumping factor A (ClfA) reduced adhesion of S. aureus to vWbp, VWF and activated endothelial cells. ADAMTS-13 and an anti-VWF A1 domain antibody, when combined, reduced S. aureus adhesion to activated endothelial cells by 90%. Selective overexpression of ClfA in the membrane of Lactococcus lactis enabled these bacteria to bind to VWF and activated endothelial cells but only in the presence of vWbp. Absence of ClfA abolished bacterial adhesion to the activated murine vessel wall. Conclusions vWbp interacts with VWF and with the SrtA-dependent staphylococcal surface protein ClfA. The complex formed by

  4. Dynamic anchoring transitions at aqueous-liquid crystal interfaces induced by specific and non-specific binding of vesicles to proteins.

    PubMed

    Tan, Lie Na; Abbott, Nicholas L

    2015-07-01

    This paper reports on the dynamics of continuous anchoring transitions at interfaces formed between nematic liquid crystals (LCs, 4'-pentyl-4-cyanobiphenyl (5CB)) and immiscible aqueous phases that are induced by either non-specific or specific interactions between phospholipid vesicles and proteins adsorbed at the LC interfaces. By analyzing the dynamic response of LCs to non-specific adsorption of lipids onto bovine serum albumin (BSA)-decorated LC interfaces, we provide evidence that the LC anchoring transitions are slower than diffusion-controlled accumulation of lipid at the interface, consistent with the hypothesis that the LC transition involves lateral reorganization of proteins and lipids at the interface. Significantly, optical measurements of the tilt angle of the LC as a function of the amount of lipid captured at the interface were found to be quantitatively consistent with theoretical predictions of LC anchoring directed by nanoscopic domains of molecules that cause planar (protein) and homeotropic (lipid) anchoring of the LC. Finally, specific binding interactions between the antibody-decorated LC interfaces and vesicles (through antibody-antigen recognition) greatly accelerated the continuous LC anchoring transitions, with dynamics that were measured to scale with the logarithm of the ligand composition of the vesicles (over four orders of magnitude). The latter dynamics were found to be strongly influenced by addition of synthetic surfactants, consistent with our proposal that the rate-limiting step underlying the response of the LC was the transfer of lipids from captured vesicles into the protein-decorated LC interface. Overall, the results presented in this paper provide quantitative insight into the origin of continuous anchoring transitions triggered by vesicles at protein-decorated LC interfaces and, more broadly, guidance for the design of stimuli-responsive LC systems. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Solution NMR assignment of LpoB, an outer-membrane anchored Penicillin-Binding Protein activator from Escherichia coli.

    PubMed

    Jean, Nicolas L; Bougault, Catherine M; Egan, Alexander J F; Vollmer, Waldemar; Simorre, Jean-Pierre

    2015-04-01

    Bacteria surround their cytoplasmic membrane with the essential heteropolymer peptidoglycan (PG), which is made of glycan chains cross-linked by short peptides, to maintain osmotic stability and cell shape. PG is assembled from lipid II precursor by glycosyltransferase and transpeptidase reactions catalyzed by PG synthases, which are anchored to the cytoplasmic membrane and are controlled from inside the cell by cytoskeletal elements. Recently, two lipoproteins, LpoA and LpoB, were shown to be required in Escherichia coli for activating the main peptidoglycan synthases, Penicillin-Binding Proteins 1A and 1B, from the outer membrane. Here we present the backbone and side-chain assignment of the (1)H, (13)C and (15)N resonances of LpoB from E. coli. We also provide evidence for a two-domain organization of LpoB and a largely disordered, 64 amino acid-long N-terminal domain.

  6. Disruption of non-anchored cell wall protein NCW-1 promotes cellulase production by increasing cellobiose uptake in Neurospora crassa.

    PubMed

    Lin, Liangcai; Chen, Yong; Li, Jingen; Wang, Shanshan; Sun, Wenliang; Tian, Chaoguang

    2017-04-01

    To elucidate the mechanism of cellulase signal transduction in filamentous fungi including the components of the cellulase induction pathway. Neurospora crassa ncw-1 encodes a non-anchored cell wall protein. The absence of ncw-1 increased cellulase gene expression and this is not due to relieving carbon catabolite repression mediated by the cre-1 pathway. A mutant lacking genes encoding both three major β-glucosidase enzymes and NCW-1 (Δ3βGΔncw-1) was constructed. Transcriptome analysis of the quadruple mutant demonstrated enhanced expression of cellodextrin transporters after ncw-1 deletion, indicating that ncw-1 affects cellulase expression and production by inhibiting the uptake of the cellodextrin. NCW-1 is a novel component that plays a critical role in the cellulase induction signaling pathway.

  7. Anchoring of protein kinase A by ERM (ezrin-radixin-moesin) proteins is required for proper netrin signaling through DCC (deleted in colorectal cancer).

    PubMed

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

    2015-02-27

    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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. 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

  9. Use of IPTG-inducible promoters for anchoring recombinant proteins on the Bacillus subtilis spore surface.

    PubMed

    Nguyen, Quynh Anh; Schumann, Wolfgang

    2014-03-01

    The method of surface display allows the fusion of passenger proteins to a carrier protein displayed on the outside of bioparticles such as spores. Here, we used spores of Bacillus subtilis, the outer surface proteins CotB, CotC, and CotG as carrier and the amyQ-encoded α-amylase and GFPuv as passenger proteins. The different translational fusions were fused to two different IPTG-inducible promoters, and the regulated expression level of both passenger proteins were measured in relation to the inducer concentration added to sporulating cells. It turned out that the amount of fusion protein on the outside of spores was dependent on the amount of IPTG added, but the optimal amount of inducer varied depending on the carrier and the passenger proteins. These experiments demonstrate that a regulatable expression of passenger proteins on the surface of spores is possible. This will help to adjust the amount of any passenger protein to that needed for specific purposes. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. C-terminal sequences of hsp70 and hsp90 as non-specific anchors for tetratricopeptide repeat (TPR) proteins.

    PubMed

    Ramsey, Andrew J; Russell, Lance C; Chinkers, Michael

    2009-10-12

    Steroid-hormone-receptor maturation is a multi-step process that involves several TPR (tetratricopeptide repeat) proteins that bind to the maturation complex via the C-termini of hsp70 (heat-shock protein 70) and hsp90 (heat-shock protein 90). We produced a random T7 peptide library to investigate the roles played by the C-termini of the two heat-shock proteins in the TPR-hsp interactions. Surprisingly, phages with the MEEVD sequence, found at the C-terminus of hsp90, were not recovered from our biopanning experiments. However, two groups of phages were isolated that bound relatively tightly to HsPP5 (Homo sapiens protein phosphatase 5) TPR. Multiple copies of phages with a C-terminal sequence of LFG were isolated. These phages bound specifically to the TPR domain of HsPP5, although mutation studies produced no evidence that they bound to the domain's hsp90-binding groove. However, the most abundant family obtained in the initial screen had an aspartate residue at the C-terminus. Two members of this family with a C-terminal sequence of VD appeared to bind with approximately the same affinity as the hsp90 C-12 control. A second generation pseudo-random phage library produced a large number of phages with an LD C-terminus. These sequences acted as hsp70 analogues and had relatively low affinities for hsp90-specific TPR domains. Unfortunately, we failed to identify residues near hsp90's C-terminus that impart binding specificity to individual hsp90-TPR interactions. The results suggest that the C-terminal sequences of hsp70 and hsp90 act primarily as non-specific anchors for TPR proteins.

  11. Telomere anchoring at the nuclear periphery requires the budding yeast Sad1-UNC-84 domain protein Mps3.

    PubMed

    Bupp, Jennifer M; Martin, Adriana E; Stensrud, Elizabeth S; Jaspersen, Sue L

    2007-12-03

    Positioning of telomeres at the nuclear periphery can have dramatic effects on gene expression by establishment of heritable, transcriptionally repressive subdomains. However, little is known about the integral membrane proteins that mediate telomere tethering at the nuclear envelope. Here, we find a previously unrecognized function for the Saccharomyces cerevisiae Sad1-UNC-84 domain protein Mps3 in regulating telomere positioning in mitotic cells. Our data demonstrate that the nucleoplasmic N-terminal acidic domain of Mps3 is not essential for viability. However, this acidic domain is necessary and sufficient for telomere tethering during S phase and the silencing of reporter constructs integrated at telomeres. We show that this is caused by the role of the Mps3 acidic domain in binding and localization of the silent information regulator protein Sir4 to the nuclear periphery. Thus, Mps3 functions as an integral membrane anchor for telomeres and is a novel nuclear receptor for the Sir4 pathway of telomere tethering and gene inactivation.

  12. Telomere anchoring at the nuclear periphery requires the budding yeast Sad1-UNC-84 domain protein Mps3

    PubMed Central

    Bupp, Jennifer M.; Martin, Adriana E.; Stensrud, Elizabeth S.; Jaspersen, Sue L.

    2007-01-01

    Positioning of telomeres at the nuclear periphery can have dramatic effects on gene expression by establishment of heritable, transcriptionally repressive subdomains. However, little is known about the integral membrane proteins that mediate telomere tethering at the nuclear envelope. Here, we find a previously unrecognized function for the Saccharomyces cerevisiae Sad1-UNC-84 domain protein Mps3 in regulating telomere positioning in mitotic cells. Our data demonstrate that the nucleoplasmic N-terminal acidic domain of Mps3 is not essential for viability. However, this acidic domain is necessary and sufficient for telomere tethering during S phase and the silencing of reporter constructs integrated at telomeres. We show that this is caused by the role of the Mps3 acidic domain in binding and localization of the silent information regulator protein Sir4 to the nuclear periphery. Thus, Mps3 functions as an integral membrane anchor for telomeres and is a novel nuclear receptor for the Sir4 pathway of telomere tethering and gene inactivation. PMID:18039933

  13. A Cholesterol Recognition Amino Acid Consensus Domain in GP64 Fusion Protein Facilitates Anchoring of Baculovirus to Mammalian Cells

    PubMed Central

    Luz-Madrigal, Agustin; Asanov, Alexander; Camacho-Zarco, Aldo R.; Sampieri, Alicia

    2013-01-01

    Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV. PMID:23986592

  14. Detergent-insoluble GPI-anchored proteins are apically sorted in fischer rat thyroid cells, but interference with cholesterol or sphingolipids differentially affects detergent insolubility and apical sorting.

    PubMed

    Lipardi, C; Nitsch, L; Zurzolo, C

    2000-02-01

    In contrast to Madin-Darby canine kidney cells, Fischer rat thyroid cells deliver the majority of endogenous glycosylphosphatidyl inositol (GPI)-anchored proteins to the basolateral surface. However, we report here that the GPI proteins Placental Alkaline Phosphatase (PLAP) and Neurotrophin Receptor-Placental Alkaline Phosphatase (NTR-PLAP) are apically localized in transfected Fischer rat thyroid cells. In agreement with the "raft hypothesis," which postulates the incorporation of GPI proteins into glycosphingolipids and cholesterol-enriched rafts, we found that both of these proteins were insoluble in Triton X-100 and floated into the lighter fractions of sucrose density gradients. However, disruption of lipid rafts by removal of cholesterol did not cause surface missorting of PLAP and NTR-PLAP, and the altered surface sorting of these proteins after Fumonisin B1 treatment did not correlate with reduced levels in Triton X-100 -insoluble fractions. Furthermore, in contrast to the GPI-anchored forms of both of these proteins, the secretory and transmembrane forms (in the absence of a basolateral cytoplasmic signal) were sorted to the apical surface without association with lipid microdomains. Together, these data demonstrate that the GPI anchor is required to mediate raft association but is not sufficient to determine apical sorting. They also suggest that signals present in the ectodomain of the proteins play a major role and that lipid rafts may facilitate the recognition of these signals in the trans-Golgi network, even though they are not required for apical sorting.

  15. A Glycosylphosphatidylinositol-Anchored Carbonic Anhydrase-Related Protein of Toxoplasma gondii Is Important for Rhoptry Biogenesis and Virulence

    PubMed Central

    Chasen, Nathan M.; Asady, Beejan; Lemgruber, Leandro; Vommaro, Rossiane C.; Kissinger, Jessica C.; Coppens, Isabelle

    2017-01-01

    ABSTRACT Carbonic anhydrase-related proteins (CARPs) have previously been described as catalytically inactive proteins closely related to α-carbonic anhydrases (α-CAs). These CARPs are found in animals (both vertebrates and invertebrates) and viruses as either independent proteins or domains of other proteins. We report here the identification of a new CARP (TgCA_RP) in the unicellular organism Toxoplasma gondii that is related to the recently described η-class CA found in Plasmodium falciparum. TgCA_RP is posttranslationally modified at its C terminus with a glycosylphosphatidylinositol anchor that is important for its localization in intracellular tachyzoites. The protein localizes throughout the rhoptry bulbs of mature tachyzoites and to the outer membrane of nascent rhoptries in dividing tachyzoites, as demonstrated by immunofluorescence and immunoelectron microscopy using specific antibodies. T. gondii mutant tachyzoites lacking TgCA_RP display a growth and invasion phenotype in vitro and have atypical rhoptry morphology. The mutants also exhibit reduced virulence in a mouse model. Our results show that TgCA_RP plays an important role in the biogenesis of rhoptries. IMPORTANCE Toxoplasma gondii is an intracellular pathogen that infects humans and animals. The pathogenesis of T. gondii is linked to its lytic cycle, which starts when tachyzoites invade host cells and secrete proteins from specialized organelles. Once inside the host cell, the parasite creates a parasitophorous vacuole (PV) where it divides. Rhoptries are specialized secretory organelles that contain proteins, many of which are secreted during invasion. These proteins have important roles not only during the initial interaction between parasite and host but also in the formation of the PV and in the modification of the host cell. We report here the identification of a new T. gondii carbonic anhydrase-related protein (TgCA_RP), which localizes to rhoptries of mature tachyzoites. TgCA_RP is

  16. Tic40, a membrane-anchored co-chaperone homolog in the chloroplast protein translocon

    PubMed Central

    Chou, Ming-Lun; Fitzpatrick, Lynda M.; Tu, Shuh-Long; Budziszewski, Gregory; Potter-Lewis, Sharon; Akita, Mitsuru; Levin, Joshua Z.; Keegstra, Kenneth; Li, Hsou-min

    2003-01-01

    The function of Tic40 during chloroplast protein import was investigated. Tic40 is an inner envelope membrane protein with a large hydrophilic domain located in the stroma. Arabidopsis null mutants of the atTic40 gene were very pale green and grew slowly but were not seedling lethal. Isolated mutant chloroplasts imported precursor proteins at a lower rate than wild-type chloroplasts. Mutant chloroplasts were normal in allowing binding of precursor proteins. However, during subsequent translocation across the inner membrane, fewer precursors were translocated and more precursors were released from the mutant chloroplasts. Cross-linking experiments demonstrated that Tic40 was part of the translocon complex and functioned at the same stage of import as Tic110 and Hsp93, a member of the Hsp100 family of molecular chaperones. Tertiary structure prediction and immunological studies indicated that the C-terminal portion of Tic40 contains a TPR domain followed by a domain with sequence similarity to co-chaperones Sti1p/Hop and Hip. We propose that Tic40 functions as a co-chaperone in the stromal chaperone complex that facilitates protein translocation across the inner membrane. PMID:12805212

  17. The mouse neuronal cell surface protein F3: a phosphatidylinositol- anchored member of the immunoglobulin superfamily related to chicken contactin

    PubMed Central

    1989-01-01

    Several members of the Ig superfamily are expressed on neural cells where they participate in surface interactions between cell bodies and processes. Their Ig domains are more closely related to each other than to Ig variable and constant domains and have been grouped into the C2 set. Here, we report the cloning and characterization of another member of this group, the mouse neuronal cell surface antigen F3. The F3 cDNA sequence contains an open reading frame that could encode a 1,020-amino acid protein consisting of a signal sequence, six Ig-like domains of the C2 type, a long premembrane region containing two segments that exhibit sequence similarity to fibronectin type III repeats and a moderately hydrophobic COOH-terminal sequence. The protein does not contain a typical transmembrane segment but appears to be attached to the membrane by a phosphatidylinositol anchor. Antibodies against the F3 protein recognize a prominent 135-kD protein in mouse brain. In fetal brain cultures, they stain the neuronal cell surface and, in cultures maintained in chemically defined medium, most prominently neurites and neurite bundles. The mouse f3 gene maps to band F of chromosome 15. The gene transcripts detected in the brain by F3 cDNA probes are developmentally regulated, the highest amounts being expressed between 1 and 2 wk after birth. The F3 nucleotide and deduced amino acid sequence show striking similarity to the recently published sequence of the chicken neuronal cell surface protein contactin. However, there are important differences between the two molecules. In contrast to F3, contactin has a transmembrane and a cytoplasmic domain. Whereas contactin is insoluble in nonionic detergent and is tightly associated with the cytoskeleton, about equal amounts of F3 distribute between buffer-soluble, nonionic detergent-soluble, and detergent- insoluble fractions. Among other neural cell surface proteins, F3 most resembles the neuronal cell adhesion protein L1, with 25% amino

  18. Display of alpha-amylase on the surface of Corynebacterium glutamicum cells by using NCgl1221 as the anchoring protein, and production of glutamate from starch.

    PubMed

    Yao, Wenjuan; Chu, Chunli; Deng, Xiaozhao; Zhang, Yun; Liu, Miao; Zheng, Pu; Sun, Zhihao

    2009-10-01

    We developed a new cell surface display system in Corynebacterium glutamicum based on the C-terminally truncated NCgl1221 anchor protein to increase L-glutamate production from starch directly. The C-terminally truncated NCgl1221 protein is a mutant NCgl1221 and leads to the constitutive export of L-glutamate. The N terminus of alpha-amylase (AmyA) was fused to truncated NCgl1221, and the resulting fusion protein was expressed on the cell surface by IPTG induction. Localization of the fusion protein was confirmed by immunofluorescence microscopy and flow cytometric analysis. The results of L-glutamate fermentation showed that the soluble starch was utilized to grow and produce L-glutamate by the recombinant strain displaying AmyA. The amount of soluble starch was reduced from 30.0 +/- 2.8 to 4.5 +/- 0.7 g/l under non-inducing condition and from 50.0 +/- 2.4 to 12.5 +/- 1.1 g/l under biotin limitation in 36 h. The glutamate concentration in the medium was transiently increased in 14 h under no induction, while under biotin-limiting condition, glutamate production was continuously elevated during fermentation. The amount of glutamate reached 19.3 +/- 2.1 g/l after 26 h of fermentation with biotin limitation, which was greater than that produced by the strain using PgsA, one of the poly-gamma-glutamate synthetase complexes, as the anchor protein under the same condition. Therefore, the truncated NCgl1221 anchor protein has more advantages than the PgsA anchor protein in glutamate fermentation because truncated NCgl1221 leads to the constitutive export of L-glutamate without any treatments.

  19. Involvement of sortase anchoring of cell wall proteins in biofilm formation by Streptococcus mutans.

    PubMed

    Lévesque, Céline M; Voronejskaia, Elena; Huang, Yi-Chen Cathy; Mair, Richard W; Ellen, Richard P; Cvitkovitch, Dennis G

    2005-06-01

    Streptococcus mutans is one of the best-known biofilm-forming organisms associated with humans. We investigated the role of the sortase gene (srtA) in monospecies biofilm formation and observed that inactivation of srtA caused a decrease in biofilm formation. Genes encoding three putative sortase-dependent proteins were also found to be up-regulated in biofilms versus planktonic cells and mutations in these genes resulted in reduced biofilm biomass.

  20. C-terminal sequences of hsp70 and hsp90 as non-specific anchors for TPR proteins

    PubMed Central

    Ramsey, Andrew J.; Russell, Lance C.; Chinkers, Michael

    2013-01-01

    Synopsis Steroid hormone receptor maturation is a multi-step process that involves several TPR proteins that bind to the maturation complex via the C-termini of hsp70 and hsp90. We produced a random T7 peptide library to investigate the roles played by the C-termini of the two heat shock proteins in the TPR/hsp interactions. Surprisingly, phages with the MEEVD sequence, found at the C-terminus of hsp90, were not recovered from our biopanning experiments. However, two groups of phages were isolated that bound relatively tightly to HsPP5 TPR. Multiple copies of phages with a C-terminal sequence of LFG were isolated. These phages bound specifically to the TPR domain of HsPP5 although mutation studies produced no evidence that they bound to the domain’s hsp90-binding groove. However, the most abundant family obtained in the initial screen had an aspartate at the C-terminus. Two members of this family with a C-terminal sequence of VD appeared to bind with approximately the same affinity as the hsp90 C-12 control. A second generation pseudo-random phage library produced a large number of phages with a LD C-terminus. These sequences acted as hsp70 analogues and had relatively low affinities for hsp90 specific TPR domains. Unfortunately, we failed to identify residues near hsp90’s C-terminus that impart binding specificity to individual hsp90/TPR interactions. The data suggest that the C-terminal sequences of hsp70 and hsp90 act primarily as non-specific anchors for TPR proteins. PMID:19689428

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

    DOE PAGES

    Banerjee, Ankan; Tsai, Chi -Lin; Chaudhury, Paushali; ...

    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

  2. Surface display of bacterial tyrosinase on spores of Bacillus subtilis using CotE as an anchor protein.

    PubMed

    Hosseini-Abari, Afrouzossadat; Kim, Byung-Gee; Lee, Sang-Hyuk; Emtiazi, Giti; Kim, Wooil; Kim, June-Hyung

    2016-12-01

    Tyrosinases, copper-containing monooxygenases, are widely used enzymes for industrial, medical, and environmental applications. We report the first functional surface display of Bacillus megaterium tyrosinase on Bacillus subtilis spores using CotE as an anchor protein. Flow Cytometry was used to verify surface expression of tyrosinase on the purified spores. Moreover, tyrosinase activity of the displayed enzyme on B. subtilis spores was monitored in the presence of L-tyrosine (substrate) and CuSO 4 (inducer). The stability of the spore-displayed tyrosinase was then evaluated after 15 days maintenance of the spores at room temperature, and no significant decrease in the enzyme activity was observed. In addition, the tyrosinase-expressing spores could be repeatedly used with 62% retained enzymatic activity after six times washing with Tris-HCl buffer. This genetically immobilized tyrosinase on the spores would make a new advance in industrial, medical, and environmental applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. 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

  4. Analysis of Novel Iron-Regulated, Surface-Anchored Hemin-Binding Proteins in Corynebacterium diphtheriae

    PubMed Central

    Allen, Courtni E.; Burgos, Jonathan M.

    2013-01-01

    Corynebacterium diphtheriae utilizes hemin and hemoglobin (Hb) as iron sources during growth in iron-depleted environments, and recent studies have shown that the surface-exposed HtaA protein binds both hemin and Hb and also contributes to the utilization of hemin iron. Conserved (CR) domains within HtaA and in the associated hemin-binding protein, HtaB, are required for the ability to bind hemin and Hb. In this study, we identified and characterized two novel genetic loci in C. diphtheriae that encode factors that bind hemin and Hb. Both genetic systems contain two-gene operons that are transcriptionally regulated by DtxR and iron. The gene products of these operons are ChtA-ChtB and ChtC-CirA (previously DIP0522-DIP0523). The chtA and chtB genes are carried on a putative composite transposon associated with C. diphtheriae isolates that dominated the diphtheria outbreak in the former Soviet Union in the 1990s. ChtA and ChtC each contain a single N-terminal CR domain and exhibit significant sequence similarity to each other but only limited similarity with HtaA. The chtB and htaB gene products exhibited a high level of sequence similarity throughout their sequences, and both proteins contain a single CR domain. Whole-cell binding studies as well as protease analysis indicated that all four of the proteins encoded by these two operons are surface exposed, which is consistent with the presence of a transmembrane segment in their C-terminal regions. ChtA, ChtB, and ChtC are able to bind hemin and Hb, with ChtA showing the highest affinity. Site-directed mutagenesis showed that specific tyrosine residues within the ChtA CR domain were critical for hemin and Hb binding. Hemin iron utilization assays using various C. diphtheriae mutants indicate that deletion of the chtA-chtB region and the chtC gene has no affect on the ability of C. diphtheriae to use hemin or Hb as iron sources; however, a chtB htaB double mutant exhibits a significant decrease in hemin iron use

  5. Analysis of novel iron-regulated, surface-anchored hemin-binding proteins in Corynebacterium diphtheriae.

    PubMed

    Allen, Courtni E; Burgos, Jonathan M; Schmitt, Michael P

    2013-06-01

    Corynebacterium diphtheriae utilizes hemin and hemoglobin (Hb) as iron sources during growth in iron-depleted environments, and recent studies have shown that the surface-exposed HtaA protein binds both hemin and Hb and also contributes to the utilization of hemin iron. Conserved (CR) domains within HtaA and in the associated hemin-binding protein, HtaB, are required for the ability to bind hemin and Hb. In this study, we identified and characterized two novel genetic loci in C. diphtheriae that encode factors that bind hemin and Hb. Both genetic systems contain two-gene operons that are transcriptionally regulated by DtxR and iron. The gene products of these operons are ChtA-ChtB and ChtC-CirA (previously DIP0522-DIP0523). The chtA and chtB genes are carried on a putative composite transposon associated with C. diphtheriae isolates that dominated the diphtheria outbreak in the former Soviet Union in the 1990s. ChtA and ChtC each contain a single N-terminal CR domain and exhibit significant sequence similarity to each other but only limited similarity with HtaA. The chtB and htaB gene products exhibited a high level of sequence similarity throughout their sequences, and both proteins contain a single CR domain. Whole-cell binding studies as well as protease analysis indicated that all four of the proteins encoded by these two operons are surface exposed, which is consistent with the presence of a transmembrane segment in their C-terminal regions. ChtA, ChtB, and ChtC are able to bind hemin and Hb, with ChtA showing the highest affinity. Site-directed mutagenesis showed that specific tyrosine residues within the ChtA CR domain were critical for hemin and Hb binding. Hemin iron utilization assays using various C. diphtheriae mutants indicate that deletion of the chtA-chtB region and the chtC gene has no affect on the ability of C. diphtheriae to use hemin or Hb as iron sources; however, a chtB htaB double mutant exhibits a significant decrease in hemin iron use

  6. Heme Assimilation in Schizosaccharomyces pombe Requires Cell-surface-anchored Protein Shu1 and Vacuolar Transporter Abc3.

    PubMed

    Mourer, Thierry; Normant, Vincent; Labbé, Simon

    2017-03-24

    The Schizosaccharomyces pombe shu1 + gene encodes a cell-surface protein required for assimilation of exogenous heme. In this study, shaving experiments showed that Shu1 is released from membrane preparations when spheroplast lysates are incubated with phosphoinositide-specific phospholipase C (PI-PLC). Shu1 cleavability by PI-PLC and its predicted hydropathy profile strongly suggested that Shu1 is a glycosylphosphatidylinositol-anchored protein. When heme biosynthesis is selectively blocked in hem1 Δ mutant cells, the heme analog zinc mesoporphyrin IX (ZnMP) first accumulates into vacuoles and then subsequently, within the cytoplasm in a rapid and Shu1-dependent manner. An HA 4 -tagged shu1 + allele that retained wild-type function localizes to the cell surface in response to low hemin concentrations, but under high hemin concentrations, Shu1-HA 4 re-localizes to the vacuolar membrane. Inactivation of abc3 + , encoding a vacuolar membrane transporter, results in hem1 Δ abc3 Δ mutant cells being unable to grow in the presence of hemin as the sole iron source. In hem1 Δ abc3 Δ cells, ZnMP accumulates primarily in vacuoles and does not sequentially accumulate in the cytosol. Consistent with a role for Abc3 as vacuolar hemin exporter, results with hemin-agarose pulldown assays showed that Abc3 binds to hemin. In contrast, an Abc3 mutant in which an inverted Cys-Pro motif had been replaced with Ala residues fails to bind hemin with high affinity. Taken together, these results show that Shu1 undergoes rapid hemin-induced internalization from the cell surface to the vacuolar membrane and that the transporter Abc3 participates in the mobilization of stored heme from the vacuole to the cytosol. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Anchor Modeling

    NASA Astrophysics Data System (ADS)

    Regardt, Olle; Rönnbäck, Lars; Bergholtz, Maria; Johannesson, Paul; Wohed, Petia

    Maintaining and evolving data warehouses is a complex, error prone, and time consuming activity. The main reason for this state of affairs is that the environment of a data warehouse is in constant change, while the warehouse itself needs to provide a stable and consistent interface to information spanning extended periods of time. In this paper, we propose a modeling technique for data warehousing, called anchor modeling, that offers non-destructive extensibility mechanisms, thereby enabling robust and flexible management of changes in source systems. A key benefit of anchor modeling is that changes in a data warehouse environment only require extensions, not modifications, to the data warehouse. This ensures that existing data warehouse applications will remain unaffected by the evolution of the data warehouse, i.e. existing views and functions will not have to be modified as a result of changes in the warehouse model.

  8. Tryptophan-rich basic protein (WRB) mediates insertion of the tail-anchored protein otoferlin and is required for hair cell exocytosis and hearing.

    PubMed

    Vogl, Christian; Panou, Iliana; Yamanbaeva, Gulnara; Wichmann, Carolin; Mangosing, Sara J; Vilardi, Fabio; Indzhykulian, Artur A; Pangršič, Tina; Santarelli, Rosamaria; Rodriguez-Ballesteros, Montserrat; Weber, Thomas; Jung, Sangyong; Cardenas, Elena; Wu, Xudong; Wojcik, Sonja M; Kwan, Kelvin Y; Del Castillo, Ignacio; Schwappach, Blanche; Strenzke, Nicola; Corey, David P; Lin, Shuh-Yow; Moser, Tobias

    2016-12-01

    The transmembrane recognition complex (TRC40) pathway mediates the insertion of tail-anchored (TA) proteins into membranes. Here, we demonstrate that otoferlin, a TA protein essential for hair cell exocytosis, is inserted into the endoplasmic reticulum (ER) via the TRC40 pathway. We mutated the TRC40 receptor tryptophan-rich basic protein (Wrb) in hair cells of zebrafish and mice and studied the impact of defective TA protein insertion. Wrb disruption reduced otoferlin levels in hair cells and impaired hearing, which could be restored in zebrafish by transgenic Wrb rescue and otoferlin overexpression. Wrb-deficient mouse inner hair cells (IHCs) displayed normal numbers of afferent synapses, Ca 2+ channels, and membrane-proximal vesicles, but contained fewer ribbon-associated vesicles. Patch-clamp of IHCs revealed impaired synaptic vesicle replenishment. In vivo recordings from postsynaptic spiral ganglion neurons showed a use-dependent reduction in sound-evoked spiking, corroborating the notion of impaired IHC vesicle replenishment. A human mutation affecting the transmembrane domain of otoferlin impaired its ER targeting and caused an auditory synaptopathy. We conclude that the TRC40 pathway is critical for hearing and propose that otoferlin is an essential substrate of this pathway in hair cells. © 2016 The Authors.

  9. Comprehensive behavioral analysis of voltage-gated calcium channel beta-anchoring and -regulatory protein knockout mice

    PubMed Central

    Nakao, Akito; Miki, Takafumi; Shoji, Hirotaka; Nishi, Miyuki; Takeshima, Hiroshi; Miyakawa, Tsuyoshi; Mori, Yasuo

    2015-01-01

    Calcium (Ca2+) influx through voltage-gated Ca2+ channels (VGCCs) induces numerous intracellular events such as neuronal excitability, neurotransmitter release, synaptic plasticity, and gene regulation. It has been shown that genes related to Ca2+ signaling, such as the CACNA1C, CACNB2, and CACNA1I genes that encode VGCC subunits, are associated with schizophrenia and other psychiatric disorders. Recently, VGCC beta-anchoring and -regulatory protein (BARP) was identified as a novel regulator of VGCC activity via the interaction of VGCC β subunits. To examine the role of the BARP in higher brain functions, we generated BARP knockout (KO) mice and conducted a comprehensive battery of behavioral tests. BARP KO mice exhibited greatly reduced locomotor activity, as evidenced by decreased vertical activity, stereotypic counts in the open field test, and activity level in the home cage, and longer latency to complete a session in spontaneous T-maze alteration test, which reached “study-wide significance.” Acoustic startle response was also reduced in the mutants. Interestingly, they showed multiple behavioral phenotypes that are seemingly opposite to those seen in the mouse models of schizophrenia and its related disorders, including increased working memory, flexibility, prepulse inhibition, and social interaction, and decreased locomotor activity, though many of these phenotypes are statistically weak and require further replications. These results demonstrate that BARP is involved in the regulation of locomotor activity and, possibly, emotionality. The possibility was also suggested that BARP KO mice may serve as a unique tool for investigating the pathogenesis/pathophysiology of schizophrenia and related disorders. Further evaluation of the molecular and physiological phenotypes of the mutant mice would provide new insights into the role of BARP in higher brain functions. PMID:26136667

  10. The zebrafish pinball wizard gene encodes WRB, a tail-anchored-protein receptor essential for inner-ear hair cells and retinal photoreceptors.

    PubMed

    Lin, Shuh-Yow; Vollrath, Melissa A; Mangosing, Sara; Shen, Jun; Cardenas, Elena; Corey, David P

    2016-02-15

    The zebrafish pinball wizard (pwi) mutant is deaf and blind. The pwi phenotype includes a reduced auditory startle response and reduced visual evoked potentials, suggesting fatigue of synaptic release at ribbon synapses in hair cells and photoreceptors. The gene defective in the pwi mutant is WRB, a protein homologous to the yeast protein Get1, which is involved in the insertion of 'tail-anchored' membrane proteins. Many tail-anchored proteins are associated with synaptic vesicles, and both vesicles and synaptic ribbons are reduced in synaptic regions of hair cells in pwi. Abnormal processing of synaptic vesicle proteins important for ribbon synapses can explain the pwi phenotype. In a large-scale zebrafish insertional mutagenesis screen, we identified the pinball wizard (pwi) line, which displays a deafness and blindness phenotype. Although the gross morphology and structure of the pwi larval inner ear was near normal, acoustic startle stimuli evoked smaller postsynaptic responses in afferent neurons, which rapidly fatigued. In the retina, similarly, an abnormal electroretinogram suggested reduced transmission at the photoreceptor ribbon synapse. A functional deficit in these specialized synapses was further supported by a reduction of synaptic marker proteins Rab3 and cysteine-string protein (CSP/Dnajc5) in hair cells and photoreceptors, as well as by a reduction of the number of both ribbons and vesicles surrounding the ribbons in hair cells. The pwi gene encodes a homologue of the yeast Get1 and human tryptophan-rich basic (WRB) proteins, which are receptors for membrane insertion of tail-anchored (TA) proteins. We identified more than 100 TA proteins expressed in hair cells, including many synaptic proteins. The expression of synaptobrevin and syntaxin 3, TA proteins essential for vesicle fusion, was reduced in the synaptic layers of mutant retina, consistent with a role for the pwi/WRB protein in TA-protein processing. The WRB protein was located near the

  11. Nanobiotechnologic approach to a promising vaccine prototype for immunisation against leishmaniasis: a fast and effective method to incorporate GPI-anchored proteins of Leishmania amazonensis into liposomes.

    PubMed

    Colhone, Marcelle Carolina; Silva-Jardim, Izaltina; Stabeli, Rodrigo Guerino; Ciancaglini, Pietro

    2015-01-01

    Liposomes are known to be a potent adjuvant for a wide range of antigens, as well as appropriate antigen carriers for antibody generation response in vivo. In addition, liposomes are effective vehicles for peptides and proteins, thus enhancing their immunogenicity. Considering these properties of liposomes and the antigenicity of the Leishmania membrane proteins, we evaluated if liposomes carrying glycosylphosphatidylinositol (GPI)-anchored proteins of Leishmania amazonensis promastigotes could induce protective immunity in BALB/c mice. To assay protective immunity, BALB/c mice were intraperitoneally injected with liposomes, GPI-protein extract (EPSGPI) as well as with the proteoliposomes carrying GPI-proteins. Mice inoculated with EPSGPI and total protein present in constitutive proteoliposomes displayed a post-infection protection of about 70% and 90%, respectively. The liposomes are able to work as adjuvant in the EPSGPI protection. These systems seem to be a promising vaccine prototype for immunisation against leishmaniasis.

  12. Voltage-dependent potentiation of L-type Ca2+ channels in skeletal muscle cells requires anchored cAMP-dependent protein kinase.

    PubMed Central

    Johnson, B D; Scheuer, T; Catterall, W A

    1994-01-01

    Skeletal muscle L-type Ca2+ channels respond to trains of brief depolarizations with a strong shift of the voltage dependence of channel activation toward more negative membrane potentials and slowing of channel deactivation. Increased Ca2+ entry resulting from this potentiation of channel activity may increase contractile force in response to tetanic stimuli. This voltage-dependent Ca2+ channel potentiation requires phosphorylation by cAMP-dependent protein kinase (PKA) at a rate that suggests that kinase and channel may be maintained in close proximity through kinase anchoring. A peptide derived from the conserved kinase-binding domain of a PKA-anchoring protein (AKAP) prevents potentiation by endogenous PKA as effectively as inhibition of PKA by a specific peptide inhibitor or by omission of ATP from the intracellular solution. In contrast, a proline-substituted mutant of AKAP peptide has no effect. Potentiation in the presence of 2 microM exogenous catalytic subunit of PKA is unaffected, indicating that kinase anchoring is specifically blocked by the AKAP peptide. No effects of these agents were observed on the level or voltage dependence of basal Ca2+ channel activity before potentiation, suggesting that close physical proximity between the skeletal muscle Ca2+ channel and PKA is critical for voltage-dependent potentiation of Ca2+ channel activity but not for basal activity. Images PMID:7972090

  13. Novel mechanism of gene regulation: the protein Rv1222 of Mycobacterium tuberculosis inhibits transcription by anchoring the RNA polymerase onto DNA

    PubMed Central

    Rudra, Paulami; Prajapati, Ranjit Kumar; Banerjee, Rajdeep; Sengupta, Shreya; Mukhopadhyay, Jayanta

    2015-01-01

    We propose a novel mechanism of gene regulation in Mycobacterium tuberculosis where the protein Rv1222 inhibits transcription by anchoring RNA polymerase (RNAP) onto DNA. In contrast to our existing knowledge that transcriptional repressors function either by binding to DNA at specific sequences or by binding to RNAP, we show that Rv1222-mediated transcription inhibition requires simultaneous binding of the protein to both RNAP and DNA. We demonstrate that the positively charged C-terminus tail of Rv1222 is responsible for anchoring RNAP on DNA, hence the protein slows down the movement of RNAP along the DNA during transcription elongation. The interaction between Rv1222 and DNA is electrostatic, thus the protein could inhibit transcription from any gene. As Rv1222 slows down the RNA synthesis, upon expression of the protein in Mycobacterium smegmatis or Escherichia coli, the growth rate of the bacteria is severely impaired. The protein does not possess any significant affinity for DNA polymerase, thus, is unable to inhibit DNA synthesis. The proposed mechanism by which Rv1222 inhibits transcription reveals a new repertoire of prokaryotic gene regulation. PMID:25999340

  14. Novel mechanism of gene regulation: the protein Rv1222 of Mycobacterium tuberculosis inhibits transcription by anchoring the RNA polymerase onto DNA.

    PubMed

    Rudra, Paulami; Prajapati, Ranjit Kumar; Banerjee, Rajdeep; Sengupta, Shreya; Mukhopadhyay, Jayanta

    2015-07-13

    We propose a novel mechanism of gene regulation in Mycobacterium tuberculosis where the protein Rv1222 inhibits transcription by anchoring RNA polymerase (RNAP) onto DNA. In contrast to our existing knowledge that transcriptional repressors function either by binding to DNA at specific sequences or by binding to RNAP, we show that Rv1222-mediated transcription inhibition requires simultaneous binding of the protein to both RNAP and DNA. We demonstrate that the positively charged C-terminus tail of Rv1222 is responsible for anchoring RNAP on DNA, hence the protein slows down the movement of RNAP along the DNA during transcription elongation. The interaction between Rv1222 and DNA is electrostatic, thus the protein could inhibit transcription from any gene. As Rv1222 slows down the RNA synthesis, upon expression of the protein in Mycobacterium smegmatis or Escherichia coli, the growth rate of the bacteria is severely impaired. The protein does not possess any significant affinity for DNA polymerase, thus, is unable to inhibit DNA synthesis. The proposed mechanism by which Rv1222 inhibits transcription reveals a new repertoire of prokaryotic gene regulation. © Crown copyright 2015.

  15. Anchored PKA as a gatekeeper for gap junctions.

    PubMed

    Pidoux, Guillaume; Taskén, Kjetil

    2015-01-01

    Anchored protein kinase A (PKA) bound to A Kinase Anchoring Protein (AKAP) mediates effects of localized increases in cAMP in defined subcellular microdomains and retains the specificity in cAMP-PKA signaling to distinct extracellular stimuli. Gap junctions are pores between adjacent cells constituted by connexin proteins that provide means of communication and transfer of small molecules. While the PKA signaling is known to promote human trophoblast cell fusion, the gap junction communication through connexin 43 (Cx43) is a prerequisite for this process. We recently demonstrated that trophoblast fusion is regulated by ezrin, a known AKAP, which binds to Cx43 and delivers PKA in the vicinity gap junctions. We found that disruption of the ezrin-Cx43 interaction abolished PKA-dependent phosphorylation of Cx43 as well as gap junction communication and subsequently cell fusion. We propose that the PKA-ezrin-Cx43 macromolecular complex regulating gap junction communication constitutes a general mechanism to control opening of Cx43 gap junctions by phosphorylation in response to cAMP signaling in various cell types.

  16. 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"

  17. AKAP18:PKA-RIIα structure reveals crucial anchor points for recognition of regulatory subunits of PKA.

    PubMed

    Götz, Frank; Roske, Yvette; Schulz, Maike Svenja; Autenrieth, Karolin; Bertinetti, Daniela; Faelber, Katja; Zühlke, Kerstin; Kreuchwig, Annika; Kennedy, Eileen J; Krause, Gerd; Daumke, Oliver; Herberg, Friedrich W; Heinemann, Udo; Klussmann, Enno

    2016-07-01

    A-kinase anchoring proteins (AKAPs) interact with the dimerization/docking (D/D) domains of regulatory subunits of the ubiquitous protein kinase A (PKA). AKAPs tether PKA to defined cellular compartments establishing distinct pools to increase the specificity of PKA signalling. Here, we elucidated the structure of an extended PKA-binding domain of AKAP18β bound to the D/D domain of the regulatory RIIα subunits of PKA. We identified three hydrophilic anchor points in AKAP18β outside the core PKA-binding domain, which mediate contacts with the D/D domain. Such anchor points are conserved within AKAPs that bind regulatory RII subunits of PKA. We derived a different set of anchor points in AKAPs binding regulatory RI subunits of PKA. In vitro and cell-based experiments confirm the relevance of these sites for the interaction of RII subunits with AKAP18 and of RI subunits with the RI-specific smAKAP. Thus we report a novel mechanism governing interactions of AKAPs with PKA. The sequence specificity of each AKAP around the anchor points and the requirement of these points for the tight binding of PKA allow the development of selective inhibitors to unequivocally ascribe cellular functions to the AKAP18-PKA and other AKAP-PKA interactions. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  18. Aromatic anchor at an invariant hormone-receptor interface: Function of insulin residue B24 with application to protein design

    DOE PAGES

    Pandyarajan, Vijay; Smith, Brian J.; Phillips, Nelson B.; ...

    2014-10-10

    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 Phe B24, 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, Met B24 was found to confer essentially native affinity and bioactivity. Molecular modeling suggests that this linear side chain can serve as an alternative hydrophobic anchormore » at the hormone-receptor interface. These findings motivated further substitution of Phe B24 by cyclohexanylalanine (Cha), which contains a nonplanar aliphatic ring. Contrary to expectations, [Cha B24]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 Cha B24 analog, determined as an R 6 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 Phe B24 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. Finally, 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.« less

  19. Sustained activation of a G protein-coupled receptor via "anchored" agonist binding. Molecular localization of the salmeterol exosite within the 2-adrenergic receptor.

    PubMed

    Green, S A; Spasoff, A P; Coleman, R A; Johnson, M; Liggett, S B

    1996-09-27

    An inherent therapeutic limitation of many G protein-coupled receptor agonists is a short duration of action due to rapid dissociation from receptors. Salmeterol is a modified beta-adrenergic receptor (betaAR) agonist that has a long duration of action at the beta2AR (but not the beta1AR) both in vitro and in vivo and that is persistent despite extensive washout of the agonist. It has been proposed that salmeterol binds not only to the active site of the beta2AR (localized to receptor transmembrane spanning domains (TMDs) 3 and 5) but also to another site (termed the "exosite") that anchors it to the receptor and provides for repetitive active-site binding events. To identify the location of this exosite, we used site-directed mutagenesis to replace beta2AR amino acids 149-173 (within TMD4) with beta1AR sequence. The resulting constructs were then expressed in COS-7 cells for radioligand binding studies. Using this approach, when this domain was replaced with the analogous beta1AR sequence, the ability of salmeterol to persist at the receptor under washout conditions was reduced by 67%. The results from more selective mutants (S-(149-166), S-(164-173), and S-(149-158)) indicated that a limited 10-amino acid region (beta2AR residues 149-158), localized at the interface of the cytoplasm and the transmembrane domain, contains a critical determinant for exosite binding. Whereas CHW cells stably expressing wild-type beta2AR displayed persistent salmeterol-promoted cAMP accumulation despite agonist washout, substitution of beta2AR residues 149-158 with beta1AR sequence resulted in a 56% attenuation of salmeterol-promoted cAMP accumulation under identical washout conditions. A reverse chimera was also studied, which consisted of a substitution of beta2AR residues 152-156 into the beta1AR. This substitution was found to confer exosite binding to the beta1AR. None of these mutations decreased the affinity of salmeterol for the receptor at the active site as assessed in

  20. Apical sorting of lysoGPI-anchored proteins occurs independent of association with detergent-resistant membranes but dependent on their N-glycosylation

    PubMed Central

    Castillon, Guillaume Alain; Michon, Laetitia; Watanabe, Reika

    2013-01-01

    Most glycosylphosphatidylinositol-anchored proteins (GPI-APs) are located at the apical surface of epithelial cells. The apical delivery of GPI-APs is believed to result from their association with lipid rafts. We find that overexpression of C-terminally tagged PGAP3 caused predominant production of lysoGPI-APs, an intermediate precursor in the GPI lipid remodeling process in Madin–Darby canine kidney cells. In these cells, produced lysoGPI-APs are not incorporated into detergent-resistant membranes (DRMs) but still are delivered apically, suggesting that GPI-AP association with DRMs is not necessary for apical targeting. In contrast, apical transport of both fully remodeled and lyso forms of GPI-APs is dependent on N-glycosylation, confirming a general role of N-glycans in apical protein transport. We also find that depletion of cholesterol causes apical-to-basolateral retargeting not only of fully remodeled GPI-APs, but also of lysoGPI-APs, as well as endogenous soluble and transmembrane proteins that would normally be targeted to the apical membrane. These findings confirm the essential role for cholesterol in the apical protein targeting and further demonstrate that the mechanism of cholesterol-dependent apical sorting is not related to DRM association of GPI-APs. PMID:23615438

  1. 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 yieldedmore » 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.« less

  2. Ascidian sperm glycosylphosphatidylinositol-anchored CRISP-like protein as a binding partner for an allorecognizable sperm receptor on the vitelline coat.

    PubMed

    Urayama, Satoshi; Harada, Yoshito; Nakagawa, Yoko; Ban, Susumu; Akasaka, Mari; Kawasaki, Nana; Sawada, Hitoshi

    2008-08-01

    Although ascidians are hermaphroditic, many species including Halocynthia roretzi are self-sterile. We previously reported that a vitelline coat polymorphic protein HrVC70, consisting of 12 EGF (epidermal growth factor)-like repeats, is a candidate allorecognition protein in H. roretzi, because the isolated HrVC70 shows higher affinity to nonself-sperm than to self-sperm. Here, we show that a sperm 35-kDa glycosylphosphatidylinositol-anchored CRISP (cysteine-rich secretory protein)-like protein HrUrabin in a low density detergent-insoluble membrane fraction is a physiological binding partner for HrVC70. We found that HrVC70 specifically interacts with HrUrabin, which had been separated by SDS-PAGE and transferred onto a nitrocellulose membrane. HrUrabin has an N-linked sugar chain, essential for binding to HrVC70. HrUrabin mRNA is expressed in the testis but not in the ovary, and the protein appears to be localized on the surface of sperm head and tail. Anti-HrUrabin antibody, which neutralizes the interaction between HrUrabin and HrVC70, potently inhibited fertilization and allorecognizable sperm-binding to HrVC70-agarose. However, no significant difference in the binding ability of HrUrabin to HrVC70 was observed in autologous and allogeneic combinations by Far Western analyses. These results indicate that sperm-egg binding in H. roretzi is mediated by the molecular interaction between HrUrabin on the sperm surface and HrVC70 on the vitelline coat, but that HrUrabin per se is unlikely to be a direct allorecognition protein.

  3. Immobilization of the Glycosylphosphatidylinositol-anchored Gas1 Protein into the Chitin Ring and Septum Is Required for Proper Morphogenesis in Yeast

    PubMed Central

    Rolli, Eleonora; Ragni, Enrico; Calderon, Julia; Porello, Silvia; Fascio, Umberto

    2009-01-01

    Gas1p is a glucan-elongase that plays a crucial role in yeast morphogenesis. It is predominantly anchored to the plasma membrane through a glycosylphosphatidylinositol, but a fraction was also found covalently bound to the cell wall. We have used fusions with the green fluorescent protein or red fluorescent protein (RFP) to determine its localization. Gas1p was present in microdomains of the plasma membrane, at the mother-bud neck and in the bud scars. By exploiting the instability of RFP-Gas1p, we identified mobile and immobile pools of Gas1p. Moreover, in chs3Δ cells the chitin ring and the cross-linked Gas1p were missing, but this unveiled an additional unexpected localization of Gas1p along the septum line in cells at cytokinesis. Localization of Gas1p was also perturbed in a chs2Δ mutant where a remedial septum is produced. Phenotypic analysis of cells expressing a fusion of Gas1p to a transmembrane domain unmasked new roles of the cell wall-bound Gas1p in the maintenance of the bud neck size and in cell separation. We present evidence that Crh1p and Crh2p are required for tethering Gas1p to the chitin ring and bud scar. These results reveal a new mechanism of protein immobilization at specific sites of the cell envelope. PMID:19793924

  4. 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

  5. Tat proteins as novel thylakoid membrane anchors organize a biosynthetic pathway in chloroplasts and increase product yield 5-fold.

    PubMed

    Henriques de Jesus, Maria Perestrello Ramos; Zygadlo Nielsen, Agnieszka; Busck Mellor, Silas; Matthes, Annemarie; Burow, Meike; Robinson, Colin; Erik Jensen, Poul

    2017-11-01

    Photosynthesis drives the production of ATP and NADPH, and acts as a source of carbon for primary metabolism. NADPH is also used in the production of many natural bioactive compounds. These are usually synthesized in low quantities and are often difficult to produce by chemical synthesis due to their complex structures. Some of the crucial enzymes catalyzing their biosynthesis are the cytochromes P450 (P450s) situated in the endoplasmic reticulum (ER), powered by electron transfers from NADPH. Dhurrin is a cyanogenic glucoside and its biosynthesis involves a dynamic metabolon formed by two P450s, a UDP-glucosyltransferase (UGT) and a P450 oxidoreductase (POR). Its biosynthetic pathway has been relocated to the chloroplast where ferredoxin, reduced through the photosynthetic electron transport chain, serves as an efficient electron donor to the P450s, bypassing the involvement of POR. Nevertheless, translocation of the pathway from the ER to the chloroplast creates other difficulties, such as the loss of metabolon formation and intermediate diversion into other metabolic pathways. We show here that co-localization of these enzymes in the thylakoid membrane leads to a significant increase in product formation, with a concomitant decrease in off-pathway intermediates. This was achieved by exchanging the membrane anchors of the dhurrin pathway enzymes to components of the Twin-arginine translocation pathway, TatB and TatC, which have self-assembly properties. Consequently, we show 5-fold increased titers of dhurrin and a decrease in the amounts of intermediates and side products in Nicotiana benthamiana. Further, results suggest that targeting the UGT to the membrane is a key factor to achieve efficient substrate channeling. Copyright © 2017 International Metabolic Engineering Society. All rights reserved.

  6. 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.

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

    SciTech Connect

    Kind, Barbara, E-mail: barbara.kind@uniklinikum-dresden.de; Koehler, Katrin, E-mail: katrin.koehler@uniklinikum-dresden.de; Lorenz, Mike, E-mail: mlorenz@mpi-cbg.de

    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 andmore » 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.« less

  8. 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

  9. The Mr 140,000 Intermediate Chain of Chlamydomonas Flagellar Inner Arm Dynein Is a WD-Repeat Protein Implicated in Dynein Arm Anchoring

    PubMed Central

    Yang, Pinfen; Sale, Winfield S.

    1998-01-01

    Previous structural and biochemical studies have revealed that the inner arm dynein I1 is targeted and anchored to a unique site located proximal to the first radial spoke in each 96-nm axoneme repeat on flagellar doublet microtubules. To determine whether intermediate chains mediate the positioning and docking of dynein complexes, we cloned and characterized the 140-kDa intermediate chain (IC140) of the I1 complex. Sequence and secondary structural analysis, with particular emphasis on β-sheet organization, predicted that IC140 contains seven WD repeats. Reexamination of other members of the dynein intermediate chain family of WD proteins indicated that these polypeptides also bear seven WD/β-sheet repeats arranged in the same pattern along each intermediate chain protein. A polyclonal antibody was raised against a 53-kDa fusion protein derived from the C-terminal third of IC140. The antibody is highly specific for IC140 and does not bind to other dynein intermediate chains or proteins in Chlamydomonas flagella. Immunofluorescent microscopy of Chlamydomonas cells confirmed that IC140 is distributed along the length of both flagellar axonemes. In vitro reconstitution experiments demonstrated that the 53-kDa C-terminal fusion protein binds specifically to axonemes lacking the I1 complex. Chemical cross-linking indicated that IC140 is closely associated with a second intermediate chain in the I1 complex. These data suggest that IC140 contains domains responsible for the assembly and docking of the I1 complex to the doublet microtubule cargo. PMID:9843573

  10. The maize (Zea mays L.) roothairless3 gene encodes a putative GPI-anchored, monocot-specific, COBRA-like protein that significantly affects grain yield.

    PubMed

    Hochholdinger, Frank; Wen, Tsui-Jung; Zimmermann, Roman; Chimot-Marolle, Patricia; da Costa e Silva, Oswaldo; Bruce, Wesley; Lamkey, Kendall R; Wienand, Udo; Schnable, Patrick S

    2008-06-01

    The rth3 (roothairless 3) mutant is specifically affected in root hair elongation. We report here the cloning of the rth3 gene via a PCR-based strategy (amplification of insertion mutagenized sites) and demonstrate that it encodes a COBRA-like protein that displays all the structural features of a glycosylphosphatidylinositol anchor. Genes of the COBRA family are involved in various types of cell expansion and cell wall biosynthesis. The rth3 gene belongs to a monocot-specific clade of the COBRA gene family comprising two maize and two rice genes. While the rice (Oryza sativa) gene OsBC1L1 appears to be orthologous to rth3 based on sequence similarity (86% identity at the protein level) and maize/rice synteny, the maize (Zea mays L.) rth3-like gene does not appear to be a functional homolog of rth3 based on their distinct expression profiles. Massively parallel signature sequencing analysis detected rth3 expression in all analyzed tissues, but at relatively low levels, with the most abundant expression in primary roots where the root hair phenotype is manifested. In situ hybridization experiments confine rth3 expression to root hair-forming epidermal cells and lateral root primordia. Remarkably, in replicated field trials involving near-isogenic lines, the rth3 mutant conferred significant losses in grain yield.

  11. A Kinase Divided.

    PubMed

    Karra, Aroon S; Taylor, Clinton A; Thorne, Curtis A; Cobb, Melanie H

    2015-08-10

    In this issue of Cancer Cell, Herrero and colleagues identify an anti-tumorigenic small molecule that blocks ERK dimerization, but neither its catalytic activity nor its phosphorylation by MEK. These findings demonstrate that targeting protein dimerization could be a therapeutic avenue for inhibiting kinase signaling pathways associated with lower drug resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. 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. Copyright © 2016 by the American Society of Nephrology.

  13. Functional Characterization and Localization of a Bacillus subtilis Sortase and Its Substrate and Use of This Sortase System To Covalently Anchor a Heterologous Protein to the B. subtilis Cell Wall for Surface Display

    PubMed Central

    Liew, Pei Xiong; Wang, Christopher L. C.

    2012-01-01

    Sortases catalyze the covalent anchoring of proteins to the cell surface on Gram-positive bacteria. Bioinformatic analysis suggests the presence of structural genes encoding sortases and their substrates in the Bacillus subtilis genome. In this study, a β-lactamase reporter was fused to the cell wall anchoring domain from a putative sortase substrate, YhcR. Covalent anchoring of this fusion protein to the cell wall was confirmed by using the eight-protease-deficient B. subtilis strain WB800 as the host. Inactivation of yhcS abolished the cell wall anchoring reaction. The amounts of fusion protein anchored to the cell wall were proportional to the levels of YhcS. These data demonstrate that YhcS and YhcR are the sortase and sortase substrate, respectively, in B. subtilis. Furthermore, yhcS is not essential for the survival of B. subtilis under the cultivation condition tested. YhcR fusions were distributed helically in the lateral cell wall. Interestingly, when viewed with an epifluorescence microscope, YhcS also appeared to form short helical arcs. This is the first report to illustrate such distribution of sortases in a rod-shaped bacterium. Models for the spatial distribution of both the sortase and its substrate are discussed. The amount of the reporters displayed on the surface was unambiguously quantified via a unique strategy. Under optimal conditions with the overproduction of YhcS, 47,300 YhcR fusions could be displayed per cell. Displayed reporters were biologically functional and surface accessible. Characterization of the sortase-substrate system allowed the successful development of a YhcR-based covalent surface display system. This system may have various biotechnological applications. PMID:22020651

  14. Quantitative determination of the lateral density and intermolecular correlation between proteins anchored on the membrane surfaces using grazing incidence small-angle X-ray scattering and grazing incidence X-ray fluorescence.

    PubMed

    Abuillan, Wasim; Vorobiev, Alexei; Hartel, Andreas; Jones, Nicola G; Engstler, Markus; Tanaka, Motomu

    2012-11-28

    As a physical model of the surface of cells coated with densely packed, non-crystalline proteins coupled to lipid anchors, we functionalized the surface of phospholipid membranes by coupling of neutravidin to biotinylated lipid anchors. After the characterization of fine structures perpendicular to the plane of membrane using specular X-ray reflectivity, the same membrane was characterized by grazing incidence small angle X-ray scattering (GISAXS). Within the framework of distorted wave Born approximation and two-dimensional Percus-Yevick function, we can analyze the form and structure factors of the non-crystalline, membrane-anchored proteins for the first time. As a new experimental technique to quantify the surface density of proteins on the membrane surface, we utilized grazing incidence X-ray fluorescence (GIXF). Here, the mean intermolecular distance between proteins from the sulfur peak intensities can be calculated by applying Abelé's matrix formalism. The characteristic correlation distance between non-crystalline neutravidin obtained by the GISAXS analysis agrees well with the intermolecular distance calculated by GIXF, suggesting a large potential of the combination of GISAXS and GIXF in probing the lateral density and correlation of non-crystalline proteins displayed on the membrane surface.

  15. 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…

  16. Growth arrest specific 1 (Gas1) and glial cell line-derived neurotrophic factor receptor α1 (Gfrα1), two mouse oocyte glycosylphosphatidylinositol-anchored proteins, are involved in fertilisation.

    PubMed

    Agopiantz, M; Xandre-Rodriguez, L; Jin, B; Urbistondoy, G; Ialy-Radio, C; Chalbi, M; Wolf, J-P; Ziyyat, A; Lefèvre, B

    2017-04-01

    Recently, Juno, the oocyte receptor for Izumo1, a male immunoglobulin, was discovered. Juno is an essential glycosylphosphatidylinositol (GIP)-anchored protein. This result did not exclude the participation of other GIP-anchored proteins in this process. After bibliographic and database searches we selected five GIP-anchored proteins (Cpm, Ephrin-A4, Gas1, Gfra1 and Rgmb) as potential oocyte candidates participating in fertilisation. Western blot and immunofluorescence analyses showed that only three were present on the mouse ovulated oocyte membrane and, of these, only two were clearly involved in the fertilisation process, namely growth arrest specific 1 (Gas1) and glial cell line-derived neurotrophic factor receptor α1 (Gfrα1). This was demonstrated by evaluating oocyte fertilisability after treatment of oocytes with antibodies against the selected proteins, with their respective short interference RNA or both. Gfrα1 and Gas1 seem to be neither redundant nor synergistic. In conclusion, oocyte Gas1 and Gfrα1 are both clearly involved in fertilisation.

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

    PubMed Central

    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

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

    PubMed

    Yu, Xiu-Jun; Liu, Mei; Holden, David W

    2016-07-12

    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. Upon invasion of epithelial cells, the majority of vacuoles containing Salmonella enterica migrate to the perinuclear region-located Golgi network and remain in this region of the cell during the first few rounds of bacterial replication, forming a

  19. Structure of a BAG6 (Bcl-2-associated Athanogene 6)-Ubl4a (Ubiquitin-like Protein 4a) Complex Reveals a Novel Binding Interface That Functions in Tail-anchored Protein Biogenesis*

    PubMed Central

    Kuwabara, Naoyuki; Minami, Ryosuke; Yokota, Naoto; Matsumoto, Hirofumi; Senda, Toshiya; Kawahara, Hiroyuki; Kato, Ryuichi

    2015-01-01

    BAG6 is an essential protein that functions in two distinct biological pathways, ubiquitin-mediated protein degradation of defective polypeptides and tail-anchored (TA) transmembrane protein biogenesis in mammals, although its structural and functional properties remain unknown. We solved a crystal structure of the C-terminal heterodimerization domains of BAG6 and Ubl4a and characterized their interaction biochemically. Unexpectedly, the specificity and structure of the C terminus of BAG6, which was previously classified as a BAG domain, were completely distinct from those of the canonical BAG domain. Furthermore, the tight association of BAG6 and Ubl4a resulted in modulation of Ubl4a protein stability in cells. Therefore, we propose to designate the Ubl4a-binding region of BAG6 as the novel BAG-similar (BAGS) domain. The structure of Ubl4a, which interacts with BAG6, is similar to the yeast homologue Get5, which forms a homodimer. These observations indicate that the BAGS domain of BAG6 promotes the TA protein biogenesis pathway in mammals by the interaction with Ubl4a. PMID:25713138

  20. Freely turning over palmitate in erythrocyte membrane proteins is not responsible for the anchoring of lipid rafts to the spectrin skeleton: a study with bio-orthogonal chemical probes.

    PubMed

    Ciana, Annarita; Achilli, Cesare; Hannoush, Rami N; Risso, Angela; Balduini, Cesare; Minetti, Giampaolo

    2013-03-01

    Erythrocyte lipid rafts are anchored to the underlying spectrin membrane skeleton [A. Ciana, C. Achilli, C. Balduini, G. Minetti, On the association of lipid rafts to the spectrin skeleton in human erythrocytes, Biochim. Biophys. Acta 1808 (2011) 183-190]. The nature of this linkage and the molecules involved are poorly understood. The interaction is sensitive to the increase in pH and ionic strength induced by carbonate. Given the role of palmitoylation in modulating the partitioning of certain proteins between various sub-cellular compartments and the plasma membrane, we asked whether palmitoylation of p55, a peripheral protein located at the junctional complex between spectrin-actin-protein 4.1 that anchors the membrane skeleton to the lipid bilayer via the transmembrane protein glycophorin C, could contribute to the anchoring of lipid rafts to the membrane skeleton. We adopted a new, non-radioactive method for studying protein palmitoylation, based on bio-orthogonal chemical analogues of fatty acids, containing an omega-alkynyl group, to metabolically label cell proteins, which are then revealed by a "click chemistry" reaction of the alkynyl moiety with an azide-containing reporter tag. We show that the membrane localization and palmitoylation levels of p55 did not change after carbonate treatment. 2-bromopalmitate and cerulenin, two known palmitoylation inhibitors, completely inhibited p55 palmitoylation, and protein palmitoyl thioesterase-1 (PPT1) reduced it, without affecting the association between lipid rafts and membrane-skeleton, indicating, on the one hand, that p55 palmitoylation is enzymatic, and, on the other, that it is not involved in the modulation of the linkage of lipid rafts to the membrane-skeleton. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Bivalent ligation of the collagen-binding modules of fibronectin by SFS, a non-anchored bacterial protein of Streptococcus equi.

    PubMed

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

    2015-02-20

    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 (8-9)FNI 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 (8-9)FNI or a large dimeric FN construct that is constitutively in an extended conformation. In contrast to bacterial peptides that bind (2-5)FNI in addition to (8-9)FNI, 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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. 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

  3. 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

  4. 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.

  5. Cell-Surface Displayed Expression of Trehalose Synthase from Pseudomonas putida ATCC 47054 in Pichia Pastoris Using Pir1p as an Anchor Protein

    PubMed Central

    Yang, Shaojie; Lv, Xin; Wang, Xihui; Wang, Junqing; Wang, Ruiming; Wang, Tengfei

    2017-01-01

    Yeast cell-surface display technologies have been widely applied in the fields of food, medicine, and feed enzyme production, including lipase, α-amylase, and endoglucanase. In this study, a treS gene was fused with the yeast cell-surface anchor protein gene Pir1p by overlap PCR, the Pir1p-treS fusion gene was ligated into pPICZαA and pGAPZαA and transformed into P. pastoris GS115 to obtain recombinant yeast strains that displays trehalose synthase(TreS) on its cell surface as an efficient and recyclable whole-cell biocatalyst. Firstly, the enhanced green fluorescence protein gene (egfp) was used as the reporter protein to fusion the Pir1p gene and treS gene to construct the recombinant plasmids containing treS-egfg-Pir1p fusion gene, and electrotransformed into P. pastoris GS115 to analyze the surface display characteristics of fusion gene by Western blot, fluorescence microscopy and flow cytometry. The analysis shown that the treS-egfg-Pir1p fusion protein can be successfully displayed on the surface of yeast cell, and the expression level increased with the extension of fermentation time. These results implied that the Pir1p-treS fusion gene can be well displayed on the cell surface. Secondly, in order to obtain surface active cells with high enzyme activity, the enzymatic properties of TreS displayed on the cell surface was analyzed, and the fermentation process of recombinant P. patoris GS115 containing pPICZαA-Pir1p-treS and pGAPZαA-Pir1p-treS was studied respectively. The cell surface display TreS was stable over a broad range of temperatures (10–45°C) and pH (6.0–8.5). The activity of TreS displayed on cell surface respectively reached 1,108 Ug−1 under PAOX1 control for 150 h, and 1,109 Ug−1 under PGAP control for 75h in a 5 L fermenter, respectively. Lastly, the cell-surface displayed TreS was used to product trehalose using high maltose syrup as substrate at pH 8.0 and 15°C. The surface display TreS cells can be recycled for three times and

  6. 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…

  7. 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

  8. 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.

  9. Anchored paired comparisons

    NASA Astrophysics Data System (ADS)

    Dalal, E. N.; Handley, J. C.; Wu, W.; Wang, J.

    2008-01-01

    The method of paired comparisons is often used in image quality evaluations. Psychometric scale values for quality judgments are modeled using Thurstone's Law of Comparative Judgment in which distance in a psychometric scale space is a function of the probability of preference. The transformation from psychometric space to probability is a cumulative probability distribution. The major drawback of a complete paired comparison experiment is that every treatment is compared to every other, thus the number of comparisons grows quadratically. We ameliorate this difficulty by performing paired comparisons in two stages, by precisely estimating anchors in the psychometric scale space which are spaced apart to cover the range of scale values and comparing treatments against those anchors. In this model, we employ a generalized linear model where the regression equation has a constant offset vector determined by the anchors. The result of this formulation is a straightforward statistical model easily analyzed using any modern statistics package. This enables model fitting and diagnostics. This method was applied to overall preference evaluations of color pictorial hardcopy images. The results were found to be compatible with complete paired comparison experiments, but with significantly less effort.

  10. One Single Basic Amino Acid at the ω-1 or ω-2 Site Is a Signal That Retains Glycosylphosphatidylinositol-Anchored Protein in the Plasma Membrane of Aspergillus fumigatus

    PubMed Central

    Ouyang, Haomiao; Chen, Xiaomin; Lü, Yang; Wilson, Iain B. H.; Tang, Guomin; Wang, Aoquan

    2013-01-01

    Although the plasma membrane is the terminal destination for glycosylphosphatidylinositol (GPI) proteins in higher eukaryotes, cell wall-attached GPI proteins (GPI-CWPs) are found in many fungal species. In yeast, some of the cis-requirements directing localization of GPI proteins to the plasma membrane or cell wall are now understood. However, it remains to be determined how Aspergillus fumigatus, an opportunistic fungal pathogen, signals, and sorts GPI proteins to either the plasma membrane or the cell wall. In this study, chimeric green fluorescent proteins (GFPs) were constructed as fusions with putative C-terminal GPI signal sequences from A. fumigatus Mp1p, Gel1p, and Ecm33p, as well as site-directed mutations thereof. By analyzing cellular localization of chimeric GFPs using Western blotting, electron microscopy, and fluorescence microscopy, we showed that, in contrast to yeast, a single Lys residue at the ω-1 or ω-2 site alone could retain GPI-anchored GFP in the plasma membrane. Although the signal for cell wall distribution has not been identified yet, it appeared that the threonine/serine-rich region at the C-terminal half of AfMp1 was not required for cell wall distribution. Based on our results, the cis-requirements directing localization of GPI proteins in A. fumigatus are different from those in yeast. PMID:23584992

  11. 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.

  12. Career anchors of dentist leaders.

    PubMed

    Tuononen, Tiina; Lammintakanen, Johanna; Suominen, Anna Liisa

    2016-08-01

    The work of a health care leader is demanding; in order to cope, leaders need motivation and support. The occurrence of intrinsic factors called career anchors (combination of one's competence, motives and values) could be a contributing factor in dentist leaders' career decisions. The aim of our study was to identify dentist leaders' career anchors and their association to dentist leaders' retention or turnover of the leadership position. Materials were gathered in 2014 via an electronic questionnaire from 156 current (Leaders) or former (Leavers) Finnish dentist leaders. Career anchor evaluation was conducted by the questionnaire and scoring-table taken from Edgar Schein's Career Anchors Self-Assessment. Both the most and the least important career anchors were detected by the highest and lowest scores and their occurrence reported as percentages. Associations between career anchor scores and tendency to stay were analyzed with logistic regression. 'Technical/Functional Competence' and 'Lifestyle' were most frequently reported as the most important and 'Entrepreneurial Creativity' and 'General Managerial Competence' as the least important career anchors. However, a higher level of 'General Managerial Competence' anchor was most significantly associated with staying in a leadership position. Instead, 'Pure Challenge' and 'Lifestyle' decreased the odds to stay. The knowledge of the important and essential career anchors of dentist leaders' and individuals' could perform crucial part in career choices and also in planning education, work opportunities and human resource policies promoting retention of dentist leaders and probably also other health care leaders.

  13. Oral vaccine of Lactococcus lactis harbouring pandemic H1N1 2009 haemagglutinin1 and nisP anchor fusion protein elevates anti-HA1 sIgA levels in mice.

    PubMed

    Joan, Stella Siaw Xiu; Pui-Fong, Jee; Song, Adelene Ai-Lian; Chang, Li-Yen; Yusoff, Khatijah; AbuBakar, Sazaly; Rahim, Raha Abdul

    2016-05-01

    An oral lactococcal-based vaccine which haboured the haemagglutinin1 (HA1) antigen fused to nisP anchor protein for the purpose of surface displaying the HA1 antigen was developed against H1N1 virus. Recombinant L. lactis strains expressed HA1-nisP fusion proteins when induced with nisin, as confirmed through western blotting. However, immunofluorescense did not detect any surface-displayed proteins, suggesting that the protein was either unsuccessfully translocated or improperly displayed. Despite this, oral administration of recombinant L. lactis strains to BALB/c mice revealed that significant levels of anti-HA1 sIgA antibodies were detected in mice fecal suspension samples of mice group NZ9000 (pNZ:HN) when compared to the negative control NZ9000 (pNZ8048) group. Specific anti-HA1 sIgA antibodies were locally produced and live recombinant lactococcal vaccine was able to elicit humoral response of BALB/c mice despite unsuccessful surface display of the HA1 epitope.

  14. Not all Anchors Weigh Equally.

    PubMed

    Greenstein, Michael; Velazquez, Alexandra

    2017-11-01

    The anchoring bias is a reliable effect wherein a person's judgments are affected by initially presented information, but it is unknown specifically why this effect occurs. Research examining this bias suggests that elements of both numeric and semantic priming may be involved. To examine this, the present research used a phenomenon wherein people treat numeric information presented differently in Arabic numeral or verbal formats. We presented participants with one of many forms of an anchor that represented the same value (e.g., twelve hundred or 1,200). Thus, we could examine how a concept's meaning and its absolute numeric value affect anchoring. Experiments 1 and 2 showed that people respond to Arabic and verbal anchors differently. Experiment 3 showed that these differences occurred largely because people tend to think of numbers in digit format. This suggests that one's conceptual understanding of the anchored information matters more than its strict numeric value.

  15. Extracellular glycosylphosphatidylinositol-anchored mannoproteins and proteases of Cryptococcus neoformans.

    PubMed

    Eigenheer, Richard A; Jin Lee, Young; Blumwald, Eduardo; Phinney, Brett S; Gelli, Angie

    2007-06-01

    Extracellular proteins of Cryptococcus neoformans are involved in the pathogenesis of cryptococcosis, and some are immunoreactive antigens that may potentially serve as candidates for vaccine development. To further study the extracellular proteome of the human fungal pathogen Cry. neoformans, we conducted a proteomic analysis of secreted and cell wall-bound proteins with an acapsular strain of Cry. neoformans. Proteins were identified from both intact cells and cell walls. In both cases, extracellular proteins were removed with trypsin or beta-glucanase, and then all proteins/peptides were purified by solid-phase extraction, spin dialysis, and HPLC, and identified by liquid chromatography-mass spectrometry. This study identified 29 extracellular proteins with a predicted N-terminal signal sequence and also a predicted glycosylphosphatidylinositol anchor motif in more than half. Among the novel proteins identified were five glycosylphosphatidylinositol-anchored proteins with extensive Ser/Thr-rich regions but no apparent functional domains, a glycosylphosphatidylinositol-anchored aspartic protease, and a metalloprotease with structural similarity to an elastinolytic metalloprotease of Aspergillus fumigatus. This study suggests that Cry. neoformans has the machinery required to target glycosylphosphatidylinositol-anchored proteins to the cell wall, and it confirms the extracellular proteolytic ability of Cry. neoformans.

  16. 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.

  17. cAMP Signaling Compartmentation: Adenylyl Cyclases as Anchors of Dynamic Signaling Complexes.

    PubMed

    Johnstone, Timothy B; Agarwal, Shailesh R; Harvey, Robert D; Ostrom, Rennolds S

    2018-04-01

    It is widely accepted that cAMP signaling is compartmentalized within cells. However, our knowledge of how receptors, cAMP signaling enzymes, effectors, and other key proteins form specific signaling complexes to regulate specific cell responses is limited. The multicomponent nature of these systems and the spatiotemporal dynamics involved as proteins interact and move within a cell make cAMP responses highly complex. Adenylyl cyclases, the enzymatic source of cAMP production, are key starting points for understanding cAMP compartments and defining the functional signaling complexes. Three basic elements are required to form a signaling compartment. First, a localized signal is generated by a G protein-coupled receptor paired to one or more of the nine different transmembrane adenylyl cyclase isoforms that generate the cAMP signal in the cytosol. The diffusion of cAMP is subsequently limited by several factors, including expression of any number of phosphodiesterases (of which there are 24 genes plus spice variants). Finally, signal response elements are differentially localized to respond to cAMP produced within each locale. A-kinase-anchoring proteins, of which there are 43 different isoforms, facilitate this by targeting protein kinase A to specific substrates. Thousands of potential combinations of these three elements are possible in any given cell type, making the characterization of cAMP signaling compartments daunting. This review will focus on what is known about how cells organize cAMP signaling components as well as identify the unknowns. We make an argument for adenylyl cyclases being central to the formation and maintenance of these signaling complexes. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

  18. The N-terminus of IpaB provides a potential anchor to the Shigella type III secretion system tip complex protein IpaD

    PubMed Central

    Dickenson, Nicholas E.; Arizmendi, Olivia; Patil, Mrinalini K.; Toth, Ronald T.; Middaugh, C. Russell; Picking, William D.; Picking, Wendy L.

    2014-01-01

    The type III secretion system (T3SS) is an essential virulence factor for Shigella flexneri, providing a conduit through which host-altering effectors are injected directly into a host cell to promote uptake. The type III secretion apparatus (T3SA) is comprised of a basal body, external needle, and regulatory tip complex. The nascent needle is a polymer of MxiH capped by a pentamer of invasion plasmid antigen D (IpaD). Exposure to bile salts (e.g. deoxycholate) causes a conformational change in IpaD and promotes recruitment of IpaB to the needle tip. It has been proposed that IpaB senses contact with host cell membranes, recruiting IpaC and inducing full secretion of T3SS effectors. While the steps of T3SA maturation and their external triggers have been identified, details of specific protein interactions and mechanisms have remained difficult to study due to the hydrophobic nature of the IpaB and IpaC translocator proteins. Here we explored the ability for a series of soluble N-terminal IpaB peptides to interact with IpaD. We found that DOC is required for the interaction and that a region of IpaB between residues 11–27 is required for maximum binding, which was confirmed in vivo. Furthermore, intramolecular FRET measurements indicated that movement of the IpaD distal domain away from the protein core accompanied the binding of IpaB11-226. Together these new findings provide important new insight into the interactions and potential mechanisms that define the maturation of the Shigella T3SA needle tip complex and provide a foundation for further studies probing T3SS activation. PMID:24236510

  19. N-terminus of IpaB provides a potential anchor to the Shigella type III secretion system tip complex protein IpaD.

    PubMed

    Dickenson, Nicholas E; Arizmendi, Olivia; Patil, Mrinalini K; Toth, Ronald T; Middaugh, C Russell; Picking, William D; Picking, Wendy L

    2013-12-10

    The type III secretion system (T3SS) is an essential virulence factor for Shigella flexneri , providing a conduit through which host-altering effectors are injected directly into a host cell to promote uptake. The type III secretion apparatus (T3SA) is composed of a basal body, external needle, and regulatory tip complex. The nascent needle is a polymer of MxiH capped by a pentamer of invasion plasmid antigen D (IpaD). Exposure to bile salts (e.g., deoxycholate) causes a conformational change in IpaD and promotes recruitment of IpaB to the needle tip. It has been proposed that IpaB senses contact with host cell membranes, recruiting IpaC and inducing full secretion of T3SS effectors. Although the steps of T3SA maturation and their external triggers have been identified, details of specific protein interactions and mechanisms have remained difficult to study because of the hydrophobic nature of the IpaB and IpaC translocator proteins. Here, we explored the ability for a series of soluble N-terminal IpaB peptides to interact with IpaD. We found that DOC is required for the interaction and that a region of IpaB between residues 11-27 is required for maximum binding, which was confirmed in vivo. Furthermore, intramolecular FRET measurements indicated that movement of the IpaD distal domain away from the protein core accompanied the binding of IpaB11-226. Together, these new findings provide important new insight into the interactions and potential mechanisms that define the maturation of the Shigella T3SA needle tip complex and provide a foundation for further studies probing T3SS activation.

  20. Immediate Early Genes Anchor a Biological Pathway of Proteins Required for Memory Formation, Long-Term Depression and Risk for Schizophrenia

    PubMed Central

    Marballi, Ketan K.; Gallitano, Amelia L.

    2018-01-01

    While the causes of myriad medical and infectious illnesses have been identified, the etiologies of neuropsychiatric illnesses remain elusive. This is due to two major obstacles. First, the risk for neuropsychiatric disorders, such as schizophrenia, is determined by both genetic and environmental factors. Second, numerous genes influence susceptibility for these illnesses. Genome-wide association studies have identified at least 108 genomic loci for schizophrenia, and more are expected to be published shortly. In addition, numerous biological processes contribute to the neuropathology underlying schizophrenia. These include immune dysfunction, synaptic and myelination deficits, vascular abnormalities, growth factor disruption, and N-methyl-D-aspartate receptor (NMDAR) hypofunction. However, the field of psychiatric genetics lacks a unifying model to explain how environment may interact with numerous genes to influence these various biological processes and cause schizophrenia. Here we describe a biological cascade of proteins that are activated in response to environmental stimuli such as stress, a schizophrenia risk factor. The central proteins in this pathway are critical mediators of memory formation and a particular form of hippocampal synaptic plasticity, long-term depression (LTD). Each of these proteins is also implicated in schizophrenia risk. In fact, the pathway includes four genes that map to the 108 loci associated with schizophrenia: GRIN2A, nuclear factor of activated T-cells (NFATc3), early growth response 1 (EGR1) and NGFI-A Binding Protein 2 (NAB2); each of which contains the “Index single nucleotide polymorphism (SNP)” (most SNP) at its respective locus. Environmental stimuli activate this biological pathway in neurons, resulting in induction of EGR immediate early genes: EGR1, EGR3 and NAB2. We hypothesize that dysfunction in any of the genes in this pathway disrupts the normal activation of Egrs in response to stress. This may result in

  1. 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.

  2. The RACK1 signal anchor protein from Trypanosoma brucei associates with eukaryotic elongation factor 1A: a role for translational control in cytokinesis

    PubMed Central

    Regmi, Sandesh; Rothberg, Karen G; Hubbard, James G; Ruben, Larry

    2008-01-01

    RACK1 is a WD-repeat protein that forms signal complexes at appropriate locations in the cell. RACK1 homologues are core components of ribosomes from yeast, plants and mammals. In contrast, a cryo-EM analysis of trypanosome ribosomes failed to detect RACK1, thus eliminating an important translational regulatory mechanism. Here we report that TbRACK1 from Trypanosoma brucei associates with eukaryotic translation elongation factor-1a (eEF1A) as determined by tandem MS of TAP-TbRACK1 affinity eluates, co-sedimentation in a sucrose gradient, and co-precipitation assays. Consistent with these observations, sucrose gradient purified 80S monosomes and translating polysomes each contained TbRACK1. When RNAi was used to deplete cells of TbRACK1, a shift in the polysome profile was observed, while the phosphorylation of a ribosomal protein increased. Under these conditions, cell growth became hypersensitive to the translational inhibitor anisomycin. The kinetoplasts and nuclei were misaligned in the postmitotic cells, resulting in partial cleavage furrow ingression during cytokinesis. Overall, these findings identify eEF1A as a novel TbRACK1 binding partner and establish TbRACK1 as a component of the trypanosome translational apparatus. The synergy between anisomycin and TbRACK1 RNAi suggests that continued translation is required for complete ingression of the cleavage furrow. PMID:18786142

  3. Phosphatidylinositol 3-Kinase and Rab5 GTPase Inversely Regulate the Smad Anchor for Receptor Activation (SARA) Protein Independently of Transforming Growth Factor-β1

    PubMed Central

    Runyan, Constance E.; Liu, Zongyi; Schnaper, H. William

    2012-01-01

    SARA has been shown to be a regulator of epithelial cell phenotype, with reduced expression during TGF-β1-mediated epithelial-to-mesenchymal transition. Examination of the pathways that might play a role in regulating SARA expression identified phosphatidylinositol 3-kinase (PI3K) pathway inhibition as sufficient to reduce SARA expression. The mechanism of PI3K inhibition-mediated SARA down-regulation differs from that induced by TGF-β1 in that, unlike TGF-β1, PI3K-dependent depletion of SARA was apparent within 6 h and did not occur at the mRNA or promoter level but was blocked by inhibition of proteasome-mediated degradation. This effect was independent of Akt activity because neither reducing nor enhancing Akt activity modulated the expression of SARA. Therefore, this is likely a direct effect of p85α action, and co-immunoprecipitation of SARA and p85α confirmed that these proteins interact. Both SARA and PI3K have been shown to be associated with endosomes, and either LY294002 or p85α knockdown enlarged SARA-containing endocytic vesicles. Inhibition of clathrin-mediated endocytosis blocked SARA down-regulation, and a localization-deficient mutant SARA was protected against down-regulation. As inhibiting PI3K can activate the endosomal fusion-regulatory small GTPase Rab5, we expressed GTPase-deficient Rab5 and observed endosomal enlargement and reduced SARA protein expression, similar to that seen with PI3K inhibition. Importantly, either interference with PI3K via LY294002 or p85α knockdown, or constitutive activity of the Rab5 pathway, enhanced the expression of smooth muscle α-actin. Together, these data suggest that although TGF-β1 can induce epithelial-to-mesenchymal transition through reduction in SARA expression, SARA is also basally regulated by its interaction with PI3K. PMID:22942286

  4. Phosphatidylinositol 3-kinase and Rab5 GTPase inversely regulate the Smad anchor for receptor activation (SARA) protein independently of transforming growth factor-β1.

    PubMed

    Runyan, Constance E; Liu, Zongyi; Schnaper, H William

    2012-10-19

    SARA has been shown to be a regulator of epithelial cell phenotype, with reduced expression during TGF-β1-mediated epithelial-to-mesenchymal transition. Examination of the pathways that might play a role in regulating SARA expression identified phosphatidylinositol 3-kinase (PI3K) pathway inhibition as sufficient to reduce SARA expression. The mechanism of PI3K inhibition-mediated SARA down-regulation differs from that induced by TGF-β1 in that, unlike TGF-β1, PI3K-dependent depletion of SARA was apparent within 6 h and did not occur at the mRNA or promoter level but was blocked by inhibition of proteasome-mediated degradation. This effect was independent of Akt activity because neither reducing nor enhancing Akt activity modulated the expression of SARA. Therefore, this is likely a direct effect of p85α action, and co-immunoprecipitation of SARA and p85α confirmed that these proteins interact. Both SARA and PI3K have been shown to be associated with endosomes, and either LY294002 or p85α knockdown enlarged SARA-containing endocytic vesicles. Inhibition of clathrin-mediated endocytosis blocked SARA down-regulation, and a localization-deficient mutant SARA was protected against down-regulation. As inhibiting PI3K can activate the endosomal fusion-regulatory small GTPase Rab5, we expressed GTPase-deficient Rab5 and observed endosomal enlargement and reduced SARA protein expression, similar to that seen with PI3K inhibition. Importantly, either interference with PI3K via LY294002 or p85α knockdown, or constitutive activity of the Rab5 pathway, enhanced the expression of smooth muscle α-actin. Together, these data suggest that although TGF-β1 can induce epithelial-to-mesenchymal transition through reduction in SARA expression, SARA is also basally regulated by its interaction with PI3K.

  5. Insect cell-derived cofactors become fully functional after proteinase K and heat treatment for high-fidelity amplification of glycosylphosphatidylinositol-anchored recombinant scrapie and BSE prion proteins.

    PubMed

    Imamura, Morikazu; Kato, Nobuko; Okada, Hiroyuki; Yoshioka, Miyako; Iwamaru, Yoshifumi; Shimizu, Yoshihisa; Mohri, Shirou; Yokoyama, Takashi; Murayama, Yuichi

    2013-01-01

    The central event in prion infection is the conformational conversion of host-encoded cellular prion protein (PrP(C)) into the pathogenic isoform (PrP(Sc)). Diverse mammalian species possess the cofactors required for in vitro replication of PrP(Sc) by protein-misfolding cyclic amplification (PMCA), but lower organisms, such as bacteria, yeasts, and insects, reportedly lack the essential cofactors. Various cellular components, such as RNA, lipids, and other identified cofactor molecules, are commonly distributed in both eukaryotes and prokaryotes, but the reasons for the absence of cofactor activity in lower organisms remain to be elucidated. Previously, we reported that brain-derived factors were necessary for the in vitro replication of glycosylphosphatidylinositol-anchored baculovirus-derived recombinant PrP (Bac-PrP). Here, we demonstrate that following protease digestion and heat treatment, insect cell lysates had the functional cofactor activity required for Bac-PrP replication by PMCA. Mammalian PrP(Sc) seeds and Bac-PrP(Sc) generated by PMCA using Bac-PrP and insect cell-derived cofactors showed similar pathogenicity and produced very similar lesions in the brains of inoculated mice. These results suggested that the essential cofactors required for the high-fidelity replication of mammalian PrP(Sc) were present in the insect cells but that the cofactor activity was masked or inhibited in the native state. We suggest that not only RNA, but also DNA, are the key components of PMCA, although other cellular factors were necessary for the expression of the cofactor activity of nucleic acids. PMCA using only insect cell-derived substances (iPMCA) was highly useful for the ultrasensitive detection of PrP(Sc) of some prion strains.

  6. 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

  7. 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

  8. Genome-wide in silico identification of GPI proteins in Mycosphaerella fijiensis and transcriptional analysis of two GPI-anchored β-1,3-glucanosyltransferases.

    PubMed

    Kantún-Moreno, Nuvia; Vázquez-Euán, Roberto; Tzec-Simá, Miguel; Peraza-Echeverría, Leticia; Grijalva-Arango, Rosa; Rodríguez-García, Cecilia; James, Andrew C; Ramírez-Prado, Jorge; Islas-Flores, Ignacio; Canto-Canché, Blondy

    2013-01-01

    The hemibiotrophic fungus Mycosphaerella fijiensis is the causal agent of black Sigatoka (BS), the most devastating foliar disease in banana (Musa spp.) worldwide. Little is known about genes that are important during M. fijiensis-Musa sp. interaction. The fungal cell wall is an attractive area of study because it is essential for maintenance of cellular homeostasis and it is the most external structure in the fungal cell and therefore mediates the interaction of the pathogen with the host. In this manuscript we describe the in silico identification of glycosyl phosphatidylinositol-protein (GPI) family in M. fijiensis, and the analysis of two β-1,3-glucanosyltrans-ferases (Gas), selected by homology with fungal pathogenicity factors. Potential roles in pathogenesis were evaluated through analyzing expression during different stages of black Sigatoka disease, comparing expression data with BS symptoms and fungal biomass inside leaves. Real-time quantitative RT-PCR showed nearly constant expression of MfGAS1 with slightly increases (about threefold) in conidia and at speck-necrotrophic stage during banana-pathogen interaction. Conversely, MfGAS2 expression was increased during biotrophy (about seven times) and reached a maximum at speck (about 23 times) followed by a progressive decrease in next stages, suggesting an active role in M. fijiensis pathogenesis.

  9. Anchored in the Florida Current

    NASA Astrophysics Data System (ADS)

    Brooks, David A.

    I once spent a week aboard a vessel anchored in the Florida current, in water 830 m deep, far from the sight of land. We were there to measure transport variations, and I recall the numbing hourly routine of profiling the current with an instrument that traveled down a weighted line suspended from the ship. In much sharper focus I remember the mighty current that ceaselessly swept past the vessel. The current raised a foaming bow wave and strummed the anchor cable with a note that permeated the quiet ship, giving the strange experience of being underway with silent engines while going nowhere.

  10. 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

  11. ATHLETE : Double Auger Anchoring Mechanism

    NASA Technical Reports Server (NTRS)

    Shin, Joseph

    2011-01-01

    The All-Terrain Hex-Legged Extra-Terrestrial Explorer (ATHLETE) is a six-limbed robot designed to support surface explorations on Near Earth Objects, the Moon and Mars. ATHLETE can carry large payloads on its top deck and can carry a fully equipped pressurized habitat in low gravity. The robot has wheels on each of its six articulated limbs, allowing it to actively conform to terrain while driving and to walk when driving is impractical. With the use of a tool adapter, ATHLETE limbs can be equipped with end effectors to support various mission objectives. For work on Near Earth Objects and other microgravity environments, an anchoring mechanism is needed to keep the ATHLETE from floating off the surface. My goal for this spring session at JPL was to design and build a counter rotating, double auger, anchoring mechanism. The mechanism mates to the tool adapter and is driven off the wheel motor. The double auger anchoring mechanism will be tested in a regolith simulant that will determine the uplift capacity of the anchoring mechanism.

  12. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Permanent Ground Anchors : Nicholson Design Criteria

    DOT National Transportation Integrated Search

    1982-09-01

    This study discusses the methods used by Nicholson Construction Company in the design of permanent ground anchors specifically as related to retaining walls. Basic soil parameters, design concepts, drilling and grouting methods for ground anchors are...

  14. 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

  15. Anchoring bias in online voting

    NASA Astrophysics Data System (ADS)

    Yang, Zimo; Zhang, Zi-Ke; Zhou, Tao

    2012-12-01

    Voting online with explicit ratings could largely reflect people's preferences and objects' qualities, but ratings are always irrational, because they may be affected by many unpredictable factors like mood, weather and other people's votes. By analyzing two real systems, this paper reveals a systematic bias embedding in the individual decision-making processes, namely people tend to give a low rating after a low rating, as well as a high rating following a high rating. This so-called anchoring bias is validated via extensive comparisons with null models, and numerically speaking, the extent of bias decays with voting interval in a logarithmic form. Our findings could be applied in the design of recommender systems and considered as important complementary materials to previous knowledge about anchoring effects on financial trades, performance judgments, auctions, and so on.

  16. Pharmacophore anchor models of flaviviral NS3 proteases lead to drug repurposing for DENV infection.

    PubMed

    Pathak, Nikhil; Lai, Mei-Ling; Chen, Wen-Yu; Hsieh, Betty-Wu; Yu, Guann-Yi; Yang, Jinn-Moon

    2017-12-28

    Viruses of the flaviviridae family are responsible for some of the major infectious viral diseases around the world and there is an urgent need for drug development for these diseases. Most of the virtual screening methods in flaviviral drug discovery suffer from a low hit rate, strain-specific efficacy differences, and susceptibility to resistance. It is because they often fail to capture the key pharmacological features of the target active site critical for protein function inhibition. So in our current work, for the flaviviral NS3 protease, we summarized the pharmacophore features at the protease active site as anchors (subsite-moiety interactions). For each of the four flaviviral NS3 proteases (i.e., HCV, DENV, WNV, and JEV), the anchors were obtained and summarized into 'Pharmacophore anchor (PA) models'. To capture the conserved pharmacophore anchors across these proteases, were merged the four PA models. We identified five consensus core anchors (CEH1, CH3, CH7, CV1, CV3) in all PA models, represented as the "Core pharmacophore anchor (CPA) model" and also identified specific anchors unique to the PA models. Our PA/CPA models complied with 89 known NS3 protease inhibitors. Furthermore, we proposed an integrated anchor-based screening method using the anchors from our models for discovering inhibitors. This method was applied on the DENV NS3 protease to screen FDA drugs discovering boceprevir, telaprevir and asunaprevir as promising anti-DENV candidates. Experimental testing against DV2-NGC virus by in-vitro plaque assays showed that asunaprevir and telaprevir inhibited viral replication with EC 50 values of 10.4 μM & 24.5 μM respectively. The structure-anchor-activity relationships (SAAR) showed that our PA/CPA model anchors explained the observed in-vitro activities of the candidates. Also, we observed that the CEH1 anchor engagement was critical for the activities of telaprevir and asunaprevir while the extent of inhibitor anchor occupation guided

  17. Glucose-induced sequential processing of a glycosyl-phosphatidylinositol-anchored ectoprotein in Saccharomyces cerevisiae.

    PubMed

    Müller, G; Gross, E; Wied, S; Bandlow, W

    1996-01-01

    Transfer of spheroplasts from the yeast Saccharomyces cerevisiae to glucose leads to the activation of an endogenous (glycosyl)-phosphatidylinositol-specific phospholipase C ([G]PI-PLC), which cleaves the anchor of at least one glycosyl-phosphatidylinositol (GPI)-anchored protein, the cyclic AMP (cAMP)-binding ectoprotein Gce1p (G. Müller and W. Bandlow, J. Cell Biol. 122:325-336, 1993). Analyses of the turnover of two constituents of the anchor, myo-inositol and ethanolamine, relative to the protein label as well as separation of the two differently processed versions of Gce1p by isoelectric focusing in spheroplasts demonstrate the glucose-induced conversion of amphiphilic Gce1p first into a lipolytically cleaved hydrophilic intermediate, which is then processed into another hydrophilic version lacking both myo-inositol and ethanolamine. When incubated with unlabeled spheroplasts, the lipolytically cleaved intermediate prepared in vitro is converted into the version lacking all anchor constituents, whereby the anchor glycan is apparently removed as a whole. The secondary cleavage ensues independently of the carbon source, attributing the key role in glucose-induced anchor processing to the endogenous (G)PI-PLC. The secondary processing of the lipolytically cleaved intermediate of Gce1p at the plasma membrane is correlated with the emergence of a covalently linked high-molecular-weight form of a cAMP-binding protein at the cell wall. This protein lacks anchor components, and its protein moiety appears to be identical with double-processed Gce1p detectable at the plasma membrane in spheroplasts. The data suggest that glucose-induced double processing of GPI anchors represents part of a mechanism of regulated cell wall expression of proteins in yeast cells.

  18. Handbook for Uplift-Resisting Anchors

    DTIC Science & Technology

    1975-09-01

    construction or a trihedron construction of three flat * Free-fall anchors plates (Y fluke). The plates enter the seabed edge-wise. After emplacement...variety of forms has evolved, Figure 2.1-1 neck, a flat arrowhead shape, and a "three- illustrates the general design of such anchors. The dimensional...34 arrowhead (a pointed trihedron of flat anchor-projectile includes a piston and fluke. The gun plates with serrated or nonserrated edges). Figure

  19. 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.

  20. Spatial regulation of the cAMP-dependent protein kinase during chemotactic cell migration.

    PubMed

    Howe, Alan K; Baldor, Linda C; Hogan, Brian P

    2005-10-04

    Historically, the cAMP-dependent protein kinase (PKA) has a paradoxical role in cell motility, having been shown to both facilitate and inhibit actin cytoskeletal dynamics and cell migration. In an effort to understand this dichotomy, we show here that PKA is regulated in subcellular space during cell migration. Immunofluorescence microscopy and biochemical enrichment of pseudopodia showed that type II regulatory subunits of PKA and PKA activity are enriched in protrusive cellular structures formed during chemotaxis. This enrichment correlates with increased phosphorylation of key cytoskeletal substrates for PKA, including the vasodilator-stimulated phosphoprotein (VASP) and the protein tyrosine phosphatase containing a PEST motif. Importantly, inhibition of PKA activity or its ability to interact with A kinase anchoring proteins inhibited the activity of the Rac GTPase within pseudopodia. This effect correlated with both decreased guanine nucleotide exchange factor activity and increased GTPase activating protein activity. Finally, inhibition of PKA anchoring, like inhibition of total PKA activity, inhibited pseudopod formation and chemotactic cell migration. These data demonstrate that spatial regulation of PKA via anchoring is an important facet of normal chemotactic cell movement.

  1. 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 preformed...

  2. 21 CFR 872.3130 - Preformed anchor.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 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 preformed...

  3. 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 preformed...

  4. 21 CFR 872.3130 - Preformed anchor.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 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 preformed...

  5. 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 preformed...

  6. Case Type, Anchoring Errors, and Counselor Education.

    ERIC Educational Resources Information Center

    Pain, Michelle D.; Sharpley, Christopher F.

    1988-01-01

    Graduate students (N=28) in counseling read client information that was nonemotive and factual, indicative of well-adjustment, or suggestive of need for psychological help, then rated mental health of clients. Results revealed anchoring errors for all cases regardless of severity; presence of anchoring errors was restricted to those made as result…

  7. 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.

  8. 24 CFR 3285.401 - Anchoring instructions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... foundation system. See § 3285.301. (b) For anchor assembly type installations, the installation instructions... § 3285.301(d). (c) All anchoring and foundation systems must be capable of meeting the loads that the... wind zone areas may have foundation or anchorage systems that are capable of meeting the lower design...

  9. 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…

  10. A Tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles.

    PubMed

    Zúñiga-Navarrete, Fernando; Gómez, Isabel; Peña, Guadalupe; Bravo, Alejandra; Soberón, Mario

    2013-03-01

    Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl-phosphatidyl-inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68 kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68 kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. GPI-anchored Proteins and Free GPI Glycolipids of Procyclic Form Trypanosoma brucei Are Nonessential for Growth, Are Required for Colonization of the Tsetse Fly, and Are Not the Only Components of the Surface Coat

    PubMed Central

    Güther, Maria Lucia Sampaio; Lee, Sylvia; Tetley, Laurence; Acosta-Serrano, Alvaro

    2006-01-01

    The procyclic form of Trypanosoma brucei exists in the midgut of the tsetse fly. The current model of its surface glycocalyx is an array of rod-like procyclin glycoproteins with glycosylphosphatidylinositol (GPI) anchors carrying sialylated poly-N-acetyllactosamine side chains interspersed with smaller sialylated poly-N-acetyllactosamine–containing free GPI glycolipids. Mutants for TbGPI12, deficient in the second step of GPI biosynthesis, were devoid of cell surface procyclins and poly-N-acetyllactosamine–containing free GPI glycolipids. This major disruption to their surface architecture severely impaired their ability to colonize tsetse fly midguts but, surprisingly, had no effect on their morphology and growth characteristics in vitro. Transmission electron microscopy showed that the mutants retained a cell surface glycocalyx. This structure, and the viability of the mutants in vitro, prompted us to look for non-GPI–anchored parasite molecules and/or the adsorption of serum components. Neither were apparent from cell surface biotinylation experiments but [3H]glucosamine biosynthetic labeling revealed a group of previously unidentified high apparent molecular weight glycoconjugates that might contribute to the surface coat. While characterizing GlcNAc-PI that accumulates in the TbGPI12 mutant, we observed inositolphosphoceramides for the first time in this organism. PMID:17035628

  12. MAIL1 is essential for development of the primary root but not of anchor roots.

    PubMed

    Ühlken, Christine; Hoth, Stefan; Weingartner, Magdalena

    2014-01-01

    MAIN-LIKE1 (MAIL1) is a ubiquitously expressed nuclear protein, which has a crucial function during root development. We have recently described loss of function mutants for MAIL1, in which the organization and function of the primary root meristem is lost soon after germination. Moreover cell differentiation is impaired resulting in primary root growth arrest soon after emergence. Here we show that mail1 mutants form several anchor roots from the hypocotyl to root junction. These anchor roots show similar defects in the organization of the stem cell niche as the primary root. In contrast, differentiation processes are not impaired and thus anchor roots seem to be able to compensate for the loss of primary root function. Our data show that MAIL1 is essential for specification of cell fate in the primary root but not in anchor roots.

  13. 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

  14. 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.

  15. 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

  16. Permanent Ground Anchors : Stump Design Criteria

    DOT National Transportation Integrated Search

    1982-09-01

    This document summarizes the main design methods used by the principal investigators in the design of permanent ground anchors, including basic concepts, design criteria, and analytical techniques. The application of these design methods are illustra...

  17. Performance of Adhesive and Cementitious Anchoring Systems

    DOT National Transportation Integrated Search

    2017-08-01

    This research project evaluated the behavior of adhesive and cementitious bonded anchoring systems per the approach found in the provisional standard AASHTO TP-84, in order to provide recommendations pertaining to the test method. Additional paramete...

  18. Molecular cloning of a glycosylphosphatidylinositol-anchored molecule CDw108.

    PubMed

    Yamada, A; Kubo, K; Takeshita, T; Harashima, N; Kawano, K; Mine, T; Sagawa, K; Sugamura, K; Itoh, K

    1999-04-01

    CDw108, also known as the John-Milton-Hagen human blood group Ag, is an 80-kDa glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that is preferentially expressed on activated lymphocytes and E. The molecular characteristics and biological function of the CDw108 were not clarified previously. In this manuscript, we identify the cDNA clone containing the entire coding sequence of the CDw108 gene and report its molecular characteristics. The 1998-base pairs of the open reading frame of the cloned cDNA encoded a protein of 666 amino acids (aa), including the 46 aa of the signal peptide and the 19 aa of the GPI-anchor motif. Thus, the membrane-anchoring form of CDw108 was the 602 aa, and the estimated molecular mass of the unglycosylated form was 68 kDa. The RGD (Arg-Gly-Asp) cell attachment sequence and the five potential N-linked glycosylation sites were located on the membrane-anchoring form. Flow cytometric and immunoprecipitation analyses of the CDw108 cDNA transfectants confirmed that the cloned cDNA encoded the native form of CDw108. The CDw108 mRNA was expressed in activated PBMCs as well as in the spleen, thymus, testis, placenta, and brain, but was not expressed in any other tissues tested. Radiation hybrid mapping indicated that the CDw108 gene was located in the middle of the long arm of chromosome 15 (15q23-24). This molecular information will be critical for understanding the biological function of the CDw108 Ag.

  19. Anchoring effects on early autobiographical memories.

    PubMed

    Greenberg, Daniel L; Bishara, Anthony J; Mugayar-Baldocchi, Marino A

    2017-10-01

    Studies of childhood memory typically show that our earliest memories come from between three and four years of age. This finding is not universal, however. The age estimate varies across cultures and is affected by social influences. Research from the judgments and decision-making literature suggests that these estimates might also involve a judgment under uncertainty. Therefore, they might be susceptible to less social influences such as heuristics and biases. To investigate this possibility, we conducted two experiments that used anchoring paradigms to influence participants' estimates of their age during early autobiographical memories. In Experiment 1, participants answered either a high-anchor or a low-anchor question, and were warned that the anchor was uninformative; they went on to estimate their age during their earliest autobiographical memory. In Experiment 2, we replicated Experiment 1 and extended the design to examine additional early autobiographical memories. In both experiments, participants in the low-anchor condition gave earlier age estimates than those in the high-anchor condition. These results provide new insights into the methods used to investigate autobiographical memory. Moreover, they show that reports of early autobiographical memories can be influenced by a relatively light touch - a change to a single digit in a single question.

  20. 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 vessel...

  1. 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 vessel...

  2. 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…

  3. Further Study of the Choice of Anchor Tests in Equating

    ERIC Educational Resources Information Center

    Trierweiler, Tammy J.; Lewis, Charles; Smith, Robert L.

    2016-01-01

    In this study, we describe what factors influence the observed score correlation between an (external) anchor test and a total test. We show that the anchor to full-test observed score correlation is based on two components: the true score correlation between the anchor and total test, and the reliability of the anchor test. Findings using an…

  4. An earth anchor system: installation and design guide.

    Treesearch

    R.L. Copstead; D.D. Studier

    1990-01-01

    A system for anchoring the guylines and skylines of cable yarding equipment is presented. A description of three types of tipping plate anchors is given. Descriptions of the installation equipment and methods specific to each type are given. Procedures for determining the correct number of anchors to install are included, as are guidelines for installing the anchors so...

  5. 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

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

    SciTech Connect

    Robbany, Z., E-mail: zdrobbany@gmail.com; Handayani, G., E-mail: gunawanhandayani@gmail.com

    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 validatemore » the results of the comparison method of measurement used pile integrity test.« less

  7. HSV1 MicroRNA Modulation of GPI Anchoring and Downstream Immune Evasion.

    PubMed

    Enk, Jonatan; Levi, Assi; Weisblum, Yiska; Yamin, Rachel; Charpak-Amikam, Yoav; Wolf, Dana G; Mandelboim, Ofer

    2016-10-18

    Herpes simplex virus 1 (HSV1) is a ubiquitous human pathogen that utilizes variable mechanisms to evade immune surveillance. The glycosylphosphatidylinositol (GPI) anchoring pathway is a multistep process in which a myriad of different proteins are covalently attached to a GPI moiety to be presented on the cell surface. Among the different GPI-anchored proteins there are many with immunological importance. We present evidence that the HSV1-encoded miR H8 directly targets PIGT, a member of the protein complex that covalently attaches proteins to GPI in the final step of GPI anchoring. This results in a membrane down-modulation of several different immune-related, GPI-anchored proteins, including ligands for natural killer-activating receptors and the prominent viral restriction factor tetherin. Thus, we suggest that by utilizing just one of dozens of miRNAs encoded by HSV1, the virus can counteract the host immune response at several key points. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  8. Probing the specificity of protein-protein interactions by quantitative chemical proteomics.

    PubMed

    Kovanich, Duangnapa; Aye, Thin Thin; Heck, Albert J R; Scholten, Arjen

    2012-01-01

    Chemical proteomics is a versatile tool to investigate protein-small molecule interactions, but can be extended to probe also secondary binding investigating small molecule-protein 1-protein 2 interactions, providing insight into protein scaffolds. This application of chemical proteomics has in particular been applied extensively to cyclic nucleotide (cAMP, cGMP) signaling. cAMP regulates cellular functions primarily by activating cAMP-dependent protein kinase (PKA). Compartmentalization of PKA plays an important role in the specificity of cAMP signaling events and is mediated by interaction of the regulatory subunit (PKA-R) with A-kinase anchoring proteins (AKAPs), which often form the core of even larger protein machineries. The selective binding of AKAPs to one of the major isoforms PKA-R type I (PKA-RI) and PKA-R type II (PKA-RII) is an important feature of cAMP/PKA signaling. However, this specificity is not well established for most AKAPs. Here, we describe a chemical proteomics approach that combines cAMP-based affinity chromatography with quantitative mass spectrometry to investigate PKA-R isoform/AKAP specificity directly in lysates of cells and tissues of any origin. With this tool, several novel PKA-R/AKAP specificities can be easily resolved.

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

    PubMed Central

    Wang, Jimin; Li, Yue; Modis, Yorgo

    2014-01-01

    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

  10. Roles of actin cytoskeleton for regulation of chloroplast anchoring

    PubMed Central

    Sakai, Yuuki; Takagi, Shingo

    2017-01-01

    ABSTRACT Chloroplasts are known to maintain specific intracellular distribution patterns under specific environmental conditions, enabling the optimal performance of photosynthesis. To this end, chloroplasts are anchored in the cortical cytoplasm. In leaf epidermal cells of aquatic monocot Vallisneria, we recently demonstrated that the anchored chloroplasts are rapidly de-anchored upon irradiation with high-intensity blue light and that the process is probably mediated by the blue-light receptor phototropins. Chloroplast de-anchoring is a necessary step rendering the previously anchored chloroplasts mobile to allow their migration. In this article, based on the results obtained in Vallisneria together with those in other plant species, we briefly discussed possible modes of regulation of chloroplast anchoring and de-anchoring by actin cytoskeleton. The topics include roles of photoreceptor systems, actin-filament-dependent and -independent chloroplast anchoring, and independence of chloroplast de-anchoring from actomyosin and microtubule systems. PMID:28829686

  11. Fibre-Reinforced Adhesive for Structure Anchoring

    NASA Astrophysics Data System (ADS)

    Barnat, J.; Bajer, M.

    2015-11-01

    The topic of this paper is the glue-concrete interface of bonded anchors loaded by tension force. The paper is closely focused on bond strength experiments using high strength concrete up to class C50/60 or higher together with pure epoxy resin and fibre-reinforced resin. The goal of this research is to find the limits of the effective use of such glue types in high performance concrete, and also to verify the most commonly used design methods for bonded anchors. The presented research includes experimental analysis of the glue-concrete interface and the influence of its parameters on anchor behaviour. The presented analysis shows some problems of the 'separated failure modes' approach and also presents experimentally verified bond strength values obtained for the currently most widespread glue types. Results of fibre reinforced epoxy resin are also presented in this paper.

  12. 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

  13. 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

  14. 30 CFR 57.19002 - Anchoring.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Anchoring. 57.19002 Section 57.19002 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 Personnel Hoisting Hoists § 57...

  15. 30 CFR 56.19002 - Anchoring.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Anchoring. 56.19002 Section 56.19002 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-SURFACE METAL AND NONMETAL MINES Personnel Hoisting Hoists § 56.19002...

  16. 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.

  17. 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.

  18. 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 installation...

  19. 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…

  20. Heat-Conducting Anchors for Thermocouples

    NASA Technical Reports Server (NTRS)

    Macdavid, Kenton S.

    1987-01-01

    Metal particles in adhesive aid heat transfer. Aluminum caps containing silver-filled epoxy used as high-thermal-conductance anchors for thermocouples, epoxy providing thermal path between mounting surfaces and thermocouple measuring junctions. Normally, epoxy-filled aluminum caps used when measuring steady-state temperatures. Silver-filled epoxy used when thermocouple not isolated electrically from surface measured.

  1. 30 CFR 57.7032 - Anchoring.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Anchoring. 57.7032 Section 57.7032 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 Drilling and Rotary Jet Piercing...

  2. 24 CFR 3285.401 - Anchoring instructions.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 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 installation...

  3. 30 CFR 57.7032 - Anchoring.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Anchoring. 57.7032 Section 57.7032 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 Drilling and Rotary Jet Piercing...

  4. 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.

  5. 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

  6. 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

  7. Role of GPI-anchored enzyme in liposome detergent-resistance.

    PubMed

    Morandat, S; Bortolato, M; Roux, B

    2003-02-01

    In this work, we investigated the role of a glycosylphosphatidylinositol (GPI)-anchored protein, the alkaline phosphatase, on the solubilization of detergent-resistant liposomes. In vivo, GPI-anchored proteins are clustered into sphingolipid- and cholesterol-rich membrane domains and this peculiar composition provides cold-detergent-insolubility. To better understand the mechanisms involved in the clustering of these subdomain components, we built a model, namely sphingolipid- and cholesterol-rich liposomes. We show the cold-Triton X-100 resistance of liposomes before and after insertion of GPI-anchored enzyme. When the amount of incorporated enzyme varied, significant changes in membrane stability occurred. Low protein contents into liposomes increased detergent insolubility, whereas high amounts decreased it. Furthermore, significant differences in the detergent-resistance of each lipid were exhibited between liposomes and proteoliposomes. Thus, the enzyme insertion led to a dramatic decrease of cholesterol solubilization, in line with the existence of cholesterol/GPI interactions. Effect of temperature on detergent resistance was also investigated. Liposome solubilization increased with temperature up to a threshold value of 40/45 degrees C. This was also the temperature at which a phase transition of liposome membrane occurred, as evidenced by Laurdan fluorescence. Although the GPI-anchored enzyme insertion modified membrane stability, no change was observed on phase transition. Our work highlights the importance of GPI-anchored proteins in the structure of sphingolipid- and cholesterol-rich membrane domains, in the detergent-insolubility of these peculiar domains, as well as in interaction of GPI proteins with cholesterol.

  8. Functional Anchoring Lipids for Drug Delivery Carrier Fabrication and Cell Surface Re-Engineering Applications

    NASA Astrophysics Data System (ADS)

    Vabbilisetty, Pratima

    For decades, lipid vesicular bodies such as liposomes have been widely used and explored as biomimetic models of cell membranes and as drug/gene delivery carrier systems. Similarly, micellar iron oxide nanoparticles have also been investigated as potential MRI agents as well as drug delivery carrier systems. Cell surface carbohydrate-protein interactions allow them to serve as markers for recognition of many molecular and cellular activities thereby, are exploited as attractive molecules for surface modification of nanocarrier systems with purpose for tissues specific targeting and biocompatibility. In addition, the cell lipid membrane serves as an important platform for occurrence of many biological processes that are governed and guided by cell surface receptors. Introduction of chemoselective functional groups, via bio-orthogonal conjugation strategies, at the cell surface facilitates many cellular modifications and paves path for novel and potential biomedical applications. Anchoring lipids are needed for liposome surface functionalization with ligands of interest and play important roles in ligand grafting density, liposomes stability and biological activity. On the other hand, anchoring lipids are also needed for cell surface re-engineering by lipid fusion approach and have high impact for ligand insertion efficiency and biological activity. Overall, in this dissertation study, functional anchoring lipids for glyco-functionalized carrier systems and for efficient cell surface re-engineering applications were systematically investigated, respectively. Firstly, investigation of the synthesis of glyco-functionalized liposome systems based on phosphatidylethonalamine (PE) and cholesterol (Chol) anchoring lipids, prepared by post chemically selective functionalization via Staudinger ligation were carried out. The effect of anchor lipids on the stability, encapsulation and releasing capacity of the glycosylated liposomes were investigated by dynamic light

  9. 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…

  10. 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…

  11. Liquid-phase synthesis of bridged peptides using olefin metathesis of a protected peptide with a long aliphatic chain anchor.

    PubMed

    Aihara, Keisuke; Komiya, Chiaki; Shigenaga, Akira; Inokuma, Tsubasa; Takahashi, Daisuke; Otaka, Akira

    2015-02-06

    Bridged peptides including stapled peptides are attractive tools for regulating protein-protein interactions (PPIs). An effective synthetic methodology in a heterogeneous system for the preparation of these peptides using olefin metathesis and hydrogenation of protected peptides with a long aliphatic chain anchor is reported.

  12. Measures for the Safe Operation of Anchoring in a Storm

    NASA Astrophysics Data System (ADS)

    Han, Tianding; Ai, Wanzheng

    2018-01-01

    The collision and stranding of ship other shipwreck accidents are mainly caused by the ship dragging. As the water is less in coastal areas, anchoring has less influence on cementing ship, so strong wind is the most important factor for ship anchoring. Therefore, it is very important to study the safety evaluation of mooring in strong wind. In this paper, the measures taken after the ship anchoring is come up with from the analysis on the typical accidents and causes of anchoring security. The safety measures at the time of anchoring are also studied.

  13. Comparative Study on Different Slot Forms of Prestressed Anchor Blocks

    NASA Astrophysics Data System (ADS)

    Fan, Rong; Si, Jianhui; Jian, Zheng

    2018-03-01

    In this paper, two models of prestressed pier, rectangular cavity anchor block and arch hollow anchor block are established. The ABAQUS software was used to calculate the stress of the surface of the neck of the pier and the cavity of the anchor block, through comparative analysis. The results show that compared with the rectangular cavity anchor block, the stress of the pier and the cavity can be effectively reduced when the arch hole is used, and the amount of prestressed anchor can be reduced, so as to obtain obvious economic benefits.

  14. Electrochromic mirror using viologen-anchored nanoparticles

    SciTech Connect

    Kim, Han Na; University of Science and Technology, Advanced Device Technology, 217 Gajeong-roYuseong-gu, Daejeon 305-350; Cho, Seong M.

    2016-10-15

    Highlights: • Three types of ECM device were fabricated using viologen-anchored ECDs. • The devices were investigated according to their optical structures. • The anti-reflection material affects the reflectance and the coloration efficiency. • The device design of ECMs is a crucial factor for clear reflected images. - Abstract: Electrochromic mirrors (ECMs) that are used in automobile mirrors need to have high reflectance, a high contrast ratio, and a clear image. In particular, it is critical that distortions of clear images are minimized for safety. Therefore, an ECM is fabricated using viologen-anchored nanoparticles and a magnesium fluoride (MgF{sub 2}) layermore » with an anti-reflection function. The ECM has approximately 30.42% in the reflectance dynamic range and 125 cm{sup 2}/C high coloration efficiency.« less

  15. 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.

  16. 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.

  17. Load Transfer Mechanisms in Anchored Geosynthetic Systems

    DTIC Science & Technology

    1990-12-20

    Peak Load .... 38I 18. Load-Deformation Relationship, Nonwoven Fabric ...... 40 19. Load-Deformation Relationship, Woven Fabric ....... 41 20. Load-Time...Relationship, Nonwoven Fabric ............. 42 21. Load-Time Relationship, Woven Fabric ............... 43 22. Anchor Patterns...Ottawa 20-30 and Glazier Way sand) and two3 fabrics (a Trevia 1155 nonwoven and a Nicolon 1500NC woven) were I i -31- I I tested. The soil/fabric

  18. 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.

  19. Resisting anchoring effects: The roles of metric and mapping knowledge.

    PubMed

    Smith, Andrew R; Windschitl, Paul D

    2015-10-01

    The biasing influence of anchors on numerical estimates is well established, but the relationship between knowledge level and the susceptibility to anchoring effects is less clear. In two studies, we addressed the potential mitigating effects of having knowledge in a domain on vulnerability to anchoring effects in that domain. Of critical interest was a distinction between two forms of knowledge-metric and mapping knowledge. In Study 1, participants who had studied question-relevant information-that is, high-knowledge participants-were less influenced by anchors than were participants who had studied irrelevant information. The results from knowledge measures suggested that the reduction in anchoring was tied to increases in metric rather than mapping knowledge. In Study 2, participants studied information specifically designed to influence different types of knowledge. As we predicted, increases in metric knowledge-and not mapping knowledge-led to reduced anchoring effects. Implications for debiasing anchoring effects are discussed.

  20. Nanofabricated racks of aligned and anchored DNA substrates for single-molecule imaging.

    PubMed

    Gorman, Jason; Fazio, Teresa; Wang, Feng; Wind, Shalom; Greene, Eric C

    2010-01-19

    Single-molecule studies of biological macromolecules can benefit from new experimental platforms that facilitate experimental design and data acquisition. Here we develop new strategies to construct curtains of DNA in which the molecules are aligned with respect to one another and maintained in an extended configuration by anchoring both ends of the DNA to the surface of a microfluidic sample chamber that is otherwise coated with an inert lipid bilayer. This "double-tethered" DNA substrate configuration is established through the use of nanofabricated rack patterns comprised of two distinct functional elements: linear barriers to lipid diffusion that align DNA molecules anchored by one end to the bilayer and antibody-coated pentagons that provide immobile anchor points for the opposite ends of the DNA. These devices enable the alignment and anchoring of thousands of individual DNA molecules, which can then be visualized using total internal reflection fluorescence microscopy under conditions that do not require continuous application of buffer flow to stretch the DNA. This unique strategy offers the potential for studying protein-DNA interactions on large DNA substrates without compromising measurements through application of hydrodynamic force. We provide a proof-of-principle demonstration that double-tethered DNA curtains made with nanofabricated rack patterns can be used in a one-dimensional diffusion assay that monitors the motion of quantum dot-tagged proteins along DNA.

  1. Lipid Binding of the Amphipathic Helix Serving as Membrane Anchor of Pestivirus Glycoprotein Erns.

    PubMed

    Aberle, Daniel; Oetter, Kay-Marcus; Meyers, Gregor

    2015-01-01

    Pestiviruses express a peculiar protein named Erns representing envelope glycoprotein and RNase, which is important for control of the innate immune response and persistent infection. The latter functions are connected with secretion of a certain amount of Erns from the infected cell. Retention/secretion of Erns is most likely controlled by its unusual membrane anchor, a long amphipathic helix attached in plane to the membrane. Here we present results of experiments conducted with a lipid vesicle sedimentation assay able to separate lipid-bound from unbound protein dissolved in the water phase. Using this technique we show that a protein composed of tag sequences and the carboxyterminal 65 residues of Erns binds specifically to membrane vesicles with a clear preference for compositions containing negatively charged lipids. Mutations disturbing the helical folding and/or amphipathic character of the anchor as well as diverse truncations and exchange of amino acids important for intracellular retention of Erns had no or only small effects on the proteins membrane binding. This result contrasts the dramatically increased secretion rates observed for Erns proteins with equivalent mutations within cells. Accordingly, the ratio of secreted versus cell retained Erns is not determined by the lipid affinity of the membrane anchor.

  2. 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

  3. The GPI Anchor Signal Sequence Dictates the Folding and Functionality of the Als5 Adhesin from Candida albicans

    PubMed Central

    Ahmad, Mohammad Faiz; Yadav, Bhawna; Kumar, Pravin; Puri, Amrita; Mazumder, Mohit; Ali, Anwar; Gourinath, Samudrala; Muthuswami, Rohini; Komath, Sneha Sudha

    2012-01-01

    Background Proteins destined to be Glycosylphosphatidylinositol (GPI) anchored are translocated into the ER lumen completely before the C-terminal GPI anchor attachment signal sequence (SS) is removed by the GPI-transamidase and replaced by a pre-formed GPI anchor precursor. Does the SS have a role in dictating the conformation and function of the protein as well? Methodology/Principal Findings We generated two variants of the Als5 protein without and with the SS in order to address the above question. Using a combination of biochemical and biophysical techniques, we show that in the case of Als5, an adhesin of C. albicans, the C-terminal deletion of 20 amino acids (SS) results in a significant alteration in conformation and function of the mature protein. Conclusions/Significance We propose that the locking of the conformation of the precursor protein in an alternate conformation from that of the mature protein is one probable strategy employed by the cell to control the behaviour and function of proteins intended to be GPI anchored during their transit through the ER. PMID:22509405

  4. 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.

  5. Binding equilibrium and kinetics of membrane-anchored receptors and ligands in cell adhesion: Insights from computational model systems and theory.

    PubMed

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

    2016-09-02

    The adhesion of cell membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. In this article, we review recent results from simulations and theory that lead to novel insights on how the binding equilibrium and kinetics of these proteins is affected by the membranes and by the membrane anchoring and molecular properties of the proteins. Simulations and theory both indicate that the binding equilibrium constant [Formula: see text] and the on- and off-rate constants of anchored receptors and ligands in their 2-dimensional (2D) membrane environment strongly depend on the membrane roughness from thermally excited shape fluctuations on nanoscales. Recent theory corroborated by simulations provides a general relation between [Formula: see text] and the binding constant [Formula: see text] of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in 3 dimensions (3D).

  6. Binding equilibrium and kinetics of membrane-anchored receptors and ligands in cell adhesion: Insights from computational model systems and theory

    PubMed Central

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

    2016-01-01

    ABSTRACT The adhesion of cell membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. In this article, we review recent results from simulations and theory that lead to novel insights on how the binding equilibrium and kinetics of these proteins is affected by the membranes and by the membrane anchoring and molecular properties of the proteins. Simulations and theory both indicate that the binding equilibrium constant K2D and the on- and off-rate constants of anchored receptors and ligands in their 2-dimensional (2D) membrane environment strongly depend on the membrane roughness from thermally excited shape fluctuations on nanoscales. Recent theory corroborated by simulations provides a general relation between K2D and the binding constant K3D of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in 3 dimensions (3D). PMID:27294442

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

    PubMed

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

    2015-08-27

    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.

  8. 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

  9. Anchor-based classification and type-C inhibitors for tyrosine kinases

    PubMed Central

    Hsu, Kai-Cheng; Sung, Tzu-Ying; Lin, Chih-Ta; Chiu, Yi-Yuan; Hsu, John T.-A.; Hung, Hui-Chen; Sun, Chung-Ming; Barve, Indrajeet; Chen, Wen-Liang; Huang, Wen-Chien; Huang, Chin-Ting; Chen, Chun-Hwa; Yang, Jinn-Moon

    2015-01-01

    Tyrosine kinases regulate various biological processes and are drug targets for cancers. At present, the design of selective and anti-resistant inhibitors of kinases is an emergent task. Here, we inferred specific site-moiety maps containing two specific anchors to uncover a new binding pocket in the C-terminal hinge region by docking 4,680 kinase inhibitors into 51 protein kinases, and this finding provides an opportunity for the development of kinase inhibitors with high selectivity and anti-drug resistance. We present an anchor-based classification for tyrosine kinases and discover two type-C inhibitors, namely rosmarinic acid (RA) and EGCG, which occupy two and one specific anchors, respectively, by screening 118,759 natural compounds. Our profiling reveals that RA and EGCG selectively inhibit 3% (EGFR and SYK) and 14% of 64 kinases, respectively. According to the guide of our anchor model, we synthesized three RA derivatives with better potency. These type-C inhibitors are able to maintain activities for drug-resistant EGFR and decrease the invasion ability of breast cancer cells. Our results show that the type-C inhibitors occupying a new pocket are promising for cancer treatments due to their kinase selectivity and anti-drug resistance. PMID:26077136

  10. 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.

  11. 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.

  12. GSKIP- and GSK3-mediated anchoring strengthens cAMP/PKA/Drp1 axis signaling in the regulation of mitochondrial elongation.

    PubMed

    Loh, Joon-Khim; Lin, Ching-Chih; Yang, Ming-Chang; Chou, Chia-Hua; Chen, Wan-Shia; Hong, Ming-Chang; Cho, Chung-Lung; Hsu, Ching-Mei; Cheng, Jiin-Tsuey; Chou, An-Kuo; Chang, Chung-Hsing; Tseng, Chao-Neng; Wang, Chi-Huei; Lieu, Ann-Shung; Howng, Shen-Long; Hong, Yi-Ren

    2015-08-01

    GSK3β binding of GSKIP affects neurite outgrowth, but the physiological significance of PKA binding to GSKIP remains to be determined. We hypothesized that GSKIP and GSK3β mediate cAMP/PKA/Drp1 axis signaling and modulate mitochondrial morphology by forming a working complex comprising PKA/GSKIP/GSK3β/Drp1. We demonstrated that GSKIP wild-type overexpression increased phosphorylation of Drp1 S637 by 7-8-fold compared to PKA kinase-inactive mutants (V41/L45) and a GSK3β binding-defective mutant (L130) under H2O2 and forskolin challenge in HEK293 cells, indicating that not only V41/L45, but also L130 may be involved in Drp1-associated protection of GSKIP. Interestingly, silencing either GSKIP or GSK3β but not GSK3α resulted in a dramatic decrease in Drp1 S637 phosphorylation, revealing that both GSKIP and GSK3β are required in this novel PKA/GSKIP/GSK3β/Drp1 complex. Moreover, overexpressed kinase-dead GSK3β-K85R, which retains the capacity to bind GSKIP, but not K85M which shows total loss of GSKIP-binding, has a higher Drp1 S637 phosphorylation similar to the GSKIP wt overexpression group, indicating that GSK3β recruits Drp1 by anchoring rather than in a kinase role. With further overexpression of either V41/L45P or the L130P GSKIP mutant, the elongated mitochondrial phenotype was lost; however, ectopically expressed Drp1 S637D, a phosphomimetic mutant, but not S637A, a non-phosphorylated mutant, restored the elongated mitochondrial morphology, indicating that Drp1 is a downstream effector of direct PKA signaling and possibly has an indirect GSKIP function involved in the cAMP/PKA/Drp1 signaling axis. Collectively, our data revealed that both GSKIP and GSK3β function as anchoring proteins in the cAMP/PKA/Drp1 signaling axis modulating Drp1 phosphorylation. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. 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

  14. The Empirical Selection of Anchor Items Using a Multistage Approach

    ERIC Educational Resources Information Center

    Craig, Brandon

    2017-01-01

    The purpose of this study was to determine if using a multistage approach for the empirical selection of anchor items would lead to more accurate DIF detection rates than the anchor selection methods proposed by Kopf, Zeileis, & Strobl (2015b). A simulation study was conducted in which the sample size, percentage of DIF, and balance of DIF…

  15. 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…

  16. How to Anchor Machinery in Your School Shop

    ERIC Educational Resources Information Center

    Walker, John R.

    1978-01-01

    An industrial arts teacher explains the need to mount school shop machinery securely and describes methods of mounting permanently or temporarily. Reasons for anchoring machine tools are safety, accuracy of operation, and the prevention of damage to the machine. Five figures illustrate anchoring and leveling. (MF)

  17. Retention of internal anchor tags by juvenile striped bass

    USGS Publications Warehouse

    Van Den Avyle, M.J.; Wallin, J.E.

    2001-01-01

    We marked hatchery-reared striped bass Morone saxatilis (145-265 mm total length) with internal anchor tags and monitored retention for 28 months after stocking in the Savannah River, Georgia and South Carolina. Anchor tags (with an 18-mm, T-shaped anchor and 42-mm streamer) were surgically implanted ventrally, and coded wire tags (1 mm long and 0.25 mm in diameter) were placed into the cheek muscle to help identify subsequent recaptures. The estimated probability of retention (SD) of anchor tags was 0.94 (0.05) at 4 months, 0.64 (0.13) at 16 months, and 0.33 (0.19) at 28 months. Of 10 fish recaptured with only coded wire tags, 5 showed an externally visible wound or scar near the point of anchor tag insertion. The incidence of wounds or scars, which we interpreted as evidence of tag shedding, increased to 50% in recaptures taken at 28 months (three of six fish). Our estimates for retention of anchor tags were generally lower than those in other studies of striped bass, possibly because of differences in the style of anchor or sizes of fish used. Because of its low rate of retention, the type of anchor tag we used may not be suitable for long-term assessments of stock enhancement programs that use striped bass of the sizes we evaluated.

  18. Use of Jackknifing to Evaluate Effects of Anchor Item Selection on Equating with the Nonequivalent Groups with Anchor Test (NEAT) Design. Research Report. ETS RR-15-10

    ERIC Educational Resources Information Center

    Lu, Ru; Haberman, Shelby; Guo, Hongwen; Liu, Jinghua

    2015-01-01

    In this study, we apply jackknifing to anchor items to evaluate the impact of anchor selection on equating stability. In an ideal world, the choice of anchor items should have little impact on equating results. When this ideal does not correspond to reality, selection of anchor items can strongly influence equating results. This influence does not…

  19. 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.

  20. P190RhoGAP prevents mitotic spindle fragmentation and is required to activate Aurora A kinase at acentriolar poles.

    PubMed

    Manukyan, Arkadi; Sargsyan, Lilit; Parsons, Sarah J; Stukenberg, P Todd

    2018-04-14

    Assembly of the mitotic spindle is essential for proper chromosome segregation during mitosis. Maintenance of spindle poles requires precise regulation of kinesin- and dynein-generated forces, and improper regulation of these forces disrupts pole integrity leading to pole fragmentation. The formation and function of the mitotic spindle are regulated by many proteins, including Aurora A kinase and the motor proteins Kif2a and Eg5. Here, we characterize a surprising role for the RhoA GTPase-activating protein, p190RhoGAP, in regulating the mitotic spindle. We show that cells depleted of p190RhoGAP arrest for long periods in mitosis during which cells go through multiple transitions between having bipolar and multipolar spindles. Most of the p190RhoGAP-depleted cells finally achieve a stable bipolar attachment and proceed through anaphase. The multipolar spindle phenotype can be rescued by low doses of an Eg5 inhibitor. Moreover, we show that p190RhoGAP-depleted multipolar cells localize Aurora A to all the poles, but the kinase is only activated at the two centriolar poles. Overall, our data identify an unappreciated connection between p190RhoGAP and the proteins that control spindle poles including Aurora A kinase and Eg5 that is required to prevent or correct spindle pole fragmentation.

  1. Hypothetical gene C18orf42 encodes a novel protein kinase A-binding protein.

    PubMed

    Fukuda, Makiha; Aizawa, Yasunori

    2015-04-01

    The substrate specificity of cAMP-dependent protein kinase A (PKA) is controlled by its interaction with the A-kinase anchoring protein (AKAP) family. Individual AKAP members are localized to particular intracellular sites and tether PKA specifically to the subcellular compartments where target substrates exist. Here, we report that the human hypothetical gene C18orf42 encodes a novel PKA-binding protein that potentially regulates PKA-AKAP interactions. C18orf42 is expressed preferentially in neural tissues. Functional motif searching predicted that C18orf42 may encode a short protein that contains a putative PKA-binding motif. To confirm this possibility, we applied the CRISPR/Cas9 genome-editing system to incorporate the FLAG tag into the C-terminus of the endogenous C18orf42 protein in the mouse neural cell line Neuro2a. Immunoprecipitation and immunoblotting using anti-FLAG antibody showed translation of the endogenous C18orf42 protein and the physical interaction of the C18orf42 protein with PKA subunits. Immunoprecipitation and pull-down assays showed that C18orf42 binds specifically to the type II regulatory subunits of PKA. Unlike the expression of many AKAPs, that of C18orf42 could block the AKAP-mediated subcellular localization of PKA. These findings suggest that C18orf42 may be a novel PKA signaling gene that serves as an endogenous disruptor peptide for PKA-AKAP interactions. © 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  2. Protein kinase A-dependent spinal α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate-receptor trafficking mediates capsaicin-induced colon-urethra cross-organ reflex sensitization.

    PubMed

    Peng, Hsien-Yu; Chang, Chao-Hsiang; Tsai, Shin-Jei; Lai, Cheng-Yuan; Tung, Kwong-Chung; Wu, Hsi-Chin; Lin, Tzer-Bin

    2011-01-01

    Intracellular redistribution of α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate receptors (AMPARs) is known to be induced by natural painful stimulation. We tested the hypothesis that that protein kinase A (PKA)-dependent AMPAR trafficking underlies the development of N-methyl-d-aspartate receptor-mediated cross-organ sensitization in vivo. We recorded urethra reflex activity and analyzed immunoblotting of lumbosacral (L6-S2) dorsal horn (DH) tissue obtained from animal preparations after intrathecal 8-bromo-cyclic adenosine monophosphate injection or intracolonic instillation with 8-methyl-N-vanillyl-trans-6-nonenamide (capsaicin). Intrathecal 8-bromo-cyclic adenosine monophosphate (300 μM, 10 μl) induced reflex potentiation (81.85 ± 22.21 spikes/stimulation) and increased the number of AMPAR Glu receptor 1 subunits in the membrane fraction of DH (1.8-fold increase vs. control). This process was prevented by pretreatment with the PKA inhibitor N-[2- ((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide(10 μM, 10 μl, 2.7 ± 0.8 [mean ± SE] spikes/stimulation) and human thyroid A kinase-anchoring protein (10 μM, 10 μl, 11.5 ± 4.8 spikes/stimulation), an inhibitor of PKA and PKA-A kinase-anchoring protein interactions. Intracolonic capsaicin instillation sensitized the urethra reflex (137.2 ± 62.4 spikes/stimulation) and, relative to control, simultaneously provoked an increase (2.9-fold) in the membrane fraction and a decrease (0.9-fold) in the cytosolic fraction of Glu receptor 1 subunits in DH. Inhibition of PKA activity and disruption of PKA-A kinase-anchoring protein interaction in the DH (2.0 ± 0.6 and 16.7 ± 2.8 spikes/stimulation, respectively) are sufficient to prevent capsaicin-dependent reflex sensitization and AMPAR trafficking in the membrane fraction (0.6- and 0.5-fold increase capsaicin). Delivery of AMPAR-containing Glu receptor 1 subunits to the membranes of lumbosacral DH neurons through a PKA-dependent pathway contributes to

  3. Biomechanical strength analysis of mini anchors for the temporomandibular joint.

    PubMed

    Lauria, Andrezza; Rodrigues, Danillo Costa; Sato, Fábio Ricardo Loureiro; Moreira, Roger William Fernandes

    2014-12-01

    The usage of mini anchors for the treatment of internal derangements of the temporomandibular joint (TMJ) is considered an innovative treatment, and it has presented excellent clinical results in the stabilization of the articular disc. This study aimed to evaluate, through mechanical tensile testing, the resistance of mini anchors for TMJ articular disc repositioning. Ten thermoplastic polymer poly-ether-ether-ketone (PEEK) mini anchors were tested in artificial polyurethane blocks with a pullout test in a mechanical Instron™ machine, model 4411. The mini anchors were pulled out until failure and the force and displacement were recorded for each specimen. A small standard deviation was noted, which indicated reproducibility. The findings indicate that Cillen™ mini anchors presented a mean force at failure up to 46.1 N with similar values in the scientific literature. Nevertheless, further studies should be conducted for a comparison of PEEK mini anchors with other commercially available anchors, in addition to studies related to PEEK, a scarcely known material in the dentistry field.

  4. Anchoring Groups for Dye-Sensitized Solar Cells

    SciTech Connect

    Zhang, Lei; Cole, Jacqueline M.

    2015-02-03

    The dyes in dye-sensitized solar cells (DSSCs) require one or more chemical substituents that can act as an anchor, enabling their adsorption onto a metal oxide substrate. This adsorption provides a means for electron injection, which is the process that initiates the electrical circuit in a DSSC. Understanding the structure of various DSSC anchors and the search for new anchors are critical factors for the development of improved DSSCs. Traditionally, carboxylic acid and cyanoacrylic acid groupsare employed as dye anchors in DSSCs. In recent years, novel anchor groups have emerged, which make a larger pool of materials available for DSSCmore » dyes, and their associated physical and chemical characteristics offer interesting effects at the interface between dye and metal oxide. This review focuses especially on the structural aspects of these novel dye anchors for TiO2-based DSSCs, including pyridine, phosphonic acid, tetracyanate, perylene dicarboxylic acid anhydride, 2-hydroxylbenzonitrile, 8-hydroxylquinoline, pyridine-N-oxide, hydroxylpyridium, catechol, hydroxamate, sulfonic acid, acetylacetanate, boronic acid, nitro, tetrazole, rhodanine, and salicylic acid substituents. We anticipate that further exploration and understanding of these new types of anchoring groups for TiO2 substrates will not only contribute to the development of advanced DSSCs, but also of quantum dot-sensitized solar cells, water splitting systems, and other self-assembled monolayer-based technologies.« less

  5. A magnetically actuated anchoring system for a wireless endoscopic capsule.

    PubMed

    Zhou, Hao; Alici, Gursel; Munoz, Fredy

    2016-12-01

    In this study, we propose a new magnetically actuated anchoring system for wireless capsule endoscopes (WCE) by employing the principle of a switchable magnetic spring. A force model is derived to predict the magnetic force needed to support the interaction between the anchors and the intestinal lumen. The theoretical and experimental analysis conducted shows that the magnetic spring is capable of providing the force needed to activate the anchoring mechanism, which consists of four foldable legs. A prototype capsule with a size comparable with the size of a commercial WCE was designed, fabricated, and tested. The in-vitro tests with a real small intestine show that the proposed anchoring mechanism is able to raise the friction force between the anchoring legs and inner wall of the intestine by more than two times after its activation using an external magnetic field. Experimental results presented demonstrate that the proposed anchoring system, which has a low foot-print not taking up too much space on the capsule, can provide a reliable anchoring capability with the capsule inside the intestinal lumen.

  6. The use of acrylic bone cement for suture anchoring.

    PubMed

    Meyer, D C; Jacob, H A C; Pistoia, W; von Roll, A; Gerber, C

    2003-05-01

    Healing of tendon or ligament sutured to bone depends among other parameters on the mechanical stability of the suture fixation in or to the bone. The authors propose a method of anchoring suture material using bone cement as a substitute for conventional suture anchors. Conditions for secure fixation of suture material in bone cement were assessed and the technique of anchoring suture material with acrylic cement in bone was developed. Mechanical testing and microcomputed tomography of the suture-cement-bone compound were done. It was found that the suture always should be knotted before embedding it at least 2 mm deep in the bone cement. The holes drilled into the bone in which the sutures are secured with cement should be at least 3.5 mm in diameter and 10 mm deep; in cortical bone a tapped thread is required. Sutures can be secured safely using cement anchors which provide higher pull-out strength of a factor two to five than conventional metallic suture anchors of comparable size. They also adapt to anatomic situations where conventional anchors cannot be used and are more favorable in osteoporotic bone. Cement anchoring of sutures seems to be a cost-effective and valuable alternative when there is poor bone quality or extraordinarily high mechanical load.

  7. Quantifying Heuristic Bias: Anchoring, Availability, and Representativeness.

    PubMed

    Richie, Megan; Josephson, S Andrew

    2018-01-01

    Construct: Authors examined whether a new vignette-based instrument could isolate and quantify heuristic bias. Heuristics are cognitive shortcuts that may introduce bias and contribute to error. There is no standardized instrument available to quantify heuristic bias in clinical decision making, limiting future study of educational interventions designed to improve calibration of medical decisions. This study presents validity data to support a vignette-based instrument quantifying bias due to the anchoring, availability, and representativeness heuristics. Participants completed questionnaires requiring assignment of probabilities to potential outcomes of medical and nonmedical scenarios. The instrument randomly presented scenarios in one of two versions: Version A, encouraging heuristic bias, and Version B, worded neutrally. The primary outcome was the difference in probability judgments for Version A versus Version B scenario options. Of 167 participants recruited, 139 enrolled. Participants assigned significantly higher mean probability values to Version A scenario options (M = 9.56, SD = 3.75) than Version B (M = 8.98, SD = 3.76), t(1801) = 3.27, p = .001. This result remained significant analyzing medical scenarios alone (Version A, M = 9.41, SD = 3.92; Version B, M = 8.86, SD = 4.09), t(1204) = 2.36, p = .02. Analyzing medical scenarios by heuristic revealed a significant difference between Version A and B for availability (Version A, M = 6.52, SD = 3.32; Version B, M = 5.52, SD = 3.05), t(404) = 3.04, p = .003, and representativeness (Version A, M = 11.45, SD = 3.12; Version B, M = 10.67, SD = 3.71), t(396) = 2.28, p = .02, but not anchoring. Stratifying by training level, students maintained a significant difference between Version A and B medical scenarios (Version A, M = 9.83, SD = 3.75; Version B, M = 9.00, SD = 3.98), t(465) = 2.29, p = .02, but not residents or attendings. Stratifying by heuristic and training level, availability maintained

  8. 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

  9. 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

  10. 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.

  11. 21. CATENARY ANCHOR BRIDGE 310, COS COB POWER PLANT, LOOKING ...

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

    21. CATENARY ANCHOR BRIDGE 310, COS COB POWER PLANT, LOOKING EASTBOUND - New York, New Haven & Hartford Railroad, Automatic Signalization System, Long Island Sound shoreline between Stamford & New Haven, Stamford, Fairfield County, CT

  12. 24. CATENARY ANCHOR BRIDGE 310, COS COB POWER PLANT, NORTH ...

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

    24. CATENARY ANCHOR BRIDGE 310, COS COB POWER PLANT, NORTH TRUSS FOR POWER LEAD-IN WIRES - New York, New Haven & Hartford Railroad, Automatic Signalization System, Long Island Sound shoreline between Stamford & New Haven, Stamford, Fairfield County, CT

  13. 23. OBLIQUE VIEW, CATENARY ANCHOR BRIDGE 310, COS COB POWER ...

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

    23. OBLIQUE VIEW, CATENARY ANCHOR BRIDGE 310, COS COB POWER PLANT - New York, New Haven & Hartford Railroad, Automatic Signalization System, Long Island Sound shoreline between Stamford & New Haven, Stamford, Fairfield County, CT

  14. 33. VIEW OF ADJACENT SUBSTATION AND TRANSFORMER, CATENARY ANCHOR BRIDGE ...

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

    33. VIEW OF ADJACENT SUBSTATION AND TRANSFORMER, CATENARY ANCHOR BRIDGE 524 - New York, New Haven & Hartford Railroad, Automatic Signalization System, Long Island Sound shoreline between Stamford & New Haven, Stamford, Fairfield County, CT

  15. 22. CATENARY ANCHOR BRIDGE 310, COS COB POWER PLANT, LOOKING ...

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

    22. CATENARY ANCHOR BRIDGE 310, COS COB POWER PLANT, LOOKING WESTBOUND - New York, New Haven & Hartford Railroad, Automatic Signalization System, Long Island Sound shoreline between Stamford & New Haven, Stamford, Fairfield County, CT

  16. 25. CATENARY ANCHOR BRIDGE 310 AND COS COB POWER PLANT ...

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

    25. CATENARY ANCHOR BRIDGE 310 AND COS COB POWER PLANT STRUCTURES, LOOKING WESTBOUND - New York, New Haven & Hartford Railroad, Automatic Signalization System, Long Island Sound shoreline between Stamford & New Haven, Stamford, Fairfield County, CT

  17. 32. OBLIQUE VIEW SHOWING ASHAPED SUPPORT, CATENARY ANCHOR BRIDGE 524, ...

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

    32. OBLIQUE VIEW SHOWING A-SHAPED SUPPORT, CATENARY ANCHOR BRIDGE 524, NEAR SOUTH NORWALK SWITCH TOWER - New York, New Haven & Hartford Railroad, Automatic Signalization System, Long Island Sound shoreline between Stamford & New Haven, Stamford, Fairfield County, CT

  18. Anchored but not internalized: shape dependent endocytosis of nanodiamond

    PubMed Central

    Zhang, Bokai; Feng, Xi; Yin, Hang; Ge, Zhenpeng; Wang, Yanhuan; Chu, Zhiqin; Raabova, Helena; Vavra, Jan; Cigler, Petr; Liu, Renbao; Wang, Yi; Li, Quan

    2017-01-01

    Nanoparticle-cell interactions begin with the cellular uptake of the nanoparticles, a process that eventually determines their cellular fate. In the present work, we show that the morphological features of nanodiamonds (NDs) affect both the anchoring and internalization stages of their endocytosis. While a prickly ND (with sharp edges/corners) has no trouble of anchoring onto the plasma membrane, it suffers from difficult internalization afterwards. In comparison, the internalization of a round ND (obtained by selective etching of the prickly ND) is not limited by its lower anchoring amount and presents a much higher endocytosis amount. Molecular dynamics simulation and continuum modelling results suggest that the observed difference in the anchoring of round and prickly NDs likely results from the reduced contact surface area with the cell membrane of the former, while the energy penalty associated with membrane curvature generation, which is lower for a round ND, may explain its higher probability of the subsequent internalization. PMID:28406172

  19. Anchored but not internalized: shape dependent endocytosis of nanodiamond.

    PubMed

    Zhang, Bokai; Feng, Xi; Yin, Hang; Ge, Zhenpeng; Wang, Yanhuan; Chu, Zhiqin; Raabova, Helena; Vavra, Jan; Cigler, Petr; Liu, Renbao; Wang, Yi; Li, Quan

    2017-04-13

    Nanoparticle-cell interactions begin with the cellular uptake of the nanoparticles, a process that eventually determines their cellular fate. In the present work, we show that the morphological features of nanodiamonds (NDs) affect both the anchoring and internalization stages of their endocytosis. While a prickly ND (with sharp edges/corners) has no trouble of anchoring onto the plasma membrane, it suffers from difficult internalization afterwards. In comparison, the internalization of a round ND (obtained by selective etching of the prickly ND) is not limited by its lower anchoring amount and presents a much higher endocytosis amount. Molecular dynamics simulation and continuum modelling results suggest that the observed difference in the anchoring of round and prickly NDs likely results from the reduced contact surface area with the cell membrane of the former, while the energy penalty associated with membrane curvature generation, which is lower for a round ND, may explain its higher probability of the subsequent internalization.

  20. Anchored but not internalized: shape dependent endocytosis of nanodiamond

    NASA Astrophysics Data System (ADS)

    Zhang, Bokai; Feng, Xi; Yin, Hang; Ge, Zhenpeng; Wang, Yanhuan; Chu, Zhiqin; Raabova, Helena; Vavra, Jan; Cigler, Petr; Liu, Renbao; Wang, Yi; Li, Quan

    2017-04-01

    Nanoparticle-cell interactions begin with the cellular uptake of the nanoparticles, a process that eventually determines their cellular fate. In the present work, we show that the morphological features of nanodiamonds (NDs) affect both the anchoring and internalization stages of their endocytosis. While a prickly ND (with sharp edges/corners) has no trouble of anchoring onto the plasma membrane, it suffers from difficult internalization afterwards. In comparison, the internalization of a round ND (obtained by selective etching of the prickly ND) is not limited by its lower anchoring amount and presents a much higher endocytosis amount. Molecular dynamics simulation and continuum modelling results suggest that the observed difference in the anchoring of round and prickly NDs likely results from the reduced contact surface area with the cell membrane of the former, while the energy penalty associated with membrane curvature generation, which is lower for a round ND, may explain its higher probability of the subsequent internalization.

  1. 2. VIEW EAST TOWARD DARIEN ANCHOR BRIDGE #465 OF CIRCUIT ...

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

    2. VIEW EAST TOWARD DARIEN ANCHOR BRIDGE #465 OF CIRCUIT BREAKER INSTALLATION ON CROSS BEAM OF BRIDGE. - New York, New Haven & Hartford Railroad, Bridge-Type Circuit Breakers, Long Island shoreline between Stamford & New Haven, Cos Cob, Fairfield County, CT

  2. 3. VIEW EAST TOWARD STAMFORD ANCHOR BRIDGE #374 OF CIRCUIT ...

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

    3. VIEW EAST TOWARD STAMFORD ANCHOR BRIDGE #374 OF CIRCUIT BREAKER INSTALLATION ON CROSS BEAM OF BRIDGE. - New York, New Haven & Hartford Railroad, Bridge-Type Circuit Breakers, Long Island shoreline between Stamford & New Haven, Cos Cob, Fairfield County, CT

  3. 27. CIRCUIT BREAKERS AT SOUTH END OF BRIDGE, CATENARY ANCHOR ...

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

    27. CIRCUIT BREAKERS AT SOUTH END OF BRIDGE, CATENARY ANCHOR BRIDGE 310, COS COB POWER PLANT - New York, New Haven & Hartford Railroad, Automatic Signalization System, Long Island Sound shoreline between Stamford & New Haven, Stamford, Fairfield County, CT

  4. 4. INTERIOR VIEW OF NORTH SECTION, SHOWING STEEL DOOR ANCHORS, ...

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

    4. INTERIOR VIEW OF NORTH SECTION, SHOWING STEEL DOOR ANCHORS, LOOKING SOUTH-SOUTHEAST - Marvine Colliery, Oil House, West side Boulevard Avenue, between East Parker Street & Route 380, Scranton, Lackawanna County, PA

  5. Genes Required for Glycolipid Synthesis and Lipoteichoic Acid Anchoring in Staphylococcus aureus▿

    PubMed Central

    Gründling, Angelika; Schneewind, Olaf

    2007-01-01

    Staphylococcus aureus lipoteichoic acid (LTA) is composed of a linear 1,3-linked polyglycerolphosphate chain and is tethered to the bacterial membrane by a glycolipid (diglucosyl-diacylglycerol [Glc2-DAG]). Glc2-DAG is synthesized in the bacterial cytoplasm by YpfP, a processive enzyme that transfers glucose to diacylglycerol (DAG), using UDP-glucose as its substrate. Here we present evidence that the S. aureus α-phosphoglucomutase (PgcA) and UTP:α-glucose 1-phosphate uridyltransferase (GtaB) homologs are required for the synthesis of Glc2-DAG. LtaA (lipoteichoic acid protein A), a predicted membrane permease whose structural gene is located in an operon with ypfP, is not involved in Glc2-DAG synthesis but is required for synthesis of glycolipid-anchored LTA. Our data suggest a model in which LtaA facilitates the transport of Glc2-DAG from the inner (cytoplasmic) leaflet to the outer leaflet of the plasma membrane, delivering Glc2-DAG as a substrate for LTA synthesis, thereby generating glycolipid-anchored LTA. Glycolipid anchoring of LTA appears to play an important role during infection, as S. aureus variants lacking ltaA display defects in the pathogenesis of animal infections. PMID:17209021

  6. Null Mutation in PGAP1 Impairing Gpi-Anchor Maturation in Patients with Intellectual Disability and Encephalopathy

    PubMed Central

    Murakami, Yoshiko; Tawamie, Hasan; Maeda, Yusuke; Büttner, Christian; Buchert, Rebecca; Radwan, Farah; Schaffer, Stefanie; Sticht, Heinrich; Aigner, Michael; Reis, André; Kinoshita, Taroh; Jamra, Rami Abou

    2014-01-01

    Many eukaryotic cell-surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). There are at least 26 genes involved in biosynthesis and remodeling of GPI anchors. Hypomorphic coding mutations in seven of these genes have been reported to cause decreased expression of GPI anchored proteins (GPI-APs) on the cell surface and to cause autosomal-recessive forms of intellectual disability (ARID). We performed homozygosity mapping and exome sequencing in a family with encephalopathy and non-specific ARID and identified a homozygous 3 bp deletion (p.Leu197del) in the GPI remodeling gene PGAP1. PGAP1 was not described in association with a human phenotype before. PGAP1 is a deacylase that removes an acyl-chain from the inositol of GPI anchors in the endoplasmic reticulum immediately after attachment of GPI to proteins. In silico prediction and molecular modeling strongly suggested a pathogenic effect of the identified deletion. The expression levels of GPI-APs on B lymphoblastoid cells derived from an affected person were normal. However, when those cells were incubated with phosphatidylinositol-specific phospholipase C (PI-PLC), GPI-APs were cleaved and released from B lymphoblastoid cells from healthy individuals whereas GPI-APs on the cells from the affected person were totally resistant. Transfection with wild type PGAP1 cDNA restored the PI-PLC sensitivity. These results indicate that GPI-APs were expressed with abnormal GPI structure due to a null mutation in the remodeling gene PGAP1. Our results add PGAP1 to the growing list of GPI abnormalities and indicate that not only the cell surface expression levels of GPI-APs but also the fine structure of GPI-anchors is important for the normal neurological development. PMID:24784135

  7. Mutations in PGAP3 impair GPI-anchor maturation, causing a subtype of hyperphosphatasia with mental retardation.

    PubMed

    Howard, Malcolm F; Murakami, Yoshiko; Pagnamenta, Alistair T; Daumer-Haas, Cornelia; Fischer, Björn; Hecht, Jochen; Keays, David A; Knight, Samantha J L; Kölsch, Uwe; Krüger, Ulrike; Leiz, Steffen; Maeda, Yusuke; Mitchell, Daphne; Mundlos, Stefan; Phillips, John A; Robinson, Peter N; Kini, Usha; Taylor, Jenny C; Horn, Denise; Kinoshita, Taroh; Krawitz, Peter M

    2014-02-06

    Glycosylphophatidylinositol (GPI)-anchored proteins play important roles in many biological processes, and mutations affecting proteins involved in the synthesis of the GPI anchor are reported to cause a wide spectrum of intellectual disabilities (IDs) with characteristic additional phenotypic features. Here, we describe a total of five individuals (from three unrelated families) in whom we identified mutations in PGAP3, encoding a protein that is involved in GPI-anchor maturation. Three siblings in a consanguineous Pakistani family presented with profound developmental delay, severe ID, no speech, psychomotor delay, and postnatal microcephaly. A combination of autozygosity mapping and exome sequencing identified a 13.8 Mb region harboring a homozygous c.275G>A (p.Gly92Asp) variant in PGAP3 region 17q11.2-q21.32. Subsequent testing showed elevated serum alkaline phosphatase (ALP), a GPI-anchored enzyme, in all three affected children. In two unrelated individuals in a cohort with developmental delay, ID, and elevated ALP, we identified compound-heterozygous variants c.439dupC (p.Leu147Profs(∗)16) and c.914A>G (p.Asp305Gly) and homozygous variant c.314C>G (p.Pro105Arg). The 1 bp duplication causes a frameshift and nonsense-mediated decay. Further evidence supporting pathogenicity of the missense mutations c.275G>A, c.314C>G, and c.914A>G was provided by the absence of the variants from ethnically matched controls, phylogenetic conservation, and functional studies on Chinese hamster ovary cell lines. Taken together with recent data on PGAP2, these results confirm the importance of the later GPI-anchor remodelling steps for normal neuronal development. Impairment of PGAP3 causes a subtype of hyperphosphatasia with ID, a congenital disorder of glycosylation that is also referred to as Mabry syndrome. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  8. A communal bacterial adhesin anchors biofilm and bystander cells to surfaces.

    PubMed

    Absalon, Cedric; Van Dellen, Katrina; Watnick, Paula I

    2011-08-01

    While the exopolysaccharide component of the biofilm matrix has been intensively studied, much less is known about matrix-associated proteins. To better understand the role of these proteins, we undertook a proteomic analysis of the V. cholerae biofilm matrix. Here we show that the two matrix-associated proteins, Bap1 and RbmA, perform distinct roles in the biofilm matrix. RbmA strengthens intercellular attachments. In contrast, Bap1 is concentrated on surfaces where it serves to anchor the biofilm and recruit cells not yet committed to the sessile lifestyle. This is the first example of a biofilm-derived, communally synthesized conditioning film that stabilizes the association of multilayer biofilms with a surface and facilitates recruitment of planktonic bystanders to the substratum. These studies define a novel paradigm for spatial and functional differentiation of proteins in the biofilm matrix and provide evidence for bacterial cooperation in maintenance and expansion of the multilayer biofilm.

  9. Poor anchoring limits dyslexics' perceptual, memory, and reading skills.

    PubMed

    Oganian, Yulia; Ahissar, Merav

    2012-07-01

    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, Putter-Katz, & Banai, 2006), which suggests that dyslexics have a general difficulty in automatic extraction of stimulus regularities from auditory inputs. This hypothesis explained a broad range of dyslexics' verbal and non-verbal difficulties. However, it was not directly tested in the context of reading and verbal memory, which poses the main stumbling blocks to dyslexics. Here we assessed the abilities of adult dyslexics to efficiently benefit from ("anchor to") regularities embedded in repeated tones, orally presented syllables, and written words. We also compared dyslexics' performance to that of individuals with attention disorder (ADHD), but no reading disability. We found an anchoring effect in all groups: all gained from stimulus repetition. However, in line with the anchoring-deficit hypothesis, controls and ADHD participants showed a significantly larger anchoring effect in all tasks. This study is the first that directly shows that the same domain-general deficit, poor anchoring, characterizes dyslexics' performance in perceptual, working memory and reading tasks. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. 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.

  11. 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.

  12. 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.

  13. Installation and use of epoxy-grouted rock anchors for skyline logging in southeast Alaska.

    Treesearch

    W.L. Schroeder; D.N. Swanston

    1992-01-01

    Field tests of the load-carrying capacity of epoxy-grouted rock anchors in poor quality bedrock on Wrangel Island in southeast Alaska demonstrated the effectiveness of rock anchors as substitutes for stump anchors for logging system guylines. Ultimate capacity depends mainly on rock hardness or strength and length of the imbedded anchor.

  14. The in silico identification of small molecules for protein-protein interaction inhibition in AKAP-Lbc-RhoA signaling complex.

    PubMed

    Khan, Asifullah; Munir, Mehwish; Aiman, Sara; Wadood, Abdul; Khan, Arif-Ullah

    2017-04-01

    The rational design of small molecules that mimic key residues at the interface of interacting proteins can be a successful approach to target certain biological signaling cascades causing pathophysiological outcome. The A-Kinase Anchoring Protein, i.e. AKAP-Lbc, catalyses nucleotide exchange on RhoA and is involved in cardiac repolarization. The oncogenic AKAP-Lbc induces the RhoA GTPase hyperactivity and aberrantly amplifies the signaling pathway leading to hypertrophic cardiomyocytes. We took advantage of the AKAP-Lbc-RhoA complex crystal structure to design in silico small molecules predicted to inhibit the associated pathological signaling cascade. We adopted the strategies of pharmacophore building, virtual screening and molecular docking to identify the small molecules capable to target AKAP-Lbc and RhoA interactions. The pharmacophore model based virtual screening unveils two lead compounds from the TIMBAL database of small molecules modulating the targeted protein-protein interactions. The molecular docking analysis revealed the lead compounds' potentialities to establish the essential chemical interactions with the key interactive residues of the complex. These features provided a road map for designing additional potent chemical derivatives and fragments of the original lead compounds to perturb the AKAP-Lbc and RhoA interactions. Experimental validations may elucidate the therapeutic potential of these lead chemical scaffolds to deal with aberrant AKAP-Lbc signaling based cardiac hypertrophy. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. The Glycosylphosphatidylinositol Anchor Biosynthesis Genes GPI12, GAA1, and GPI8 Are Essential for Cell-Wall Integrity and Pathogenicity of the Maize Anthracnose Fungus Colletotrichum graminicola.

    PubMed

    Oliveira-Garcia, Ely; Deising, Holger B

    2016-11-01

    Glycosylphosphatidylinositol (GPI) anchoring of proteins is one of the most common posttranslational modifications of proteins in eukaryotic cells and is important for associating proteins with the cell surface. In fungi, GPI-anchored proteins play essential roles in cross-linking of β-glucan cell-wall polymers and cell-wall rigidity. GPI-anchor synthesis is successively performed at the cytoplasmic and the luminal face of the ER membrane and involves approximately 25 proteins. While mutagenesis of auxiliary genes of this pathway suggested roles of GPI-anchored proteins in hyphal growth and virulence, essential genes of this pathway have not been characterized. Taking advantage of RNA interference (RNAi) we analyzed the function of the three essential genes GPI12, GAA1 and GPI8, encoding a cytoplasmic N-acetylglucosaminylphosphatidylinositol deacetylase, a metallo-peptide-synthetase and a cystein protease, the latter two representing catalytic components of the GPI transamidase complex. RNAi strains showed drastic cell-wall defects, resulting in exploding infection cells on the plant surface and severe distortion of in planta-differentiated infection hyphae, including formation of intrahyphal hyphae. Reduction of transcript abundance of the genes analyzed resulted in nonpathogenicity. We show here for the first time that the GPI synthesis genes GPI12, GAA1, and GPI8 are indispensable for vegetative development and pathogenicity of the causal agent of maize anthracnose, Colletotrichum graminicola.

  16. 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

    2008-01-04

    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.

  17. Molecular mechanism of Aurora A kinase autophosphorylation and its allosteric activation by TPX2

    DOE PAGES

    Zorba, Adelajda; Buosi, Vanessa; Kutter, Steffen; ...

    2014-05-27

    We elucidate the molecular mechanisms of two distinct activation strategies (autophosphorylation and TPX2-mediated activation) in human Aurora A kinase. Classic allosteric activation is in play where either activation loop phosphorylation or TPX2 binding to a conserved hydrophobic groove shifts the equilibrium far towards the active conformation. We resolve the controversy about the mechanism of autophosphorylation by demonstrating intermolecular autophosphorylation in a long-lived dimer by combining X-ray crystallography with functional assays. We then address the allosteric activation by TPX2 through activity assays and the crystal structure of a domain-swapped dimer of dephosphorylated Aurora A and TPX21−25. While autophosphorylation is the keymore » regulatory mechanism in the centrosomes in the early stages of mitosis, allosteric activation by TPX2 of dephosphorylated Aurora A could be at play in the spindle microtubules. The mechanistic insights into autophosphorylation and allosteric activation by TPX2 binding proposed here, may have implications for understanding regulation of other protein kinases.« less

  18. Assessing tether anchor labeling and usability in pickup trucks.

    PubMed

    Klinich, Kathleen D; Manary, Miriam A; Malik, Laura A; Flannagan, Carol A; Jermakian, Jessica S

    2018-04-03

    The objective of this study was to investigate vehicle factors associated with child restraint tether use and misuse in pickup trucks and evaluate 4 labeling interventions designed to educate consumers on proper tether use. Volunteer testing was performed with 24 subjects and 4 different pickup trucks. Each subject performed 8 child restraint installations among the 4 pickups using 2 forward-facing restraints: a Britax Marathon G4.1 and an Evenflo Triumph. Vehicles were selected to represent 4 different implementations of tether anchors among pickups: plastic loop routers (Chevrolet Silverado), webbing routers (Ram), back wall anchors (Nissan Frontier), and webbing routers plus metal anchors (Toyota Tundra). Interventions included a diagram label, Quick Response (QR) Code linked to video instruction, coordinating text label, and contrasting text tag. Subjects used the child restraint tether in 93% of trials. However, tether use was completely correct in only 9% of trials. An installation was considered functional if the subject attached the tether to a tether anchor and had a tight installation (ignoring routing and head restraint position); 28% of subjects achieved a functional installation. The most common installation error was attaching the tether hook to the anchor/router directly behind the child restraint (near the top of the seatback) rather than placing the tether through the router and attaching it to the anchor in the adjacent seating position. The Nissan Frontier, with the anchor located on the back wall of the cab, had the highest rate of correct installations but also had the highest rate of attaching the tether to components other than the tether anchor (seat adjustor, child restraint storage hook, around head restraint). None of the labeling interventions had a significant effect on correct installation; not a single subject scanned the QR Code to access the video instruction. Subjects with the most successful installations spent extensive time

  19. 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

    2015-12-03

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Interactome of the negative regulator of nuclear import BRCA1-binding protein 2.

    PubMed

    Fatima, Shadma; Wagstaff, Kylie M; Loveland, Kate L; Jans, David A

    2015-03-30

    Although the negative regulator of nuclear import (NRNI) BRCA1 binding protein 2 (BRAP2) is highly expressed in testis, its role is largely unknown. Here we address this question by documenting the BRAP2 interactome from human testis, using the yeast 2-hybrid system to identify BRAP2-interacting proteins with roles in diverse cellular processes, including regulation of the actin cytoskeleton, ubiquitinylation, cell cycle/apoptosis and transcription. Interaction with BRAP2 in adult mouse testis with three of these, PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1), A-Kinase anchor protein (AKAP3) and DNA methyl transferase 1 (DNMT1), was confirmed by coimmunoprecipitation assays. BRAP2's ability to inhibit PHLPP1 and DNMT1 nuclear localisation was also confirmed by quantitative confocal microscopy. Importantly, the physiological relevance thereof was implied by the cytoplasmic localisation of PHLPP1, AKAP3 and DNMT1 in pachytene spermatocytes/round spermatids where BRAP2 is present at high levels, and nuclear localisation of PHLPP1 and DNMT1 in spermatogonia concomitant with lower levels of BRAP2. Interestingly, BRAP2 was also present in murine spermatozoa, in part colocalised with AKAP3. Together the results indicate for the first time that BRAP2 may play an important NRNI role in germ cells of the testis, with an additional, scaffold/structural role in mature spermatozoa.

  1. Vesicle Fusion Mediated by Solanesol-Anchored DNA.

    PubMed

    Flavier, Kristina M; Boxer, Steven G

    2017-09-19

    Fusion between two lipid bilayers is one of the central processes in cell biology, playing a key role in endocytosis, exocytosis, and vesicle transport. We have previously developed a model system that uses the hybridization of complementary DNA strands to model the formation of the SNARE four-helix bundle that mediates synaptic vesicle fusion and used it to study vesicle fusion to a tethered lipid bilayer. Using single vesicle assays, 70% of observed fusion events in the DNA-lipid system are arrested at the hemifusion stage, whereas only 5% eventually go to full fusion. This may be because the diglycerol ether that anchors the DNA in the membrane spans only half the bilayer: upon hemifusion and mixing of the outer leaflets, the DNA-lipid is free to diffuse into the target membrane and away from the vesicle. Here, we test the hypothesis that the length of the membrane anchor may impact the outcome by comparing single leaflet-spanning DNA-lipid mediated vesicle fusion with fusion mediated by DNA anchored by solanesol, a C45 isoprenoid of sufficient length to span the bilayer. When the solanesol anchor was present on the incoming vesicles, target membrane, or both, ∼2-3 times as much full fusion was observed as in the DNA-lipid mediated system, as measured by lipid mixing or content transfer. These results indicate that a transmembrane anchor increases the efficiency of full fusion. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  2. Ectoenzymes of the kidney microvillar membrane. Differential solubilization by detergents can predict a glycosyl-phosphatidylinositol membrane anchor.

    PubMed Central

    Hooper, N M; Turner, A J

    1988-01-01

    The pattern of solubilization of nine kidney microvillar ectoenzymes by a range of detergents distinguished two classes of membrane proteins: those released from the membrane by bacterial phosphatidylinositol-specific phospholipase C and those not so released. The latter group of transmembrane proteins were solubilized efficiently (greater than 80%) by all the detergents examined. In contrast, proteins released by phosphatidylinositol-specific phospholipase C were solubilized effectively only by octyl glucoside, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate and sodium deoxycholate. Octyl glucoside solubilized the amphipathic forms of the ectoenzymes examined, suggesting that this may be a useful detergent in the purification of glycosyl-phosphatidylinositol-anchored ectoenzymes. PMID:2839148

  3. Anchoring of nematic liquid crystals at a solid substrate

    NASA Astrophysics Data System (ADS)

    Poniewierski, A.; Samborski, A.

    1996-03-01

    A nematic liquid crystal in contact with a solid substrate is studied by means of the Landau-de Gennes formalism. The free-energy functional is expanded around the bulk nematic order parameter up to the second-order terms. This approximation is used to obtain an explicit condition for the anchoring direction in terms of the surface and bulk coupling constants, in a semi-infinite system. Then a finite system is studied and the equilibrium free energy is found as a function of the angular deviation from the anchoring direction and the sample thickness. A geometrical measure of the anchoring strength, resembling the de Gennes extrapolation length, is obtained from the asymptotic behavior of the free energy for large sample thicknesses.

  4. Anchoring effect on first passage process in Taiwan financial market

    NASA Astrophysics Data System (ADS)

    Liu, Hsing; Liao, Chi-Yo; Ko, Jing-Yuan; Lih, Jiann-Shing

    2017-07-01

    Empirical analysis of the price fluctuations of financial markets has received extensive attention because a substantial amount of financial market data has been collected and because of advances in data-mining techniques. Price fluctuation trends can help investors to make informed trading decisions, but such decisions may also be affected by a psychological factors-the anchoring effect. This study explores the intraday price time series of Taiwan futures, and applies diffusion model and quantitative methods to analyze the relationship between the anchoring effect and price fluctuations during first passage process. Our results indicate that power-law scaling and anomalous diffusion for stock price fluctuations are related to the anchoring effect. Moreover, microscopic price fluctuations before switching point in first passage process correspond with long-term price fluctuations of Taiwan's stock market. We find that microscopic trends could provide useful information for understanding macroscopic trends in stock markets.

  5. Protein

    USDA-ARS?s Scientific Manuscript database

    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...

  6. 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)

  7. 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.

  8. 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

  9. A reusable suture anchor for arthroscopy psychomotor skills training.

    PubMed

    Tillett, Edward D; Rogers, Rainie; Nyland, John

    2003-03-01

    For residents to adequately develop the early arthroscopy psychomotor skills required to better learn how to manage the improvisational situations they will encounter during actual patient cases, they need to experience sufficient practice repetitions within a contextually relevant environment. Unfortunately, the cost of suture anchors can be a practice repetition-limiting factor in learning arthroscopic knot-tying techniques. We describe a technique for creating inexpensive reusable suture anchors and provide an example of their application to repair the anterior glenoid labrum during an arthroscopy psychomotor skills laboratory training session.

  10. Doc Toxin Is a Kinase That Inactivates Elongation Factor Tu*

    PubMed Central

    Cruz, Jonathan W.; Rothenbacher, Francesca P.; Maehigashi, Tatsuya; Lane, William S.; Dunham, Christine M.; Woychik, Nancy A.

    2014-01-01

    The Doc toxin from bacteriophage P1 (of the phd-doc toxin-antitoxin system) has served as a model for the family of Doc toxins, many of which are harbored in the genomes of pathogens. We have shown previously that the mode of action of this toxin is distinct from the majority derived from toxin-antitoxin systems: it does not cleave RNA; in fact P1 Doc expression leads to mRNA stabilization. However, the molecular triggers that lead to translation arrest are not understood. The presence of a Fic domain, albeit slightly altered in length and at the catalytic site, provided a clue to the mechanism of P1 Doc action, as most proteins with this conserved domain inactivate GTPases through addition of an adenylyl group (also referred to as AMPylation). We demonstrated that P1 Doc added a single phosphate group to the essential translation elongation factor and GTPase, elongation factor (EF)-Tu. The phosphorylation site was at a highly conserved threonine, Thr-382, which was blocked when EF-Tu was treated with the antibiotic kirromycin. Therefore, we have established that Fic domain proteins can function as kinases. This distinct enzymatic activity exhibited by P1 Doc also solves the mystery of the degenerate Fic motif unique to the Doc family of toxins. Moreover, we have established that all characterized Fic domain proteins, even those that phosphorylate, target pivotal GTPases for inactivation through a post-translational modification at a single functionally critical acceptor site. PMID:24448800

  11. Doc toxin is a kinase that inactivates elongation factor Tu.

    PubMed

    Cruz, Jonathan W; Rothenbacher, Francesca P; Maehigashi, Tatsuya; Lane, William S; Dunham, Christine M; Woychik, Nancy A

    2014-03-14

    The Doc toxin from bacteriophage P1 (of the phd-doc toxin-antitoxin system) has served as a model for the family of Doc toxins, many of which are harbored in the genomes of pathogens. We have shown previously that the mode of action of this toxin is distinct from the majority derived from toxin-antitoxin systems: it does not cleave RNA; in fact P1 Doc expression leads to mRNA stabilization. However, the molecular triggers that lead to translation arrest are not understood. The presence of a Fic domain, albeit slightly altered in length and at the catalytic site, provided a clue to the mechanism of P1 Doc action, as most proteins with this conserved domain inactivate GTPases through addition of an adenylyl group (also referred to as AMPylation). We demonstrated that P1 Doc added a single phosphate group to the essential translation elongation factor and GTPase, elongation factor (EF)-Tu. The phosphorylation site was at a highly conserved threonine, Thr-382, which was blocked when EF-Tu was treated with the antibiotic kirromycin. Therefore, we have established that Fic domain proteins can function as kinases. This distinct enzymatic activity exhibited by P1 Doc also solves the mystery of the degenerate Fic motif unique to the Doc family of toxins. Moreover, we have established that all characterized Fic domain proteins, even those that phosphorylate, target pivotal GTPases for inactivation through a post-translational modification at a single functionally critical acceptor site.

  12. A novel membrane anchor for FtsZ is linked to cell wall hydrolysis in Caulobacter crescentus.

    PubMed

    Meier, Elizabeth L; Razavi, Shiva; Inoue, Takanari; Goley, Erin D

    2016-07-01

    In most bacteria, the tubulin-like GTPase FtsZ forms an annulus at midcell (the Z-ring) which recruits the division machinery and regulates cell wall remodeling. Although both activities require membrane attachment of FtsZ, few membrane anchors have been characterized. FtsA is considered to be the primary membrane tether for FtsZ in bacteria, however in Caulobacter crescentus, FtsA arrives at midcell after stable Z-ring assembly and early FtsZ-directed cell wall synthesis. We hypothesized that additional proteins tether FtsZ to the membrane and demonstrate that in C. crescentus, FzlC is one such membrane anchor. FzlC associates with membranes directly in vivo and in vitro and recruits FtsZ to membranes in vitro. As for most known membrane anchors, the C-terminal peptide of FtsZ is required for its recruitment to membranes by FzlC in vitro and midcell recruitment of FzlC in cells. In vivo, overproduction of FzlC causes cytokinesis defects whereas deletion of fzlC causes synthetic defects with dipM, ftsE and amiC mutants, implicating FzlC in cell wall hydrolysis. Our characterization of FzlC as a novel membrane anchor for FtsZ expands our understanding of FtsZ regulators and establishes a role for membrane-anchored FtsZ in the regulation of cell wall hydrolysis. © 2016 John Wiley & Sons Ltd.

  13. Probing the Huntingtin 1-17 membrane anchor on a phospholipid bilayer by using all-atom simulations.

    PubMed

    Côté, Sébastien; Binette, Vincent; Salnikov, Evgeniy S; Bechinger, Burkhard; Mousseau, Normand

    2015-03-10

    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. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  14. The effect of suture anchor design and orientation on suture abrasion: An in vitro study.

    PubMed

    Bardana, Davide D; Burks, Robert T; West, John R; Greis, Patrick E

    2003-03-01

    To evaluate the effects of suture anchor design and orientation on suture abrasion in a cyclic model. In vitro. Biomechanical studies have shown suture breakage to be a predominant mode of failure in a suture anchor repair construct. It is possible that suture abrasion during knot tying or in vivo cyclic loading may contribute to early failure. This study specifically investigates suture abrasion caused by 17 commonly used suture anchors and demonstrates the effects of suture anchor angulation and rotation on suture abrasion. To eliminate target tissue as a source of failure, all anchors were implanted into a solid block of sawbones material and tested with No. 2 Ethibond Excel sutures (Ethicon, Somerville, NJ). The testing model focused on 3 variables: suture anchor type, suture pull angle (SA) and angle of anchor rotation (RA). Abrasion testing was then performed on a servohydraulic materials testing system by continually cycling the suture back and forth through each anchor with an excursion of 4 cm at a rate of 0.5 Hz under a load of 10 N until suture failure occurred. Sutures performed significantly better when cycled in line with the anchor at 0 degrees SA with 0 degrees RA than they did at 45 degrees SA with 0 degrees RA or 45 degrees SA with 90 degrees RA. We found no significant difference between anchors tested at 45 degrees SA with 0 degrees RA and 45 degrees SA with 90 degrees RA. For tests performed using metallic suture anchors, all constructs failed by fraying of the suture. Constructs using biopolymer anchors and nonabsorbable polymeric anchors experienced a mixture of suture and anchor eyelet failures. In addition to the statistically significant detrimental effects of suture anchor angulation and rotation on suture abrasion, suture anchor eyelet design may also influence suture abrasion. Surgeons should be aware of the effects of anchor angulation, suture position in the eyelet, and design and composition of the eyelet to maximize the durability

  15. A kinase sequence database: sequence alignments and family assignment.

    PubMed

    Buzko, Oleksandr; Shokat, Kevan M

    2002-09-01

    The Kinase Sequence Database (KSD) located at http://kinase.ucsf.edu/ksd contains information on 290 protein kinase families derived by profile-based clustering of the non-redundant list of sequences obtained from a GenBank-wide search. Included in the database are a total of 5,041 protein kinases from over 100 organisms. Clustering into families is based on the extent of homology within the kinase catalytic domain (250-300 residues in length). Alignments of the families are viewed by interactive Excel-based sequence spreadsheets. In addition, KSD features evolutionary trees derived for each family and detailed information on each sequence as well as links to the corresponding GenBank entries. Sequence manipulation tools, such as evolutionary tree generation, novel sequence assignment, and statistical analysis, are also provided. The kinase sequence database is a web-based service accessible at http://kinase.ucsf.edu/ksd buzko@cmp.ucsf.edu; shokat@cmp.ucsf.edu/ksd

  16. 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…

  17. Promises and Pitfalls of Anchoring Vignettes in Health Survey Research

    PubMed Central

    Verdes-Tennant, Emese; McEniry, Mary; Ispány, Márton

    2016-01-01

    Data harmonization is a topic of growing importance to demographers, who increasingly conduct domestic or international comparative research. Many self-reported survey items cannot be directly compared across demographic groups or countries because these groups differ in how they use subjective response categories. Anchoring vignettes, already appearing in numerous surveys worldwide, promise to overcome this problem. However, many anchoring vignettes have not been formally evaluated for adherence to the key measurement assumptions of vignette equivalence and response consistency. This article tests these assumptions in some of the most widely fielded anchoring vignettes in the world: the health vignettes in the World Health Organization (WHO) Study on Global AGEing and Adult Health (SAGE) and World Health Survey (WHS) (representing 10 countries; n = 52,388), as well as similar vignettes in the Health and Retirement Study (HRS) (n = 4,528). Findings are encouraging regarding adherence to response consistency, but reveal substantial violations of vignette equivalence both cross-nationally and across socioeconomic groups. That is, members of different sociocultural groups appear to interpret vignettes as depicting fundamentally different levels of health. The evaluated anchoring vignettes do not fulfill their promise of providing interpersonally comparable measures of health. Recommendations for improving future implementations of vignettes are discussed. PMID:26335547

  18. 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

  19. 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.…

  20. 15. DETAIL OF ANCHOR BOLT WHICH FORMERLY SECURED A TIMBER ...

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

    15. DETAIL OF ANCHOR BOLT WHICH FORMERLY SECURED A TIMBER SILL BEAM TO THE GRANITE SILL ALONG THE EASTERLY EDGE OF THE SPILLWAY APRON (NOTE 3" SWISS ARMY KNIFE TO RIGHT OF BOLT FOR SCALE); VIEW TO WEST. - Blackstone Canal Millbury Segment, Beginning northwest of State Route 146 & McCracken Road, running along west side of Route 146, Millbury, Worcester County, MA

  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 explicit…

  2. 48 CFR 1812.7000 - Anchor tenancy contracts.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... ADMINISTRATION COMPETITION AND ACQUISITION PLANNING ACQUISITION OF COMMERCIAL ITEMS Commercial Space Hardware or... a multi-year anchor tenancy contract; and (2) Specifies the commercial space product or service to... United States Government agrees to procure sufficient quantities of a commercial space product or service...

  3. End Anchoring in Short-Term Order Memory

    ERIC Educational Resources Information Center

    Farrell, Simon; Lelievre, Anna

    2009-01-01

    Temporally grouping lists has systematic effects on immediate serial recall accuracy, order errors, and recall latencies, and is generally taken to reflect the use of multiple dimensions of ordering in short-term memory. It has been argued that these representations are fully relative, in that all sequence positions are anchored to both the start…

  4. Empirical evidence for resource-rational anchoring and adjustment.

    PubMed

    Lieder, Falk; Griffiths, Thomas L; M Huys, Quentin J; Goodman, Noah D

    2018-04-01

    People's estimates of numerical quantities are systematically biased towards their initial guess. This anchoring bias is usually interpreted as sign of human irrationality, but it has recently been suggested that the anchoring bias instead results from people's rational use of their finite time and limited cognitive resources. If this were true, then adjustment should decrease with the relative cost of time. To test this hypothesis, we designed a new numerical estimation paradigm that controls people's knowledge and varies the cost of time and error independently while allowing people to invest as much or as little time and effort into refining their estimate as they wish. Two experiments confirmed the prediction that adjustment decreases with time cost but increases with error cost regardless of whether the anchor was self-generated or provided. These results support the hypothesis that people rationally adapt their number of adjustments to achieve a near-optimal speed-accuracy tradeoff. This suggests that the anchoring bias might be a signature of the rational use of finite time and limited cognitive resources rather than a sign of human irrationality.

  5. Modeling Adhesive Anchors in a Discrete Element Framework.

    PubMed

    Marcon, Marco; Vorel, Jan; Ninčević, Krešimir; Wan-Wendner, Roman

    2017-08-08

    In recent years, post-installed anchors are widely used to connect structural members and to fix appliances to load-bearing elements. A bonded anchor typically denotes a threaded bar placed into a borehole filled with adhesive mortar. The high complexity of the problem, owing to the multiple materials and failure mechanisms involved, requires a numerical support for the experimental investigation. A reliable model able to reproduce a system's short-term behavior is needed before the development of a more complex framework for the subsequent investigation of the lifetime of fasteners subjected to various deterioration processes can commence. The focus of this contribution is the development and validation of such a model for bonded anchors under pure tension load. Compression, modulus, fracture and splitting tests are performed on standard concrete specimens. These serve for the calibration and validation of the concrete constitutive model. The behavior of the adhesive mortar layer is modeled with a stress-slip law, calibrated on a set of confined pull-out tests. The model validation is performed on tests with different configurations comparing load-displacement curves, crack patterns and concrete cone shapes. A model sensitivity analysis and the evaluation of the bond stress and slippage along the anchor complete the study.

  6. 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…

  7. 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,…

  8. 24 CFR 3285.402 - Ground anchor installations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 5 2010-04-01 2010-04-01 false Ground anchor installations. 3285.402 Section 3285.402 Housing and Urban Development Regulations Relating to Housing and Urban... HOUSING AND URBAN DEVELOPMENT MODEL MANUFACTURED HOME INSTALLATION STANDARDS Anchorage Against Wind § 3285...

  9. 24 CFR 3285.402 - Ground anchor installations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 24 Housing and Urban Development 5 2012-04-01 2012-04-01 false Ground anchor installations. 3285.402 Section 3285.402 Housing and Urban Development Regulations Relating to Housing and Urban... HOUSING AND URBAN DEVELOPMENT MODEL MANUFACTURED HOME INSTALLATION STANDARDS Anchorage Against Wind § 3285...

  10. 24 CFR 3285.402 - Ground anchor installations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 24 Housing and Urban Development 5 2013-04-01 2013-04-01 false Ground anchor installations. 3285.402 Section 3285.402 Housing and Urban Development Regulations Relating to Housing and Urban... HOUSING AND URBAN DEVELOPMENT MODEL MANUFACTURED HOME INSTALLATION STANDARDS Anchorage Against Wind § 3285...

  11. 24 CFR 3285.402 - Ground anchor installations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 24 Housing and Urban Development 5 2014-04-01 2014-04-01 false Ground anchor installations. 3285.402 Section 3285.402 Housing and Urban Development Regulations Relating to Housing and Urban... HOUSING AND URBAN DEVELOPMENT MODEL MANUFACTURED HOME INSTALLATION STANDARDS Anchorage Against Wind § 3285...

  12. 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…

  13. 1. VIEW EAST TOWARD COS COB ANCHOR BRIDGE #310 OF ...

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

    1. VIEW EAST TOWARD COS COB ANCHOR BRIDGE #310 OF CIRCUIT BREAKER INSTALLATION ON CROSS BEAM OF BRIDGE. ABANDONED COS COB POWER STATION IS TO THE SOUTH OF THIS BRIDGE. - New York, New Haven & Hartford Railroad, Bridge-Type Circuit Breakers, Long Island shoreline between Stamford & New Haven, Cos Cob, Fairfield County, CT

  14. Learning to Hash With Optimized Anchor Embedding for Scalable Retrieval.

    PubMed

    Guo, Yuchen; Ding, Guiguang; Liu, Li; Han, Jungong; Shao, Ling

    2017-03-01

    Sparse representation and image hashing are powerful tools for data representation and image retrieval respectively. The combinations of these two tools for scalable image retrieval, i.e., sparse hashing (SH) methods, have been proposed in recent years and the preliminary results are promising. The core of those methods is a scheme that can efficiently embed the (high-dimensional) image features into a low-dimensional Hamming space, while preserving the similarity between features. Existing SH methods mostly focus on finding better sparse representations of images in the hash space. We argue that the anchor set utilized in sparse representation is also crucial, which was unfortunately underestimated by the prior art. To this end, we propose a novel SH method that optimizes the integration of the anchors, such that the features can be better embedded and binarized, termed as Sparse Hashing with Optimized Anchor Embedding. The central idea is to push the anchors far from the axis while preserving their relative positions so as to generate similar hashcodes for neighboring features. We formulate this idea as an orthogonality constrained maximization problem and an efficient and novel optimization framework is systematically exploited. Extensive experiments on five benchmark image data sets demonstrate that our method outperforms several state-of-the-art related methods.

  15. Synthesis of a Glycosylphosphatidylinositol Anchor Bearing Unsaturated Lipid Chains

    PubMed Central

    Swarts, Benjamin M.; Guo, Zhongwu

    2010-01-01

    A GPI anchor bearing unsaturated fatty acid lipid chains (1) was synthesized by a highly convergent strategy employing the para-methoxybenzyl group for permanent hydroxyl protection. The final global deprotection was achieved by an efficient three-step, one-pot procedure to give an 81% isolated yield of the target structure. PMID:20423078

  16. Modeling Adhesive Anchors in a Discrete Element Framework

    PubMed Central

    Marcon, Marco; Vorel, Jan; Ninčević, Krešimir; Wan-Wendner, Roman

    2017-01-01

    In recent years, post-installed anchors are widely used to connect structural members and to fix appliances to load-bearing elements. A bonded anchor typically denotes a threaded bar placed into a borehole filled with adhesive mortar. The high complexity of the problem, owing to the multiple materials and failure mechanisms involved, requires a numerical support for the experimental investigation. A reliable model able to reproduce a system’s short-term behavior is needed before the development of a more complex framework for the subsequent investigation of the lifetime of fasteners subjected to various deterioration processes can commence. The focus of this contribution is the development and validation of such a model for bonded anchors under pure tension load. Compression, modulus, fracture and splitting tests are performed on standard concrete specimens. These serve for the calibration and validation of the concrete constitutive model. The behavior of the adhesive mortar layer is modeled with a stress-slip law, calibrated on a set of confined pull-out tests. The model validation is performed on tests with different configurations comparing load-displacement curves, crack patterns and concrete cone shapes. A model sensitivity analysis and the evaluation of the bond stress and slippage along the anchor complete the study. PMID:28786964

  17. 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…

  18. MASH test 3-37 of the TxDOT 31-inch W-beam downstream anchor terminal.

    DOT National Transportation Integrated Search

    2011-12-01

    The objective of this study was to develop a suitable replacement for the downstream turndown : guardrail anchor system. The turndown guardrail anchor system does not meet mandated test requirements : under MASH for upstream anchor applic...

  19. Steel shear strength of anchors with stand-off base plates.

    DOT National Transportation Integrated Search

    2013-09-01

    Sign and signal structures are often connected to concrete foundations through a stand-off annular base plate with a double-nut anchor bolt connection, which leaves exposed anchor bolt lengths below leveling nuts used in these connections. Cantilever...

  20. Study on effective anchorage length of anchor cable based on Gauss's function

    NASA Astrophysics Data System (ADS)

    Ren, Feng; WuShuai; Liu, Yan; Liu, Jun-yan

    2017-11-01

    In the current relevant codes and technical standards, the design of anchor cables (bars) is based on the assumption that the shear stress is distributed uniformly along the anchor segment. However, according to this assumption, the super-deep pit need a large uplift bearing capacity, which will lead to the long anchor segment and has great hidden danger in the actual engineering design. Therefore, this paper, based on the previous derived shear stress function between τ and the anchor length, we get a research suitable model-Gauss curve distribution model through the analysis of saturated clay drawing data, and strata anchor shear stress distribution is more suitable for the Gauss function model in saturated clay; at the same time, through the FLAC numerical simulation of Jinan Government Affairs Center project, we obtained the optimal anchor effective anchor length is about 10~12m, the foundation pit anchor is optimized.

  1. 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

    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 sitemore » 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.« less

  2. Protein

    MedlinePlus

    ... each meal. How much do we need? Adult men and women usually need 2-3 servings of ... 1 cup bean soup = 2-oz equivalents • 1 soy or bean burger patty = 2-oz equivalents Note: Milk, cheese, and yogurt are also good sources of protein. Refer to the “Dairy” MOVE! ® Handout for more ...

  3. Essential role for SUN5 in anchoring sperm head to the tail

    PubMed Central

    Wang, Lina; Ouyang, Ying-Chun; Dong, Ming-Zhe; Liu, Chao; Zhao, Haichao; Cui, Xiuhong; Ma, Dongyuan; Zhang, Zhiguo; Yang, Xiaoyu; Guo, Yueshuai; Liu, Feng; Yuan, Li

    2017-01-01

    SUN (Sad1 and UNC84 domain containing)-domain proteins are reported to reside on the nuclear membrane playing distinct roles in nuclear dynamics. SUN5 is a new member of the SUN family, with little knowledge regarding its function. Here, we generated Sun5−/− mice and found that male mice were infertile. Most Sun5-null spermatozoa displayed a globozoospermia-like phenotype but they were actually acephalic spermatozoa. Additional studies revealed that SUN5 was located in the neck of the spermatozoa, anchoring sperm head to the tail, and without functional SUN5 the sperm head to tail coupling apparatus was detached from nucleus during spermatid elongation. Finally, we found that healthy heterozygous offspring could be obtained via intracytoplasmic injection of Sun5-mutated sperm heads for both male mice and patients. Our studies reveal the essential role of SUN5 in anchoring sperm head to the tail and provide a promising way to treat this kind of acephalic spermatozoa-associated male infertility. PMID:28945193

  4. Multivalent anchoring and cross-linking of mussel-inspired antifouling surface coatings.

    PubMed

    Wei, Qiang; Becherer, Tobias; Mutihac, Radu-Cristian; Noeske, Paul-Ludwig Michael; Paulus, Florian; Haag, Rainer; Grunwald, Ingo

    2014-08-11

    In this work, we combine nature's amazing bioadhesive catechol with the excellent bioinert synthetic macromolecule hyperbranched polyglycerol (hPG) to prepare antifouling surfaces. hPG can be functionalized by different amounts of catechol groups for multivalent anchoring and cross-linking because of its highly branched architecture. The catecholic hPGs can be immobilized on various surfaces including metal oxides, noble metals, ceramics, and polymers via simple incubation procedures. The effect of the catechol amount on the immobilization, surface morphology, stability, and antifouling performance of the coatings was studied. Both anchoring and cross-linking interactions provided by catechols can enhance the stability of the coatings. When the catechol groups on the hPG are underrepresented, the tethering of the coating is not effective; while an overrepresentation of catechol groups leads to protein adsorption and cell adhesion. Thus, only a well-balanced amount of catechols as optimized and described in this work can supply the coatings with both good stability and antifouling ability.

  5. Vibrator and PI4KIIIα govern neuroblast polarity by anchoring non-muscle myosin II.

    PubMed

    Koe, Chwee Tat; Tan, Ye Sing; Lönnfors, Max; Hur, Seong Kwon; Low, Christine Siok Lan; Zhang, Yingjie; Kanchanawong, Pakorn; Bankaitis, Vytas A; Wang, Hongyan

    2018-02-27

    A central feature of most stem cells is the ability to self-renew and undergo differentiation via asymmetric division. However, during asymmetric division the role of phosphatidylinositol (PI) lipids and their regulators is not well established. Here, we show that the sole type I PI transfer protein, Vibrator, controls asymmetric division of Drosophila neural stem cells (NSCs) by physically anchoring myosin II regulatory light chain, Sqh, to the NSC cortex. Depletion of vib or disruption of its lipid binding and transfer activities disrupts NSC polarity. We propose that Vib stimulates PI4KIIIα to promote synthesis of a plasma membrane pool of phosphatidylinositol 4-phosphate [PI(4)P] that, in turn, binds and anchors myosin to the NSC cortex. Remarkably, Sqh also binds to PI(4)P in vitro and both Vib and Sqh mediate plasma membrane localization of PI(4)P in NSCs. Thus, reciprocal regulation between Myosin and PI(4)P likely governs asymmetric division of NSCs. © 2018, Koe et al.

  6. Distribution, mobility, and anchoring of lignin-related oxidative enzymes in Arabidopsis secondary cell walls.

    PubMed

    Yi Chou, Eva; Schuetz, Mathias; Hoffmann, Natalie; Watanabe, Yoichiro; Sibout, Richard; Samuels, A Lacey

    2018-04-09

    Lignin is an important phenolic biopolymer that provides strength and rigidity to the secondary cell walls of tracheary elements, sclereids, and fibers in vascular plants. Lignin precursors, called monolignols, are synthesized in the cell and exported to the cell wall where they are polymerized into lignin by oxidative enzymes such as laccases and peroxidases. In Arabidopsis thaliana, a peroxidase (PRX64) and laccase (LAC4) are shown to localize differently within cell wall domains in interfascicular fibers: PRX64 localizes to the middle lamella whereas LAC4 localizes throughout the secondary cell wall layers. Similarly, laccases localized to, and are responsible for, the helical depositions of lignin in protoxylem tracheary elements. In addition, we tested the mobility of laccases in the cell wall using fluorescence recovery after photobleaching. mCHERRY-tagged LAC4 was immobile in secondary cell wall domains, but mobile in the primary cell wall when ectopically expressed. A small secreted red fluorescent protein (sec-mCHERRY) was engineered as a control and was found to be mobile in both the primary and secondary cell walls. Unlike sec-mCHERRY, the tight anchoring of LAC4 to secondary cell wall domains indicated that it cannot be remobilized once secreted, and this anchoring underlies the spatial control of lignification.

  7. The cutting edge: membrane-anchored serine protease activities in the pericellular microenvironment.

    PubMed

    Antalis, Toni M; Buzza, Marguerite S; Hodge, Kathryn M; Hooper, John D; Netzel-Arnett, Sarah

    2010-06-15

    The serine proteases of the trypsin-like (S1) family play critical roles in many key biological processes including digestion, blood coagulation, and immunity. Members of this family contain N- or C-terminal domains that serve to tether the serine protease catalytic domain directly to the plasma membrane. These membrane-anchored serine proteases are proving to be key components of the cell machinery for activation of precursor molecules in the pericellular microenvironment, playing vital functions in the maintenance of homoeostasis. Substrates activated by membrane-anchored serine proteases include peptide hormones, growth and differentiation factors, receptors, enzymes, adhesion molecules and viral coat proteins. In addition, new insights into our understanding of the physiological functions of these proteases and their involvement in human pathology have come from animal models and patient studies. The present review discusses emerging evidence for the diversity of this fascinating group of membrane serine proteases as potent modifiers of the pericellular microenvironment through proteolytic processing of diverse substrates. We also discuss the functional consequences of the activities of these proteases on mammalian physiology and disease.

  8. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Maximum load to failure and tensile displacement of an all-suture glenoid anchor compared with a screw-in glenoid anchor.

    PubMed

    Dwyer, Tim; Willett, Thomas L; Dold, Andrew P; Petrera, Massimo; Wasserstein, David; Whelan, Danny B; Theodoropoulos, John S

    2016-02-01

    The purpose of this study was to evaluate the biomechanical behavior of an all-suture glenoid anchor in comparison with a more conventional screw-in glenoid anchor, with regard to maximum load to failure and tensile displacement. All mechanical testing was performed using an Instron ElectroPuls E1000 mechanical machine, with a 10 N pre-load and displacement rate of 10 mm/min. Force-displacement curves were generated, with calculation of maximum load, maximum displacement, displacement at 50 N and stiffness. Pretesting of handset Y-Knots in bone analog models revealed low force displacement below 60 N of force. Subsequently, three groups of anchors were tested for pull out strength in bovine bone and cadaver glenoid bone: a bioabsorbable screw-in anchor (Bio Mini-Revo, ConMed Linvatec), a handset all-suture anchor (Y-Knot, ConMed Linvatec) and a 60 N pre-tensioned all-suture anchor (Y-Knot). A total of 8 anchors from each group was tested in proximal tibia of bovine bone and human glenoids (age range 50-90). In bovine bone, the Bio Mini-Revo displayed greater maximum load to failure (206 ± 77 N) than both the handset (140 ± 51 N; P = 0.01) and the pre-tensioned Y-Knot (135 ± 46 N; P = 0.001); no significant difference was seen between the three anchor groups in glenoid bone. Compared to the screw-in anchors, the handset all-suture anchor displayed inferior fixation, early displacement and greater laxity in the bovine bone and cadaveric bone (P < 0.05). Pre-tensioning the all-suture anchor to 60 N eliminated this behavior in all bone models. Handset Y-Knots display low force anchor displacement, which is likely due to slippage in the pilot hole. Pre-tensioning the Y-Knot to 60 N eliminates this behavior. I.

  10. Arthroscopic biceps tenodesis using suture anchors through the subclavian portal.

    PubMed

    Nord, Keith D; Smith, Garrison B; Mauck, Benjamin M

    2005-02-01

    Biceps tenodesis is typically performed through an open anterior incision. Even when an arthroscopic rotator cuff repair is performed, an open procedure is typically performed to address the biceps rupture or subluxation. Recently, there has been great interest in performing this procedure arthroscopically. Techniques have included using an interference screw or 2 suture anchors through an anterior cannula. If the biceps is partially ruptured or subluxated and the proximal end is still visible in the joint, a biceps tenodesis can be performed using standard arthroscopic techniques and suture anchors. The senior author (K.D.N.) developed the subclavian portal in 1997 for arthroscopic repair of rotator cuff tears using a pointed suture grasper. This portal is located 1 to 2 cm medial to the acromioclavicular joint line, directly above and slightly medial to the coracoid. It provides an optimal angle for suture anchor placement directly through the anterior supraspinatus or coracohumeral ligament and into the humeral head at the edge of the articular cartilage. Anchors inserted through the subclavian portal reproduce the 45 degrees Deadman's angle, which was described for placing anchors during rotator cuff repair. Using a burr or shaver through the lateral portal, the articular and bony surface under the biceps tendon and just proximal to the bicipital groove are abraded. Suture anchors are inserted through the subclavian portal, then through the biceps tendon, and into the bone. Sutures are retrieved and tied through the lateral cannula if there is a tear of the supraspinatus. If the supraspinatus is intact, the sutures can be tied intra-articularly through the anterior cannula. Release of the biceps is not performed until the repair is accomplished, which prevents the tendon from retracting down the bicipital groove. The anatomy of the subclavian portal is reviewed and the technique of the arthroscopic biceps tenodesis is presented. Preliminary results of 11 cases

  11. A Glycosylphosphatidylinositol Anchor Is Required for Membrane Localization but Dispensable for Cell Wall Association of Chitin Deacetylase 2 in Cryptococcus neoformans

    PubMed Central

    Gilbert, Nicole M.; Baker, Lorina G.; Specht, Charles A.; Lodge, Jennifer K.

    2012-01-01

    ABSTRACT Cell wall proteins (CWPs) mediate important cellular processes in fungi, including adhesion, invasion, biofilm formation, and flocculation. The current model of fungal cell wall organization includes a major class of CWPs covalently bound to β-1,6-glucan via a remnant of a glycosylphosphatidylinositol (GPI) anchor. This model was established by studies of ascomycetes more than a decade ago, and relatively little work has been done with other fungi, although the presumption has been that proteins identified in the cell wall which contain a predicted GPI anchor are covalently linked to cell wall glucans. The pathogenic basidiomycete Cryptococcus neoformans encodes >50 putatively GPI-anchored proteins, some of which have been identified in the cell wall. One of these proteins is chitin deacetylase 2 (Cda2), an enzyme responsible for converting chitin to chitosan, a cell wall polymer recently established as a virulence factor for C. neoformans infection of mammalian hosts. Using a combination of biochemistry, molecular biology, and genetics, we show that Cda2 is GPI anchored to membranes but noncovalently associated with the cell wall by means independent of both its GPI anchor and β-1,6-glucan. We also show that Cda2 produces chitosan when localized to the plasma membrane, but association with the cell wall is not essential for this process, thereby providing insight into the mechanism of chitosan biosynthesis. These results increase our understanding of the surface of C. neoformans and provide models of cell walls likely applicable to other undercharacterized basidiomycete pathogenic fungi. PMID:22354955

  12. Constrained Active Learning for Anchor Link Prediction Across Multiple Heterogeneous Social Networks

    PubMed Central

    Zhu, Junxing; Zhang, Jiawei; Wu, Quanyuan; Jia, Yan; Zhou, Bin; Wei, Xiaokai; Yu, Philip S.

    2017-01-01

    Nowadays, people are usually involved in multiple heterogeneous social networks simultaneously. Discovering the anchor links between the accounts owned by the same users across different social networks is crucial for many important inter-network applications, e.g., cross-network link transfer and cross-network recommendation. Many different supervised models have been proposed to predict anchor links so far, but they are effective only when the labeled anchor links are abundant. However, in real scenarios, such a requirement can hardly be met and most anchor links are unlabeled, since manually labeling the inter-network anchor links is quite costly and tedious. To overcome such a problem and utilize the numerous unlabeled anchor links in model building, in this paper, we introduce the active learning based anchor link prediction problem. Different from the traditional active learning problems, due to the one-to-one constraint on anchor links, if an unlabeled anchor link a=(u,v) is identified as positive (i.e., existing), all the other unlabeled anchor links incident to account u or account v will be negative (i.e., non-existing) automatically. Viewed in such a perspective, asking for the labels of potential positive anchor links in the unlabeled set will be rewarding in the active anchor link prediction problem. Various novel anchor link information gain measures are defined in this paper, based on which several constraint active anchor link prediction methods are introduced. Extensive experiments have been done on real-world social network datasets to compare the performance of these methods with state-of-art anchor link prediction methods. The experimental results show that the proposed Mean-entropy-based Constrained Active Learning (MC) method can outperform other methods with significant advantages. PMID:28771201

  13. Constrained Active Learning for Anchor Link Prediction Across Multiple Heterogeneous Social Networks.

    PubMed

    Zhu, Junxing; Zhang, Jiawei; Wu, Quanyuan; Jia, Yan; Zhou, Bin; Wei, Xiaokai; Yu, Philip S

    2017-08-03

    Nowadays, people are usually involved in multiple heterogeneous social networks simultaneously. Discovering the anchor links between the accounts owned by the same users across different social networks is crucial for many important inter-network applications, e.g., cross-network link transfer and cross-network recommendation. Many different supervised models have been proposed to predict anchor links so far, but they are effective only when the labeled anchor links are abundant. However, in real scenarios, such a requirement can hardly be met and most anchor links are unlabeled, since manually labeling the inter-network anchor links is quite costly and tedious. To overcome such a problem and utilize the numerous unlabeled anchor links in model building, in this paper, we introduce the active learning based anchor link prediction problem. Different from the traditional active learning problems, due to the one-to-one constraint on anchor links, if an unlabeled anchor link a = ( u , v ) is identified as positive (i.e., existing), all the other unlabeled anchor links incident to account u or account v will be negative (i.e., non-existing) automatically. Viewed in such a perspective, asking for the labels of potential positive anchor links in the unlabeled set will be rewarding in the active anchor link prediction problem. Various novel anchor link information gain measures are defined in this paper, based on which several constraint active anchor link prediction methods are introduced. Extensive experiments have been done on real-world social network datasets to compare the performance of these methods with state-of-art anchor link prediction methods. The experimental results show that the proposed Mean-entropy-based Constrained Active Learning (MC) method can outperform other methods with significant advantages.

  14. Failure mode of suture anchors as a function of insertion depth.

    PubMed

    Bynum, C Kelly; Lee, Steven; Mahar, Andrew; Tasto, James; Pedowitz, Robert

    2005-07-01

    Surgeons can control not only the angle but also the depth of suture anchor placement during arthroscopic rotator cuff repair, although the tendency may be to place suture anchors on the deep side to avoid damage from prominent anchor eyelets. However, little information is available regarding possible effects of suture anchor depth on construct failure mechanisms. Anchor depth affects the mode of suture failure with physiologically relevant cyclic loads. Controlled laboratory study. Metallic screw-in suture anchors loaded with No. 2 braided polyester sutures were inserted into the bovine infra-spinatus footprint with the eyelet proud, standard, or deep. Sutures were hand tied to create a closed loop. Constructs were cyclically loaded from 10 to 90 N and, if still intact at 500 cycles, taken to ultimate failure (maximum load). When clinical failure was defined as greater than 3-mm construct elongation, anchors placed with the eyelet deep experienced statistically earlier clinical failure via cutting of the suture through the bone (P < .02). However, anchors placed at this level did not experience catastrophic failure during cyclic loading. The standard and proud anchors experienced 3 mm of elongation at a greater number of cycles, but the suture material degraded at the anchor eyelet, and a majority of these constructs broke during cyclic physiologic loading. At failure testing, the deep anchors had a significantly increased failure load (164 N) compared to standard (133 N) (P < .04) and proud (113 N) anchors (P < .005). Varying the depth of suture anchor insertion changes the mechanical properties and mode of failure of suture anchor constructs. Surgeons should be aware of the effects of suture anchor depth and abrasive eyelet wear on construct failure during arthroscopic rotator cuff repair.

  15. The roles of the glycosylphosphatidylinositol anchor on the production and immunogenicity of recombinant ookinete surface antigen Pbs21 of Plasmodium berghei when prepared in a baculovirus expression system.

    PubMed

    Martínez, A P; Margos, G; Barker, G; Sinden, R E

    2000-10-01

    Malarial ookinetes express an immunodominant surface protein (P28) that is a priority candidate for the development of transmission-blocking vaccines. The full length P28 gene from Plasmodium berghei [Pbs21(1-213)] and a deletion construct [Pbs21(1-188)] encoding a protein that lacks the 25 C-terminal amino acids, including the glycosylphosphatidylinositol (GPI) anchor signal, were expressed in insect cells using baculovirus vectors. Pbs21(1-213) protein is strongly hydrophobic, found in the cytoplasm and on the surface of Spodoptera Sf21 cells, and in the culture medium. Pbs21(1-188) protein was largely found in the aqueous phase of the medium and in the cytoplasm of Sf21 cells, but was not detected on the cell surface. The presence of 25 C-terminal amino acids is therefore critical to the attachment of recombinant Pbs21 to the parasite plasma membrane. Mice were immunized subcutaneously or intramuscularly with affinity purified recombinant Pbs21(1-213), Pbs21(1-188) or native Pbs21 proteins. Following two immunizations, native Pbs21 induces higher titres when administered by either route, than the recombinant protein bearing an insect GPI anchor, which in turn is markedly more immunogenic than the recombinant polypeptide lacking a GPI anchor. When specific anti Pbs21 antibody titres exceeded 1 mg/ml all three antigens were capable of inducing transmission blockade > or = 90%, below 1 mg/ml blockade did not correlate with antibody concentration.

  16. Activation of Aurora A kinase through the FGF1/FGFR signaling axis sustains the stem cell characteristics of glioblastoma cells

    SciTech Connect

    Hsu, Yi-Chao; Institute of Biomedical Sciences, Mackay Medical College, New Taipei City, Taiwan; Kao, Chien-Yu

    2016-06-10

    Fibroblast growth factor 1 (FGF1) binds and activates FGF receptors, thereby regulating cell proliferation and neurogenesis. Human FGF1 gene 1B promoter (−540 to +31)-driven SV40 T antigen has been shown to result in tumorigenesis in the brains of transgenic mice. FGF1B promoter (−540 to +31)-driven green fluorescent protein (F1BGFP) has also been used in isolating neural stem cells (NSCs) with self-renewal and multipotency from developing and adult mouse brains. In this study, we provide six lines of evidence to demonstrate that FGF1/FGFR signaling is implicated in the expression of Aurora A (AurA) and the activation of its kinase domain (Thr288more » phosphorylation) in the maintenance of glioblastoma (GBM) cells and NSCs. First, treatment of FGF1 increases AurA expression in human GBM cell lines. Second, using fluorescence-activated cell sorting, we observed that F1BGFP reporter facilitates the isolation of F1BGFP(+) GBM cells with higher expression levels of FGFR and AurA. Third, both FGFR inhibitor (SU5402) and AurA inhibitor (VX680) could down-regulate F1BGFP-dependent AurA activity. Fourth, inhibition of AurA activity by two different AurA inhibitors (VX680 and valproic acid) not only reduced neurosphere formation but also induced neuronal differentiation of F1BGFP(+) GBM cells. Fifth, flow cytometric analyses demonstrated that F1BGFP(+) GBM cells possessed different NSC cell surface markers. Finally, inhibition of AurA by VX680 reduced the neurosphere formation of different types of NSCs. Our results show that activation of AurA kinase through FGF1/FGFR signaling axis sustains the stem cell characteristics of GBM cells. Implications: This study identified a novel mechanism for the malignancy of GBM, which could be a potential therapeutic target for GBM. - Highlights: • We report that FGF1 treatment can stimulate AurA kinase expression in human GBM cells. • FGF1/FGFR signaling is involved in the activation of AurA kinase. • FGF1 sustains the self

  17. Anomalous temperature-dependent anchoring in liquid crystals mediated by thermodynamic smectic wetting sheets

    NASA Astrophysics Data System (ADS)

    Aya, Satoshi; Araoka, Fumito

    2017-11-01

    We present an unusual temperature dependence of polar anchoring torque in a nematic liquid crystal contacting with a perfluoropolymer surface. Whereas the anchoring torque is conventionally expected to increase with decreasing temperature, we show that the polar anchoring torque in the present system decreases with decreasing temperature. This causes an orientational transition from planar to vertical during cooling. We explain the origin of this phenomenon as the result of thermodynamic growth and spreading of surface-localized smectic wetting sheets. We clearly show that the coverage of these wetting sheets on the surface creates a vertical state that violates the initial planar anchoring in the bulk, thereby reducing the effective polar anchoring torque.

  18. 19. VIEW OF ANCHOR BRIDGE NUMBER 310 LOOKING NORTHEAST FROM ...

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

    19. VIEW OF ANCHOR BRIDGE NUMBER 310 LOOKING NORTHEAST FROM THE ROOF OF THE NORTH SIDE OF THE EAST BOILER ROOM. THE ROOF OF THE LOAD DISPATCHER'S TOWER IS DIRECTLY BEHIND THE FEEDER TOWER ON THE RIGHT SIDE OF THE PHOTOGRAPH. THIS TERMINAL IS THE JUNCTION BETWEEN NORTHEAST UTILITIES LINES AND THE RAILROAD CATENARY. - New York, New Haven & Hartford Railroad, Cos Cob Power Plant, Sound Shore Drive, Greenwich, Fairfield County, CT

  19. [Treatment of calcaneal avulsion fractures with twinfix suture anchors fixation].

    PubMed

    Zhao, Bin-xiu; Wang, Kun-zheng; Wang, Chun-sheng; Xie, Yue; Dai, Zhi-tang; Liu, Gang; Liu, Wei-dong

    2011-06-01

    For the calcaneal avulsion fracture, the current method is more commonly used screws or Kirschner wire to fix fracture fragment. This article intended to explore the feasibility and clinical efficacy for the treatment of avulsion fractures with TwinFix suture anchors. From July 2007 to November 2010, 21 patients were reviewed, including 15 males and 6 females, ranging in age from 49 to 65 years,with a mean of 58.7 years. Twelve patients had nodules in the right heel and 9 patients had nodules in the left heel. All the patients had closed fractures. The typical preoperative symptoms of the patients included pain in the upper heel and weak in heel lift. Body examination results: palpable sense of bone rubbing in the back of the heel, and swelling in the heel. Surgery treatment with TwinFix suture anchors performed as follows : to fix TwinFix suture anchors into the calcaneal body, then to drill the fracture block, to make the double strand suture through the fracture holes, to knot the suture eachother to fix the block, and to use stitch to fix the remaining suture in the Achilles tendon in order to improve the block fixation. The criteria of the AOFAS Foot and Ankle Surgery by the United States Association of ankle-rear foot functional recovery was used to evaluate the Achilles tendon. Total average score was (95.5 +/- 3.12) points, including pain items of(38.5 +/- 2.18) points,the average score of functional items of (49.5 +/- 3.09) points,and power lines of 10 points in all patients. Twenty-one patients got an excellent result, 16 good and 5 poor. The methods of treatment for the calcaneal avulsion fractures with TwinFix suture anchors is a simple operation, and have excellent clinical effect, which is worthy of promotion.

  20. Anchoring the Self to the Body in Bilateral Vestibular Failure

    PubMed Central

    Toupet, Michel; van Nechel, Christian; Duquesne, Ulla; Hautefort, Charlotte; Lopez, Christophe

    2017-01-01

    Recent findings suggest that vestibular information plays a significant role in anchoring the self to the body. Out-of-body experiences of neurological origin are frequently associated with vestibular sensations, and galvanic vestibular stimulation in healthy participants anchors the self to the body. Here, we provide the first objective measures of anchoring the self to the body in chronic bilateral vestibular failure (BVF). We compared 23 patients with idiopathic BVF to 23 healthy participants in a series of experiments addressing several aspects of visuo-spatial perspective taking and embodiment. In Experiment 1, participants were involved in a virtual “dot-counting task” from their own perspective or the perspective of a distant avatar, to measure implicit and explicit perspective taking, respectively. In both groups, response times increased similarly when the avatar’s and participant’s viewpoint differed, for both implicit and explicit perspective taking. In Experiment 2, participants named ambiguous letters (such as “b” or “q”) traced on their forehead that could be perceived from an internal or external perspective. The frequency of perceiving ambiguous letters from an internal perspective was similar in both groups. In Experiment 3, participants completed a questionnaire measuring the experienced self/body and self/environment “closeness”. Both groups reported a similar embodied experience. Altogether, our data show that idiopathic BVF does not change implicit and explicit perspective taking nor subjective anchoring of the self to the body. Our negative findings offer insight into the multisensory mechanisms of embodiment. Only acute peripheral vestibular disorders and neurological disorders in vestibular brain areas (characterized by strong multisensory conflicts) may evoke disembodied experiences. PMID:28107424

  1. Pullout strength of standard vs. cement-augmented rotator cuff repair anchors in cadaveric bone.

    PubMed

    Aziz, Keith T; Shi, Brendan Y; Okafor, Louis C; Smalley, Jeremy; Belkoff, Stephen M; Srikumaran, Uma

    2018-05-01

    We evaluate a novel method of rotator cuff repair that uses arthroscopic equipment to inject bone cement into placed suture anchors. A cadaver model was used to assess the pullout strength of this technique versus anchors without augmentation. Six fresh-frozen matched pairs of upper extremities were screened to exclude those with prior operative procedures, fractures, or neoplasms. One side from each pair was randomized to undergo standard anchor fixation with the contralateral side to undergo anchor fixation augmented with bone cement. After anchor fixation, specimens were mounted on a servohydraulic testing system and suture anchors were pulled at 90° to the insertion to simulate the anatomic pull of the rotator cuff. Sutures were pulled at 1 mm/s until failure. The mean pullout strength was 540 N (95% confidence interval, 389 to 690 N) for augmented anchors and 202 N (95% confidence interval, 100 to 305 N) for standard anchors. The difference in pullout strength was statistically significant (P < 0.05). This study shows superior pullout strength of a novel augmented rotator cuff anchor technique. The described technique, which is achieved by extruding polymethylmethacrylate cement through a cannulated in situ suture anchor with fenestrations, significantly increased the ultimate failure load in cadaveric human humeri. This novel augmented fixation technique was simple and can be implemented with existing instrumentation. In osteoporotic bone, it may substantially reduce the rate of anchor failure. Copyright © 2018 Elsevier Ltd. All rights reserved.

  2. Suture strength and angle of load application in a suture anchor eyelet.

    PubMed

    Deakin, Mark; Stubbs, David; Bruce, Warwick; Goldberg, Jerome; Gillies, Ronald M; Walsh, William R

    2005-12-01

    To assess the effect of suture material, anchor orientation, and anchor eyelet design on the static loading properties of suture anchors. Biomechanical bench study. Two metallic suture anchors, Mitek GII (Mitek, Westwood, MA) and Corkscrew (Arthrex, Naples, FL) and a bioabsorbable anchor (Biocorkscrew; Arthrex) were tested with single strand of No. 2 Ethibond (Ethicon, Norderstedt, Germany) or No. 2 FiberWire (Arthrex) suture. Suture pull angle was varied through 0 degrees, 45 degrees, and 90 degrees with the anchor rotation angle in either a sagittal or coronal plane. Constructs were tested to failure using an MTS 858 Bionix testing machine (Material Testing Systems, Eden Prairie, MN). Peak loads, stiffness, energy to peak load, and failure modes were determined for all samples. FiberWire showed superior static mechanical properties when compared with single-strand Ethibond over all testing conditions (P < .05). Suture pull angle had a significant effect on load to failure with both metallic anchors but not on the bioabsorbable anchor (P < .05). Suture pull angle and anchor rotation angle play an important role in the failure load of suture when placed in an eyelet. The polyaxial nature of the Biocorkscrew eyelet allows for increased degrees of freedom but introduces failure of the suture eyelet as a new failure mode. The loading direction and placement of the suture anchor plays a role in the performance of the suture anchor-suture complex.

  3. Copper acetylacetonate anchored onto amine-functionalised clays.

    PubMed

    Pereira, Clara; Patrício, Sónia; Silva, Ana Rosa; Magalhães, Alexandre L; Carvalho, Ana Paula; Pires, João; Freire, Cristina

    2007-12-15

    Copper (II) acetylacetonate was immobilised directly onto two clays, laponite (Lap) and K10-montmorillonite (K10), and after their amine functionalisation with (3-aminopropyl)triethoxysilane (APTES). All the materials were characterised by nitrogen adsorption isotherms at -196 degrees C, elemental analysis, TG-DSC, XRD, and IR spectroscopy. The K10-based materials were also characterised by XPS. The APTES-functionalised K10 showed higher copper loading than K10, indicating that the clay functionalisation enhanced the complex immobilisation; on the contrary, in Lap-based materials higher metal content was obtained by direct complex anchoring, probably due to the delaminated nature of Lap which induced the particles aggregation on functionalisation with APTES. All the results pointed out that the Cu complex was anchored onto the amine-functionalised clays by Schiff condensation between the amine groups of anchored APTES and the carbonyl groups of the acetylacetonate ligand, whereas direct immobilisation proceeded mostly through interaction between the metal centre and the clay surface hydroxyl groups.

  4. A legged anchoring mechanism for capsule endoscopes using micropatterned adhesives.

    PubMed

    Glass, Paul; Cheung, Eugene; Sitti, Metin

    2008-12-01

    This paper presents a new concept for an anchoring mechanism to enhance existing capsule endoscopes. The mechanism consists of three actuated legs with compliant feet lined with micropillar adhesives to be pressed into the intestine wall to anchor the device at a fixed location. These adhesive systems are inspired by gecko and beetle foot hairs. Single-leg and full capsule mathematical models of the forces generated by the legs are analyzed to understand capsule performance. Empirical friction models for the interaction of the adhesives with an intestinal substrate were experimentally determined in vitro using dry and oil-coated elastomer micropillar arrays with 140 microm pillar diameter, 105 microm spacing between pillars, and an aspect ratio of 1:1 on fresh porcine small intestine specimens. Capsule prototypes were also tested in a simulated intestine environment and compared with predicted peristaltic loads to assess the viability of the proposed design. The experimental results showed that a deployed 10 gr capsule robot can withstand axial peristaltic loads and anchor reliably when actuation forces are greater than 0.27 N using dry micropillars. Required actuation forces may be reduced significantly by using micropillars coated with a thin silicone oil layer.

  5. Change in Stripes for Cholesteric Shells via Anchoring in Moderation

    NASA Astrophysics Data System (ADS)

    Tran, Lisa; Lavrentovich, Maxim O.; Durey, Guillaume; Darmon, Alexandre; Haase, Martin F.; Li, Ningwei; Lee, Daeyeon; Stebe, Kathleen J.; Kamien, Randall D.; Lopez-Leon, Teresa

    2017-10-01

    Chirality, ubiquitous in complex biological systems, can be controlled and quantified in synthetic materials such as cholesteric liquid crystal (CLC) systems. In this work, we study spherical shells of CLC under weak anchoring conditions. We induce anchoring transitions at the inner and outer boundaries using two independent methods: by changing the surfactant concentration or by raising the temperature close to the clearing point. The shell confinement leads to new states and associated surface structures: a state where large stripes on the shell can be filled with smaller, perpendicular substripes, and a focal conic domain (FCD) state, where thin stripes wrap into at least two, topologically required, double spirals. Focusing on the latter state, we use a Landau-de Gennes model of the CLC to simulate its detailed configurations as a function of anchoring strength. By abruptly changing the topological constraints on the shell, we are able to study the interconversion between director defects and pitch defects, a phenomenon usually restricted by the complexity of the cholesteric phase. This work extends the knowledge of cholesteric patterns, structures that not only have potential for use as intricate, self-assembly blueprints but are also pervasive in biological systems.

  6. Editorial Commentary: All-Suture Anchors, Foam Blocks, and Biomechanical Testing.

    PubMed

    Brand, Jefferson C

    2017-06-01

    Barber's biomechanical work is well known to Arthroscopy's readers as thorough, comprehensive, and inclusive of new designs as they become available. In "All-Suture Anchors: Biomechanical Analysis of Pullout Strength, Displacement, and Failure Mode," the latest iteration, Barber and Herbert test all-suture anchors in both porcine femurs and biphasic foam. While we await in vivo clinical trials that compare all-suture anchors to currently used anchors, Barber and Herbert have provided data to inform anchor choice, and using their biomechanical data at time zero from all-suture anchor trials in an animal model, we can determine the anchors' feasibility for human clinical investigations. Copyright © 2017 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

  7. 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.

  8. Clustering and functional coupling of diverse ion channels and signaling proteins revealed by super-resolution STORM microscopy in neurons

    PubMed Central

    Zhang, Jie; Carver, Chase M.; Choveau, Frank S.; Shapiro, Mark S.

    2017-01-01

    Summary The fidelity of neuronal signaling requires organization of signaling molecules into macromolecular complexes, whose components are in intimate proximity. The intrinsic diffraction limit of light makes visualization of individual signaling complexes using visible light extremely difficult. However, using super-resolution stochastic optical reconstruction microscopy (STORM), we observed intimate association of individual molecules within signaling complexes containing ion channels (M-type K+, L-type Ca2+, or TRPV1 channels) and G protein-coupled receptors coupled by the scaffolding protein, A-kinase-anchoring protein (AKAP)79/150. Some channels assembled as multi-channel super-complexes. Surprisingly, we identified novel layers of interplay within macromolecular complexes containing diverse channel types at the single-complex level in sensory neurons, dependent on AKAP79/150. Electrophysiological studies revealed that such ion channels are functionally coupled as well. Our findings illustrate the novel role of AKAP79/150 as a molecular coupler of different channels which conveys cross-talk between channel activities within single microdomains in tuning the physiological response of neurons. PMID:27693258

  9. Convergent regulation of skeletal muscle Ca2+ channels by dystrophin, the actin cytoskeleton, and cAMP-dependent protein kinase

    NASA Astrophysics Data System (ADS)

    Johnson, Barry D.; Scheuer, Todd; Catterall, William A.

    2005-03-01

    The skeletal muscle L-type Ca2+ channel (CaV1.1), which is responsible for initiating muscle contraction, is regulated by phosphorylation by cAMP-dependent protein kinase (PKA) in a voltage-dependent manner that requires direct physical association between the channel and the kinase mediated through A-kinase anchoring proteins (AKAPs). The role of the actin cytoskeleton in channel regulation was investigated in skeletal myocytes cultured from wild-type mice, mdx mice that lack the cytoskeletal linkage protein dystrophin, and a skeletal muscle cell line, 129 CB3. Voltage dependence of channel activation was shifted positively, and potentiation was greatly diminished in mdx myocytes and in 129 CB3 cells treated with the microfilament stabilizer phalloidin. Voltage-dependent potentiation by strong depolarizing prepulses was reduced in mdx myocytes but could be restored by positively shifting the stimulus potentials to compensate for the positive shift in the voltage dependence of gating. Inclusion of PKA in the pipette caused a negative shift in the voltage dependence of activation and restored voltage-dependent potentiation in mdx myocytes. These results show that skeletal muscle Ca2+ channel activity and voltage-dependent potentiation are controlled by PKA and microfilaments in a convergent manner. Regulation of Ca2+ channel activity by hormones and neurotransmitters that use the PKA signal transduction pathway may interact in a critical way with the cytoskeleton and may be impaired by deletion of dystrophin, contributing to abnormal regulation of intracellular calcium concentrations in dystrophic muscle.

  10. Electrolyte gated TFT biosensors based on the Donnan's capacitance of anchored biomolecules

    NASA Astrophysics Data System (ADS)

    Manoli, Kyriaki; Palazzo, Gerardo; Macchia, Eleonora; Tiwari, Amber; Di Franco, Cinzia; Scamarcio, Gaetano; Favia, Pietro; Mallardi, Antonia; Torsi, Luisa

    2017-08-01

    Biodetection using electrolyte gated field effect transistors has been mainly correlated to charge modulated transduction. Therefore, such platforms are designed and studied for limited applications involving relatively small charged species and much care is taken in the operating conditions particularly pH and salt concentration (ionic strength). However, there are several reports suggesting that the device conductance can also be very sensitive towards variations in the capacitance coupling. Understanding the sensing mechanism is important for further exploitation of these promising sensors in broader range of applications. In this paper, we present a thorough and in depth study of a multilayer protein system coupled to an electrolyte gated transistor. It is demonstrated that detection associated to a binding event taking place at a distance of 30 nm far from the organic semiconductor-electrolyte interface is possible and the device conductance is dominated by Donnan's capacitance of anchored biomolecules.

  11. Overexpression of a kinase-deficient form of the EDR1 gene enhances powdery mildew resistance and ethylene-induced senescence in Arabidopsis.

    PubMed

    Tang, Dingzhong; Innes, Roger W

    2002-12-01

    The EDR1 gene of Arabidopsis has previously been reported to encode a Raf-like mitogen-activated protein kinase kinase (MAPKK) kinase, and to function as a negative regulator of disease resistance. A phylogenetic analysis of plant and animal protein kinases revealed, however, that plant Raf-like kinases are more closely related to animal mixed lineage kinases (MLKs) than Raf-like kinases, and are deeply divergent from both classes of animal kinases, making inferences of substrate specificity questionable. We, therefore, assayed the kinase activity of recombinant EDR1 protein in vitro. The EDR1 kinase domain displayed autophosphorylation activity and phosphorylated the common MAP kinase substrate myelin basic protein. The EDR1 kinase domain also phosphorylated a kinase-deficient EDR1 protein, indicating that EDR1 autophosphorylation can occur via an intermolecular mechanism. Overexpression of a kinase-deficient full-length EDR1 gene (35S::dnEDR1) in wild-type Arabidopsis plants caused a dominant negative phenotype, conferring resistance to powdery mildew (Erysiphe cichoracearum) and enhancing ethylene-induced senescence. RNA-gel blot analyses showed that the 35S::dnEDR1 transgene was highly transcribed in transgenic plants. Western blot analysis, however, revealed that neither the wild-type nor mutant EDR1 protein could be detected in these lines, indicating that the dominant negative phenotype may be caused by a translational inhibition mechanism rather than by a protein level effect. Overexpression of orthologous dnEDR1 constructs may provide a novel strategy for controlling powdery mildew disease in crops.

  12. 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

  13. Actin Migration Driven by Directional Assembly and Disassembly of Membrane-Anchored Actin Filaments.

    PubMed

    Katsuno, Hiroko; Toriyama, Michinori; Hosokawa, Yoichiroh; Mizuno, Kensaku; Ikeda, Kazushi; Sakumura, Yuichi; Inagaki, Naoyuki

    2015-07-28

    Actin and actin-associated proteins migrate within various cell types. To uncover the mechanism of their migration, we analyzed actin waves, which translocate actin and actin-associated proteins along neuronal axons toward the growth cones. We found that arrays of actin filaments constituting waves undergo directional assembly and disassembly, with their polymerizing ends oriented toward the axonal tip, and that the lateral side of the filaments is mechanically anchored to the adhesive substrate. A combination of live-cell imaging, molecular manipulation, force measurement, and mathematical modeling revealed that wave migration is driven by directional assembly and disassembly of actin filaments and their anchorage to the substrate. Actin-associated proteins co-migrate with actin filaments by interacting with them. Furthermore, blocking this migration, by creating an adhesion-free gap along the axon, disrupts axonal protrusion. Our findings identify a molecular mechanism that translocates actin and associated proteins toward the cell's leading edge, thereby promoting directional cell motility. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  14. 33 CFR 150.905 - Why are safety zones, no anchoring areas, and areas to be avoided established?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... anchoring areas, and areas to be avoided established? 150.905 Section 150.905 Navigation and Navigable...: OPERATIONS Safety Zones, No Anchoring Areas, and Areas To Be Avoided § 150.905 Why are safety zones, no anchoring areas, and areas to be avoided established? (a) Safety zones, no anchoring areas (NAAs) and areas...

  15. 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.

  16. Understanding the low uptake of bone-anchored hearing aids: a review.

    PubMed

    Powell, R; Wearden, A; Pardesi, S M; Green, K

    2017-03-01

    Bone-anchored hearing aids improve hearing for patients for whom conventional behind-the-ear aids are problematic. However, uptake of bone-anchored hearing aids is low and it is important to understand why this is the case. A narrative review was conducted. Studies examining why people accept or decline bone-anchored hearing aids and satisfaction levels of people with bone-anchored hearing aids were reviewed. Reasons for declining bone-anchored hearing aids included limited perceived benefits, concerns about surgery, aesthetic concerns and treatment cost. No studies providing in-depth analysis of the reasons for declining or accepting bone-anchored hearing aids were identified. Studies of patient satisfaction showed that most participants reported benefits with bone-anchored hearing aids. However, most studies used cross-sectional and/or retrospective designs and only included people with bone-anchored hearing aids. Important avenues for further research are in-depth qualitative research designed to fully understand the decision-making process for bone-anchored hearing aids and rigorous quantitative research comparing satisfaction of people who receive bone-anchored hearing aids with those who receive alternative (or no) treatments.

  17. A novel role of Rab11 in trafficking GPI-anchored trans-sialidase to the plasma membrane of Trypanosoma cruzi.

    PubMed

    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.

  18. 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

  19. 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.

  20. Novel Bistable Device Using Anchoring Transition and Command Surface

    NASA Astrophysics Data System (ADS)

    Ikeda, Takuya; Aya, Satoshi; Araoka, Fumito; Ishikawa, Ken; Haba, Osamu; Yonetake, Koichiro; Momoi, Yuichi; Takezoe, Hideo

    2013-06-01

    A light-driven bistable device was fabricated using nematic liquid crystal (NLC) cells with bare glass and perfluoro-polymer (CYTOP) surfaces. The CYTOP surface induces a discontinuous anchoring transition, when a particular NLC (CCN-47) is introduced into the cells. By doping CCN-47 with a small amount of photoisomerizable azo-dendrimer molecules, the bare glass surface serves as a command surface, since the azo-dendrimer is spontaneously adsorbed on the surface. Using hybrid cells with such different surfaces, a homeotropic orientation was successfully converted to a hybrid orientation by very weak ultraviolet-light irradiation. Because of the bistability at a CYTOP surface, the hybrid orientation is retained.

  1. 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

  2. Cell invasion through basement membranes: an anchor of understanding.

    PubMed

    Sherwood, David R

    2006-05-01

    To metastasize, cancer cells must acquire the ability to breach several basement membrane barriers. Cell invasions through basement membranes also occur during normal development and immune system function, enabling organ formation and cell dispersal. The mechanisms that cells use to cross basement membranes in vivo remain elusive. In cancer and development, these invasions occur in complex and inaccessible environments, which are difficult to study in vivo. Anchor-cell invasion in Caenorhabditis elegans is a simple, visually and experimentally accessible model of basement membrane invasion that is beginning to reveal a network of cellular and molecular control mechanisms that regulate the fundamental cellular process of invasion through basement membranes.

  3. An anchoring system for fish habitat structures: field technique, evaluation, and application.

    Treesearch

    Barbara L. Fontaine; Thomas D. Merritt

    1988-01-01

    Steel cable can be used to bind rocks and logs together to construct fish habitat structures in streams. Cables must be securely anchored if structures are to withstand floods. This paper describes a way to anchor cables into bedrock or ballast boulders. Anchor tensile strength ranged from 7,500 to 36,500 pounds and was related to type of resin and embedment depth....

  4. Self-tapping ability of carbon fibre reinforced polyetheretherketone suture anchors.

    PubMed

    Feerick, Emer M; Wilson, Joanne; Jarman-Smith, Marcus; Ó'Brádaigh, Conchur M; McGarry, J Patrick

    2014-10-01

    An experimental and computational investigation of the self-tapping ability of carbon fibre reinforced polyetheretherketone (CFR-PEEK) has been conducted. Six CFR-PEEK suture anchor designs were investigated using PEEK-OPTIMA® Reinforced, a medical grade of CFR-PEEK. Experimental tests were conducted to investigate the maximum axial force and torque required for self-taping insertion of each anchor design. Additional experimental tests were conducted for some anchor designs using pilot holes. Computational simulations were conducted to determine the maximum stress in each anchor design at various stages of insertion. Simulations also were performed to investigate the effect of wall thickness in the anchor head. The maximum axial force required to insert a self-tapping CFR-PEEK suture anchor did not exceed 150 N for any anchor design. The maximum torque required to insert a self-tapping CFR-PEEK suture anchor did not exceed 0.8 Nm. Computational simulations reveal significant stress concentrations in the region of the anchor tip, demonstrating that a re-design of the tip geometry should be performed to avoid fracture during self-tapping, as observed in the experimental component of this study. This study demonstrates the ability of PEEK-OPTIMA Reinforced suture anchors to self-tap polyurethane foam bone analogue. This provides motivation to further investigate the self-tapping ability of CFR-PEEK suture anchors in animal/cadaveric bone. An optimised design for CFR-PEEK suture anchors offers the advantages of radiolucency, and mechanical properties similar to bone with the ability to self-tap. This may have positive implications for reducing surgery times and the associated costs with the procedure. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  5. SNP Discovery and Chromosome Anchoring Provide the First Physically-Anchored Hexaploid Oat Map and Reveal Synteny with Model Species

    PubMed Central

    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

    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

  6. Genomic positional conservation identifies topological anchor point RNAs linked to developmental loci.

    PubMed

    Amaral, Paulo P; Leonardi, Tommaso; Han, Namshik; Viré, Emmanuelle; Gascoigne, Dennis K; Arias-Carrasco, Raúl; Büscher, Magdalena; Pandolfini, Luca; Zhang, Anda; Pluchino, Stefano; Maracaja-Coutinho, Vinicius; Nakaya, Helder I; Hemberg, Martin; Shiekhattar, Ramin; Enright, Anton J; Kouzarides, Tony

    2018-03-15

    The mammalian genome is transcribed into large numbers of long noncoding RNAs (lncRNAs), but the definition of functional lncRNA groups has proven difficult, partly due to their low sequence conservation and lack of identified shared properties. Here we consider promoter conservation and positional conservation as indicators of functional commonality. We identify 665 conserved lncRNA promoters in mouse and human that are preserved in genomic position relative to orthologous coding genes. These positionally conserved lncRNA genes are primarily associated with developmental transcription factor loci with which they are coexpressed in a tissue-specific manner. Over half of positionally conserved RNAs in this set are linked to chromatin organization structures, overlapping binding sites for the CTCF chromatin organiser and located at chromatin loop anchor points and borders of topologically associating domains (TADs). We define these RNAs as topological anchor point RNAs (tapRNAs). Characterization of these noncoding RNAs and their associated coding genes shows that they are functionally connected: they regulate each other's expression and influence the metastatic phenotype of cancer cells in vitro in a similar fashion. Furthermore, we find that tapRNAs contain conserved sequence domains that are enriched in motifs for zinc finger domain-containing RNA-binding proteins and transcription factors, whose binding sites are found mutated in cancers. This work leverages positional conservation to identify lncRNAs with potential importance in genome organization, development and disease. The evidence that many developmental transcription factors are physically and functionally connected to lncRNAs represents an exciting stepping-stone to further our understanding of genome regulation.

  7. Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity

    PubMed Central

    Grover, Rahul; Fischer, Janine; Schwarz, Friedrich W.; Walter, Wilhelm J.; Schwille, Petra; Diez, Stefan

    2016-01-01

    In eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors, such as kinesin-1, have been well characterized in vitro as single molecules or as ensembles rigidly attached to nonbiological substrates. However, the collective transport by membrane-anchored motors, that is, motors attached to a fluid lipid bilayer, is poorly understood. Here, we investigate the influence of motors’ anchorage to a lipid bilayer on the collective transport characteristics. We reconstituted “membrane-anchored” gliding motility assays using truncated kinesin-1 motors with a streptavidin-binding peptide tag that can attach to streptavidin-loaded, supported lipid bilayers. We found that the diffusing kinesin-1 motors propelled the microtubules in the presence of ATP. Notably, we found the gliding velocity of the microtubules to be strongly dependent on the number of motors and their diffusivity in the lipid bilayer. The microtubule gliding velocity increased with increasing motor density and membrane viscosity, reaching up to the stepping velocity of single motors. This finding is in contrast to conventional gliding motility assays where the density of surface-immobilized kinesin-1 motors does not influence the microtubule velocity over a wide range. We reason that the transport efficiency of membrane-anchored motors is reduced because of their slippage in the lipid bilayer, an effect that we directly observed using single-molecule fluorescence microscopy. Our results illustrate the importance of motor–cargo coupling, which potentially provides cells with an additional means of regulating the efficiency of cargo transport. PMID:27803325

  8. Surface engineering of microparticles by novel protein transfer for targeted antigen/drug delivery.

    PubMed

    Bumgarner, Gary W; Shashidharamurthy, Rangaiah; Nagarajan, Shanmugam; D'Souza, Martin J; Selvaraj, Periasamy

    2009-07-20

    Biodegradable microparticles can function as an adjuvant by targeting antigens to professional antigen presenting cells such as dendritic cells and macrophages. To enhance targeting of microparticles, we have developed a novel method of attaching immunostimulatory molecules such as B7-1 to the surface of albumin microparticles utilizing the glycosylphosphatidyl inositol (GPI) anchor. GPI-B7-1 attaches to the surface of albumin microparticles in a protein transfer mediated process and is functionally active. This protein transfer was dependent on the concentration of the GPI-anchored protein, and independent of temperature and incubation time. Results show that the binding of the GPI-anchored protein is specifically occurring through an interaction between the GPI-anchor and the albumin microparticle surface. Stability studies indicate that the GPI-anchored protein can remain attached to the surface of the microparticle up to 7 days, with storage at 4 degrees C providing the optimal stability. Finally, we were able to simultaneously attach two different GPI-anchored proteins, GPI-B7-1 and GPI-ICAM-1, to the microparticles, demonstrating the capability of attaching more than one GPI-anchored protein to the microparticle surface. This novel method of attaching proteins to the surface of microparticles has potential implications in using microparticles as an antigen delivery device in vaccines as well as in targeted drug delivery.

  9. A STUDY OF LIFT-OFF TEST METHOD OF GROUND ANCHORS

    NASA Astrophysics Data System (ADS)

    Fujiwara, Yuu; Sakai, Toshinori

    Ground anchors (hereinafter anchors) are important to do the maintenance after construction. Residual tensile strength of anchors are generally confirmed by lift-off test that pulls an anchor using a hydraulic jack. However, the lift-off test has not been established a proper testing method. Therefore, it may not have been proper maintenance from measurement variability. In this paper, we conducted various lift-off test at cut slope of the expressway, and made proposals on proper lift-off test method.

  10. 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.

  11. Reinforcing mechanism of anchors in slopes: a numerical comparison of results of LEM and FEM

    NASA Astrophysics Data System (ADS)

    Cai, Fei; Ugai, Keizo

    2003-06-01

    This paper reports the limitation of the conventional Bishop's simplified method to calculate the safety factor of slopes stabilized with anchors, and proposes a new approach to considering the reinforcing effect of anchors on the safety factor. The reinforcing effect of anchors can be explained using an additional shearing resistance on the slip surface. A three-dimensional shear strength reduction finite element method (SSRFEM), where soil-anchor interactions were simulated by three-dimensional zero-thickness elasto-plastic interface elements, was used to calculate the safety factor of slopes stabilized with anchors to verify the reinforcing mechanism of anchors. The results of SSRFEM were compared with those of the conventional and proposed approaches for Bishop's simplified method for various orientations, positions, and spacings of anchors, and shear strengths of soil-grouted body interfaces. For the safety factor, the proposed approach compared better with SSRFEM than the conventional approach. The additional shearing resistance can explain the influence of the orientation, position, and spacing of anchors, and the shear strength of soil-grouted body interfaces on the safety factor of slopes stabilized with anchors.

  12. Development and Assessment of a New CFRP Rod Anchor System for Prestressed Concrete

    NASA Astrophysics Data System (ADS)

    Al-Mayah, A.; Soudki, K.; Plumtree, A.

    2006-09-01

    Design concepts and experimental assessment of a new wedge anchor system for prestressing CFRP rods are presented. This compact and reusable anchor consists of an outer cylinder (barrel), a number of wedges, and a soft metal sleeve. The contacting surfaces of the wedges and barrel have a circular profile along the length of the anchor. Tensile testing using different presetting loads, geometric configurations, and rod sizes was carried out. The relationship of the tensile load and displacement of the rod was established. Presetting was found unnecessary since the anchor system was found to be capable of carrying the full design strength of the rods.

  13. Pash: efficient genome-scale sequence anchoring by Positional Hashing.

    PubMed

    Kalafus, Ken J; Jackson, Andrew R; Milosavljevic, Aleksandar

    2004-04-01

    Pash is a computer program for efficient, parallel, all-against-all comparison of very long DNA sequences. Pash implements Positional Hashing, a novel parallelizable method for sequence comparison based on k-mer representation of sequences. The Positional Hashing method breaks the comparison problem in a unique way that avoids the quadratic penalty encountered with other sensitive methods and confers inherent low-level parallelism. Furthermore, Positional Hashing allows one to readily and predictably trade between sensitivity and speed. In a simulated comparison task, anchoring computationally mutated reads onto a genome, the sensitivity of Pash was equal to or greater than that of BLAST and BLAT, with Pash outperforming these programs as the reads became shorter and less similar to the genome. Using modest computing resources, we employed Pash for two large-scale sequence comparison tasks: comparison of three mammalian genomes, and anchoring millions of chimpanzee whole-genome shotgun sequencing reads onto the human genome. The results of these comparisons by Pash agree with those computed by other methods that use more than an order of magnitude more computing resources. These results confirm the sensitivity of Positional Hashing.

  14. Automatic detection of anchor points for multiple sequence alignment

    PubMed Central

    2010-01-01

    Background Determining beforehand specific positions to align (anchor points) has proved valuable for the accuracy of automated multiple sequence alignment (MSA) software. This feature can be used manually to include biological expertise, or automatically, usually by pairwise similarity searches. Multiple local similarities are be expected to be more adequate, as more biologically relevant. However, even good multiple local similarities can prove incompatible with the ordering of an alignment. Results We use a recently developed algorithm to detect multiple local similarities, which returns subsets of positions in the sequences sharing similar contexts of appearence. In this paper, we describe first how to get, with the help of this method, subsets of positions that could form partial columns in an alignment. We introduce next a graph-theoretic algorithm to detect (and remove) positions in the partial columns that are inconsistent with a multiple alignment. Partial columns can be used, for the time being, as guide only by a few MSA programs: ClustalW 2.0, DIALIGN 2 and T-Coffee. We perform tests on the effect of introducing these columns on the popular benchmark BAliBASE 3. Conclusions We show that the inclusion of our partial alignment columns, as anchor points, improve on the whole the accuracy of the aligner ClustalW on the benchmark BAliBASE 3. PMID:20813050

  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. 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.

  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. ABNORMAL POLLEN TUBE GUIDANCE1, an Endoplasmic Reticulum-Localized Mannosyltransferase Homolog of GLYCOSYLPHOSPHATIDYLINOSITOL10 in Yeast and PHOSPHATIDYLINOSITOL GLYCAN ANCHOR BIOSYNTHESIS B in Human, Is Required for Arabidopsis Pollen Tube Micropylar Guidance and Embryo Development1[W][OPEN

    PubMed Central

    Dai, Xin Ren; Gao, Xin-Qi; Chen, Guang Hui; Tang, Li Li; Wang, Hao; Zhang, Xian Sheng

    2014-01-01

    The perception and response of pollen tubes to the female guidance signals are crucial for directional pollen tube growth inside female tissues, which leads to successful reproduction. In pursuing the mechanisms underlying this biological process, we identified the Arabidopsis (Arabidopsis thaliana) abnormal pollen tube guidance1 (aptg1) mutant, whose pollen tubes showed compromised micropylar guidance. In addition to its male defect, the aptg1 mutant showed embryo lethality. APTG1 encodes a putative mannosyltransferase homolog to human PHOSPHATIDYLINOSITOL GLYCAN ANCHOR BIOSYNTHESIS B and yeast (Saccharomyces cerevisiae) GLYCOSYLPHOSPHATIDYLINOSITOL10 (GPI10), both of which are involved in the biosynthesis of GPI anchors. We found that APTG1 was expressed in most plant tissues, including mature pollen, pollen tubes, mature embryo sacs, and developing embryos. By fluorescence colabeling, we showed that APTG1 was localized in the endoplasmic reticulum, where GPI anchors are synthesized. Disruption of APTG1 affected the localization of COBRA-LIKE10, a GPI-anchored protein important for pollen tube growth and guidance. The results shown here demonstrate that APTG1 is involved in both vegetative and reproductive development in Arabidopsis, likely through processing and proper targeting of GPI-anchored proteins. PMID:24963069

  19. CDK13, a Kinase Involved in Pre-mRNA Splicing, Is a Component of the Perinucleolar Compartment

    PubMed Central

    Fayet, Claire; Genevière, Anne-Marie

    2016-01-01

    The perinucleolar compartment (PNC) is a subnuclear stucture forming predominantly in cancer cells; its prevalence positively correlates with metastatic capacity. Although several RNA-binding proteins have been characterized in PNC, the molecular function of this compartment remains unclear. Here we demonstrate that the cyclin–dependent kinase 13 (CDK13) is a newly identified constituent of PNC. CDK13 is a kinase involved in the regulation of gene expression and whose overexpression was found to alter pre-mRNA processing. In this study we show that CDK13 is enriched in PNC and co-localizes all along the cell cycle with the PNC component PTB. In contrast, neither the cyclins K and L, known to associate with CDK13, nor the potential kinase substrates accumulate in PNC. We further show that CDK13 overexpression increases PNC prevalence suggesting that CDK13 may be determinant for PNC formation. This result linked to the finding that CDK13 gene is amplified in different types of cancer indicate that this kinase can contribute to cancer development in human. PMID:26886422

  20. CDK13, a Kinase Involved in Pre-mRNA Splicing, Is a Component of the Perinucleolar Compartment.

    PubMed

    Even, Yasmine; Escande, Marie-Line; Fayet, Claire; Genevière, Anne-Marie

    2016-01-01

    The perinucleolar compartment (PNC) is a subnuclear stucture forming predominantly in cancer cells; its prevalence positively correlates with metastatic capacity. Although several RNA-binding proteins have been characterized in PNC, the molecular function of this compartment remains unclear. Here we demonstrate that the cyclin-dependent kinase 13 (CDK13) is a newly identified constituent of PNC. CDK13 is a kinase involved in the regulation of gene expression and whose overexpression was found to alter pre-mRNA processing. In this study we show that CDK13 is enriched in PNC and co-localizes all along the cell cycle with the PNC component PTB. In contrast, neither the cyclins K and L, known to associate with CDK13, nor the potential kinase substrates accumulate in PNC. We further show that CDK13 overexpression increases PNC prevalence suggesting that CDK13 may be determinant for PNC formation. This result linked to the finding that CDK13 gene is amplified in different types of cancer indicate that this kinase can contribute to cancer development in human.

  1. Glycosylphosphatidylinositol (GPI) anchor deficiency caused by mutations in PIGW is associated with West syndrome and hyperphosphatasia with mental retardation syndrome.

    PubMed

    Chiyonobu, Tomohiro; Inoue, Norimitsu; Morimoto, Masafumi; Kinoshita, Taroh; Murakami, Yoshiko

    2014-03-01

    Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors 150 or more kinds of proteins to the human cell surface. There are at least 26 genes involved in the biosynthesis and remodelling of GPI anchored proteins (GPI-APs). Recently, inherited GPI deficiencies (IGDs) were reported which cause intellectual disability often accompanied by epilepsy, coarse facial features and multiple anomalies that vary in severity depending upon the degree of defect and/or step in the pathway of affected gene. A patient born to non-consanguineous parents developed intractable seizures with typical hypsarrhythmic pattern in electroencephalography, and was diagnosed as having West syndrome. Because the patient showed severe developmental delay with dysmorphic facial features and hyperphosphatasia, characteristics often seen in IGDs, the patient was tested for GPI deficiency. The patient had decreased surface expression of GPI-APs on blood granulocytes and was identified to be compound heterozygous for NM_178517:c.211A>C and c.499A>G mutations in PIGW by targeted sequencing. Here we describe the first patient with deficiency of PIGW, which is involved in the addition of the acyl-chain to inositol in an early step of GPI biosynthesis. Therefore, IGD should be considered in West syndrome and flow cytometric analysis of blood cells is effective in screening IGD.

  2. Immunogenic Eimeria tenella Glycosylphosphatidylinositol-Anchored Surface Antigens (SAGs) Induce Inflammatory Responses in Avian Macrophages

    PubMed Central

    Chow, Yock-Ping; Wan, Kiew-Lian; Blake, Damer P.; Tomley, Fiona; Nathan, Sheila

    2011-01-01

    Background At least 19 glycosylphosphatidylinositol (GPI)-anchored surface antigens (SAGs) are expressed specifically by second-generation merozoites of Eimeria tenella, but the ability of these proteins to stimulate immune responses in the chicken is unknown. Methodology/Principal Findings Ten SAGs, belonging to two previously defined multigene families (A and B), were expressed as soluble recombinant (r) fusion proteins in E. coli. Chicken macrophages were treated with purified rSAGs and changes in macrophage nitrite production, and in mRNA expression profiles of inducible nitric oxide synthase (iNOS) and of a panel of cytokines were measured. Treatment with rSAGs 4, 5, and 12 induced high levels of macrophage nitric oxide production and IL-1β mRNA transcription that may contribute to the inflammatory response observed during E. tenella infection. Concomitantly, treatment with rSAGs 4, 5 and 12 suppressed the expression of IL-12 and IFN-γ and elevated that of IL-10, suggesting that during infection these molecules may specifically impair the development of cellular mediated immunity. Conclusions/Significance In summary, some E. tenella SAGs appear to differentially modulate chicken innate and humoral immune responses and those derived from multigene family A (especially rSAG 12) may be more strongly linked with E. tenella pathogenicity associated with the endogenous second generation stages. PMID:21980402

  3. 46 CFR 130.240 - Anchors and chains for OSVs of 100 or more gross tons.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... tons must be fitted with anchors and chains meeting the applicable standards set by the ABS for classed... feet) in length and with an equipment number from the ABS of less than 150 may be equipped with either... capable of heaving in either anchor. (c) Standards of classification societies other than the ABS may be...

  4. 46 CFR 130.240 - Anchors and chains for OSVs of 100 or more gross tons.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... tons must be fitted with anchors and chains meeting the applicable standards set by the ABS for classed... feet) in length and with an equipment number from the ABS of less than 150 may be equipped with either... capable of heaving in either anchor. (c) Standards of classification societies other than the ABS may be...

  5. 46 CFR 130.240 - Anchors and chains for OSVs of 100 or more gross tons.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... tons must be fitted with anchors and chains meeting the applicable standards set by the ABS for classed... feet) in length and with an equipment number from the ABS of less than 150 may be equipped with either... capable of heaving in either anchor. (c) Standards of classification societies other than the ABS may be...

  6. Arsonic acid as a robust anchor group for the surface modification of Fe3O4.

    PubMed

    Ahn, Jihoon; Moon, Doo-Sik; Lee, Jin-Kyu

    2013-12-03

    In order to use iron oxide nanoparticles (Fe3O4) in various applications, a surface modification that provides colloidal stability and additional functionality to the nanoparticles is necessary. For the modification of the nanoparticle surface with ligand molecules, the ligand molecule should contain an anchor group that has a strong affinity for the surface. However, currently used anchor groups have shown some problems such as low affinity and stability as well as reactivity with the surface. In this study, arsonic acid (RAsO(OH)2) was investigated as a novel anchor group. It was possible to introduce azide groups on the surface of iron oxide nanoparticles using 4-azidophenylarsonic acid, and the desired functional molecules could be chemically attached to the surface via copper-catalyzed azide-alkyne cycloaddition (click chemistry). By quantifying and comparing the amount of attached anchors on the surface, it was found that arsonic acid displays better affinity than other currently used anchors (catechol, carboxylic acid). Furthermore, we examined the binding reversibility, long-term anchoring stability, and anchoring stability at various pH values. It was revealed that arsonic acid is a stable anchor in various conditions.

  7. Career Anchors: Distribution and Impact on Job Satisfaction, the Israeli Case

    ERIC Educational Resources Information Center

    Danziger, Nira; Valency, Rony

    2006-01-01

    Purpose: The purpose of this paper is to examine the career anchor concept developed by Edgar Schein. Design/methodology/approach: The paper focuses on the distribution of the eight career anchors, on a large heterogeneous sample and the differences in the distribution by gender and type of employment; and the impact of the congruence on job…

  8. Management of subluxated capsular bag-fixated intraocular lenses using a capsular anchor.

    PubMed

    Ton, Yokrat; Naftali, Modi; Gortzak, Ruth Lapid; Assia, Ehud I

    2016-05-01

    We describe the use of the capsular anchor (AssiAnchor) to manage a subluxated intraocular lens (IOL) in the capsular bag. The anchor comprises 2 prongs that hold the anterior lens capsule and a central rod that is sutured to the scleral wall, enabling centration of the IOL-capsular bag complex. Six pseudophakic patients presenting with subluxated posterior chamber IOLs in the capsular bag were operated on using the device. The anchor was used successfully in all cases, although in 2 cases only 1 prong was placed under the capsulorhexis edge. In 1 eye, 2 anchors were used 1 month apart following repeated traumatic zonular injury. The capsular bag holding the IOL remained centered and stable throughout the follow-up period. The anchoring device, which was originally designed to preserve the lens capsule and stabilize subluxated crystalline lenses, can also be used to treat subluxation of a capsular bag-fixated IOL. Dr. Assia is the inventor of the AssiAnchor, has a licensed patent of the anchor, and is consultant to Hanita Lenses. Dr. Lapid-Gortzak is a consultant to and speaker for Alcon Surgical, Inc., Hanita Lenses, Orca Surgical, and Sanoculis Ltd.; a speaker for Santen; and a consultant to Icon. Drs. Ton and Naftali have no financial or proprietary interest in any material or method mentioned. Copyright © 2016 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

  9. Anchoring Effects on Prospective and Retrospective Metacomprehension Judgments as a Function of Peer Performance Information

    ERIC Educational Resources Information Center

    Zhao, Qin; Linderholm, Tracy

    2011-01-01

    Two experiments were conducted to investigate anchoring effects on metacomprehension judgments as a function of fictitious information participants received about past peer performance. In Experiment 1 participants were randomly assigned to one of the three anchor groups that, in some cases, provided past peer performance averages in terms of a…

  10. An Exploratory Comparison of Traditional Classroom Instruction and Anchored Instruction with Secondary School Students: Turkish Experience

    ERIC Educational Resources Information Center

    Elcin, Melih; Sezer, Baris

    2014-01-01

    The purpose of this study was to investigate the impact of anchored instruction on the students in secondary school math studies classrooms. This study adopted a quasi-experimental design. This research involved both quantitative and qualitative methods to investigate the effects of anchored instruction on students' academical achievement,…

  11. 48 CFR 252.225-7019 - Restriction on acquisition of anchor and mooring chain.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... of anchor and mooring chain. 252.225-7019 Section 252.225-7019 Federal Acquisition Regulations System... and mooring chain. As prescribed in 225.7007-3, use the following clause: Restriction on Acquisition of Anchor and Mooring Chain (DEC 2009)) (a) Definition. “Component,” as used in this clause, means an...

  12. 48 CFR 252.225-7019 - Restriction on acquisition of anchor and mooring chain.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... of anchor and mooring chain. 252.225-7019 Section 252.225-7019 Federal Acquisition Regulations System... and mooring chain. As prescribed in 225.7007-3, use the following clause: Restriction on Acquisition of Anchor and Mooring Chain (DEC 2009)) (a) Definition. “Component,” as used in this clause, means an...

  13. 48 CFR 252.225-7019 - Restriction on acquisition of anchor and mooring chain.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... of anchor and mooring chain. 252.225-7019 Section 252.225-7019 Federal Acquisition Regulations System... and mooring chain. As prescribed in 225.7007-3, use the following clause: Restriction on Acquisition of Anchor and Mooring Chain (DEC 2009)) (a) Definition. “Component,” as used in this clause, means an...

  14. Model Test of Anchoring Effect on Zonal Disintegration in Deep Surrounding Rock Masses

    PubMed Central

    Chen, Xu-Guang; Zhang, Qiang-Yong; Wang, Yuan; Liu, De-Jun; Zhang, Ning

    2013-01-01

    The deep rock masses show a different mechanical behavior compared with the shallow rock masses. They are classified into alternating fractured and intact zones during the excavation, which is known as zonal disintegration. Such phenomenon is a great disaster and will induce the different excavation and anchoring methodology. In this study, a 3D geomechanics model test was conducted to research the anchoring effect of zonal disintegration. The model was constructed with anchoring in a half and nonanchoring in the other half, to compare with each other. The optical extensometer and optical sensor were adopted to measure the displacement and strain changing law in the model test. The displacement laws of the deep surrounding rocks were obtained and found to be nonmonotonic versus the distance to the periphery. Zonal disintegration occurs in the area without anchoring and did not occur in the model under anchoring condition. By contrasting the phenomenon, the anchor effect of restraining zonal disintegration was revealed. And the formation condition of zonal disintegration was decided. In the procedure of tunnel excavation, the anchor strain was found to be alternation in tension and compression. It indicates that anchor will show the nonmonotonic law during suppressing the zonal disintegration. PMID:23997683

  15. Unlocking Hospitality Managers Career Transitions through Applying Schein's Career Anchors Theory

    ERIC Educational Resources Information Center

    McGuire, David; Polla, Giovana; Heidl, Britta

    2017-01-01

    Purpose: This paper seeks to unlock the career transitions of hospitality managers through applying Schein's career anchors theory. It seeks to understand how Schein's Career Anchors help explain the career transitions of managers in the Scottish hospitality industry. Design/methodology/approach: The paper adopts a non-sequential multi-method…

  16. The Double-Anchoring Theory of Lightness Perception: A Comment on Bressan (2006)

    ERIC Educational Resources Information Center

    Howe, Piers D. L.; Sagreiya, Hersh; Curtis, Dwight L.; Zheng, Chengjie; Livingstone, Margaret S.

    2007-01-01

    Comments on an article by Bressan. Recently, a double-anchoring theory (DAT) of lightness perception was proposed (P. Bressan, 2006), which offers explanations for all the data explained by the original anchoring theory (A. Gilchrist et al., 1999), as well as a number of additional lightness phenomena. Consequently, DAT can account for an…

  17. Effects of Tagcloud-Anchored Group Discussions on Pre-Service Teachers' Collaborative Knowledge Construction

    ERIC Educational Resources Information Center

    Lin, Shu-Yuan; Xie, Ying

    2017-01-01

    Group discussions are critical for students constructing new understanding and knowledge in both classroom and distance education. Tagclouds can provide an intuitive overview about the group's collective knowledge and could potentially be used as an anchor for group discussions. The effect of using tagclouds as anchors for group discussions was…

  18. Approaches to Interactive Video Anchors in Problem-Based Science Learning

    ERIC Educational Resources Information Center

    Kumar, David Devraj

    2010-01-01

    This paper is an invited adaptation of the IEEE Education Society Distinguished Lecture Approaches to Interactive Video Anchors in Problem-Based Science Learning. Interactive video anchors have a cognitive theory base, and they help to enlarge the context of learning with information-rich real-world situations. Carefully selected movie clips and…

  19. 46 CFR 130.240 - Anchors and chains for OSVs of 100 or more gross tons.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Anchors and chains for OSVs of 100 or more gross tons... SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Miscellaneous Equipment and Systems § 130.240 Anchors and chains for OSVs of 100 or more gross tons. (a) Each OSV of 100 or more gross...

  20. 48 CFR 252.225-7019 - Restriction on acquisition of anchor and mooring chain.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... of anchor and mooring chain. 252.225-7019 Section 252.225-7019 Federal Acquisition Regulations System... and mooring chain. As prescribed in 225.7007-3, use the following clause: Restriction on Acquisition of Anchor and Mooring Chain (DEC 2009)) (a) Definition. “Component,” as used in this clause, means an...

  1. 46 CFR 130.240 - Anchors and chains for OSVs of 100 or more gross tons.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Anchors and chains for OSVs of 100 or more gross tons... SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Miscellaneous Equipment and Systems § 130.240 Anchors and chains for OSVs of 100 or more gross tons. (a) Each OSV of 100 or more gross...

  2. 48 CFR 252.225-7019 - Restriction on acquisition of anchor and mooring chain.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... of anchor and mooring chain. 252.225-7019 Section 252.225-7019 Federal Acquisition Regulations System... and mooring chain. As prescribed in 225.7007-3, use the following clause: Restriction on Acquisition of Anchor and Mooring Chain (DEC 2009)) (a) Definition. “Component,” as used in this clause, means an...

  3. Origins of the Stuttering Stereotype: Stereotype Formation through Anchoring-Adjustment

    ERIC Educational Resources Information Center

    MacKinnon, Sean P.; Hall, Shera; MacIntyre, Peter D.

    2007-01-01

    The stereotype of people who stutter is predominantly negative, holding that stutterers are excessively nervous, anxious, and reserved. The anchoring-adjustment hypothesis suggests that the stereotype of stuttering arises from a process of first anchoring the stereotype in personal feelings during times of normal speech disfluency, and then…

  4. Chemical Reactive Anchoring Lipids with Different Performance for Cell Surface Re-engineering Application.

    PubMed

    Vabbilisetty, Pratima; Boron, Mallorie; Nie, Huan; Ozhegov, Evgeny; Sun, Xue-Long

    2018-02-28

    Introduction of selectively chemical reactive groups at the cell surface enables site-specific cell surface labeling and modification opportunity, thus facilitating the capability to study the cell surface molecular structure and function and the molecular mechanism it underlies. Further, it offers the opportunity to change or improve a cell's functionality for interest of choice. In this study, two chemical reactive anchor lipids, phosphatidylethanolamine-poly(ethylene glycol)-dibenzocyclooctyne (DSPE-PEG 2000 -DBCO) and cholesterol-PEG-dibenzocyclooctyne (CHOL-PEG 2000 -DBCO) were synthesized and their potential application for cell surface re-engineering via lipid fusion were assessed with RAW 264.7 cells as a model cell. Briefly, RAW 264.7 cells were incubated with anchor lipids under various concentrations and at different incubation times. The successful incorporation of the chemical reactive anchor lipids was confirmed by biotinylation via copper-free click chemistry, followed by streptavidin-fluorescein isothiocyanate binding. In comparison, the cholesterol-based anchor lipid afforded a higher cell membrane incorporation efficiency with less internalization than the phospholipid-based anchor lipid. Low cytotoxicity of both anchor lipids upon incorporation into the RAW 264.7 cells was observed. Further, the cell membrane residence time of the cholesterol-based anchor lipid was evaluated with confocal microscopy. This study suggests the potential cell surface re-engineering applications of the chemical reactive anchor lipids.

  5. 75 FR 2152 - Certificate of Alternative Compliance for the Anchor Handling Tug Supply Vessel HOLIDAY

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-14

    ... Compliance for the Anchor Handling Tug Supply Vessel HOLIDAY AGENCY: Coast Guard, DHS. ACTION: Notice... handling tug supply vessel HOLIDAY as required by 33 U.S.C. 1605(c) and 33 CFR 81.18. DATES: The... Purpose The anchor handling tug supply vessel HOLIDAY will be used for offshore supply operations. The...

  6. Model test of anchoring effect on zonal disintegration in deep surrounding rock masses.

    PubMed

    Chen, Xu-Guang; Zhang, Qiang-Yong; Wang, Yuan; Liu, De-Jun; Zhang, Ning

    2013-01-01

    The deep rock masses show a different mechanical behavior compared with the shallow rock masses. They are classified into alternating fractured and intact zones during the excavation, which is known as zonal disintegration. Such phenomenon is a great disaster and will induce the different excavation and anchoring methodology. In this study, a 3D geomechanics model test was conducted to research the anchoring effect of zonal disintegration. The model was constructed with anchoring in a half and nonanchoring in the other half, to compare with each other. The optical extensometer and optical sensor were adopted to measure the displacement and strain changing law in the model test. The displacement laws of the deep surrounding rocks were obtained and found to be nonmonotonic versus the distance to the periphery. Zonal disintegration occurs in the area without anchoring and did not occur in the model under anchoring condition. By contrasting the phenomenon, the anchor effect of restraining zonal disintegration was revealed. And the formation condition of zonal disintegration was decided. In the procedure of tunnel excavation, the anchor strain was found to be alternation in tension and compression. It indicates that anchor will show the nonmonotonic law during suppressing the zonal disintegration.

  7. Smos Land Product Validation Activities at the Valencia Anchor Station

    NASA Astrophysics Data System (ADS)

    Lopez-Baeza, Ernesto

    ABSTRACT Soil moisture is a key parameter controlling the exchanges between the land surface and the atmosphere. In spite of being important for weather and climate modeling, this parameter is not well observed at a global scale. The SMOS (Soil Moisture and Ocean Salinity) Mission was designed by the European Space Agency (ESA) to measure soil moisture over continental surfaces as well as surface salinity over the oceans. Since 2001, the Valencia Anchor Station is currently being prepared for the validation of SMOS land products, namely soil moisture content and vegetation water content. The site has recently been selected by the Mission as a core validation site, mainly due to the reasonable homogeneous characteristics of the area which make it appropriate to undertake the validation of SMOS Level 2 land products during the Mission Commissioning Phase, before attempting more complex areas. Close to SMOS launch, ESA has defined and designed a SMOS V alidation Rehearsal C ampaign P lan which purpose is to repeat the Commissioning Phase execution with all centers, all tools, all participants, all structures, all data available, assuming all tools and structures are ready and trying to produce as close as possible the post-launch conditions. The aim is to test the readiness, the ensemble coordination and the speed of operations, and to avoid as far as possible any unexpected deficiencies of the plan and procedure during the real C ommissioning P hase campaigns. For the rehearsal activity, a control area of 10 x 10 km2 has been chosen at the Valencia Anchor Station study area where a network of ground soil moisture measuring stations is being set up based on the definition of homogeneous physio-hydrological units, attending to climatic, soil type, lithology, geology, elevation, slope and vegetation cover conditions. These stations are linked via a wireless communication system to a master post accessible via internet. The ground soil moisture stations will also be used

  8. Mechanical Characteristics Analysis of Surrounding Rock on Anchor Bar Reinforcement

    NASA Astrophysics Data System (ADS)

    Gu, Shuan-cheng; Zhou, Pan; Huang, Rong-bin

    2018-03-01

    Through the homogenization method, the composite of rock and anchor bar is considered as the equivalent material of continuous, homogeneous, isotropic and strength parameter enhancement, which is defined as reinforcement body. On the basis of elasticity, the composite and the reinforcement are analyzed, Based on strengthening theory of surrounding rock and displacement equivalent conditions, the expression of reinforcement body strength parameters and mechanical parameters is deduced. The example calculation shows that the theoretical results are close to the results of the Jia-mei Gao[9], however, closer to the results of FLAC3D numerical simulation, it is proved that the model and surrounding rock reinforcement body theory are reasonable. the model is easy to analyze and calculate, provides a new way for determining reasonable bolt support parameters, can also provides reference for the stability analysis of underground cavern bolting support.

  9. Arginoplexes: an arginine-anchored nanoliposomal carrier for gene delivery

    NASA Astrophysics Data System (ADS)

    Patel, Ketan; Tyagi, Monica; Monpara, Jasmin; Vora, Lalit; Gupta, Sanjay; Vavia, Pradeep

    2014-04-01

    There is a need of an efficient and safe non-viral gene delivery carrier due to promising future of nucleic acid-based therapeutics in the treatment of intractable diseases. Cytotoxicity and cost are the major concerns with current quaternary ammonium-based cationic liposomes. The major aim of current research work was development and in vitro evaluation of arginine-anchored nanoliposomes for gene delivery. l-Arginine-fatty acid conjugate was synthesized and characterized using IR, NMR, and mass spectroscopy. Synthesized conjugate—lauroyl arginine ethyl ester (LAE) was successfully incorporated into liposomes. Effect of nanocarrier composition on DNA binding was evaluated by preparing solid lipid nanoparticle (SLN) and self nanoemulsifying system (SNES) using same LAE concentration. Effect of cationic head on DNA binding was also evaluated. Arginine-anchored nanoliposomes—arginoplexes (APX) showed superior DNA-binding affinity. Surface PEG was expected to cause hindrance in DNA binding in SLNs and SNES. Guanidino group was found to be a better cationic head for DNA binding compared to primary amine or quaternary amine. Gel retardation assay was performed to optimize the ratio of DNA to LAE in nanocarrier. Serum stability, haemolysis, cytotoxicity, and transfection studies were carried out to evaluate APX. Binding of DNA to APX was found to be stable in the presence of serum, and no degradation of DNA was observed. APX containing 2 mg/ml LAE which exhibited particle size of 72 nm with zeta potential of +57.5 mV, showed lower cytotoxicity and better transfection. APX can be a promising carrier for gene delivery.

  10. 'Over-under' myringoplasty with umbus-anchored graft.

    PubMed

    Fiorino, F; Barbieri, F

    2008-08-01

    'Over-under' myringoplasty is a versatile and effective surgical technique for tympanic membrane repair. The main drawbacks are possible trauma to the inner ear, due to manipulation of the malleus, and graft detachment from the apical portion of the malleus during the healing process, with consequent reduction of sound transfer function. To obviate these disadvantages, we have modified the over-under myringoplasty by maintaining anchorage of the tympanic membrane to the umbus. A total of 78 umbus-anchored over-under myringoplasties were performed from 2004 to 2006 in 63 patients. After elevation of the tympanomeatal flap, the malleus was freed from the tympanic membrane in a superior to inferior direction, up to the region immediately superior to the umbus. A large graft with a radial slit was distended under the tympanic remnants and annulus, and the two tongues were positioned to surround the umbus area and overlapped under a non-perforated portion of the tympanic membrane. Graft take was obtained in 71 ears (91 per cent). The auditory results showed an average residual air-bone gap of 6.7 dB, which was significantly better (p = 0.04) in comparison to that obtained in ears undergoing traditional over-under myringoplasty (11.9 dB). Modification of the over-under myringoplasty by anchoring the graft to the umbus preserves both lever and catenary effects of the tympano-ossicular system, reduces traumatising manoeuvres during dissection of the tympanic membrane from the malleus, and yields excellent results in terms of graft take and auditory outcome.

  11. [Arthroscopic refixation of TFCC by bone screw anchor].

    PubMed

    Schmelzer-Schmied, N

    2016-08-01

    The goal of this operation technique is a stable refixation of the triangular fibrocartilage complex (TFCC) to the fovea ulnaris. The stability of the distal radio-ulnar joint (DRUJ) should be re-established. The patients pain and the feeling of instability should be reduced. Lesions of the foveal component of the TFCC resulting in DRUJ instability. Combined lesions of both components of the TFCC. Complete detachment of the TFCC from the ulna either without fracture of the styloid process of the ulna or with fracture (floating styloid). Severe lacerations of the TFCC and clinically relevant arthrosis of the DRUJ. Severely osteoporotic bone. Following diagnostic arthroscopy and performance of stability control of the TFCC with a palpation hook, reduction of the DRUJ with supination position of the wrist. Bone anchor fixation through the direct foveal portal (DF). Under arthroscopic control through the 3/4 portal, the suture from the DF portal is placed through the TFCC. Pull out and tie the strands through the 6 U portal. Restriction of rotation of the forearm in a Munster cast or special cast brace for 6 weeks. Self-controlled exercise of the wrist after 6 weeks. Physiotherapy and strength building 8 weeks postoperatively. Clinical studies of this technique showed a significant amelioration of pain perception, improved range of motion and DASH score in all patients after anchor fixation. The results are comparable to other techniques. All patients returned to work after the operation. Accordingly, using this technique a very good stabilization of the DRUJ with low complications can be achieved.

  12. Electronic medical record phenotyping using the anchor and learn framework.

    PubMed

    Halpern, Yoni; Horng, Steven; Choi, Youngduck; Sontag, David

    2016-07-01

    Electronic medical records (EMRs) hold a tremendous amount of information about patients that is relevant to determining the optimal approach to patient care. As medicine becomes increasingly precise, a patient's electronic medical record phenotype will play an important role in triggering clinical decision support systems that can deliver personalized recommendations in real time. Learning with anchors presents a method of efficiently learning statistically driven phenotypes with minimal manual intervention. We developed a phenotype library that uses both structured and unstructured data from the EMR to represent patients for real-time clinical decision support. Eight of the phenotypes were evaluated using retrospective EMR data on emergency department patients using a set of prospectively gathered gold standard labels. We built a phenotype library with 42 publicly available phenotype definitions. Using information from triage time, the phenotype classifiers have an area under the ROC curve (AUC) of infection 0.89, cancer 0.88, immunosuppressed 0.85, septic shock 0.93, nursing home 0.87, anticoagulated 0.83, cardiac etiology 0.89, and pneumonia 0.90. Using information available at the time of disposition from the emergency department, the AUC values are infection 0.91, cancer 0.95, immunosuppressed 0.90, septic shock 0.97, nursing home 0.91, anticoagulated 0.94, cardiac etiology 0.92, and pneumonia 0.97. The resulting phenotypes are interpretable and fast to build, and perform comparably to statistically learned phenotypes developed with 5000 manually labeled patients. Learning with anchors is an attractive option for building a large public repository of phenotype definitions that can be used for a range of health IT applications, including real-time decision support. © The Author 2016. Published by Oxford University Press on behalf of the American Medical Informatics Association.

  13. Modified Simple Peritoneal Wall Anchor Technique (PWAT) in Peritoneal Dialysis.

    PubMed

    Oka, Hideaki; Yamada, Shunsuke; Kamimura, Taro; Hara, Masatoshi; Hirashima, Yutaro; Matsueda, Shumei; Shukuri, Tomoya; Aihara, Seishi; Koresawa, Mai; Eriguchi, Masahiro; Harada, Atsumi; Tsuruya, Kazuhiko; Kitazono, Takanari

    ♦ BACKGROUND: Outflow obstruction, a common complication in patients with peritoneal dialysis (PD), usually results in unnecessary catheter removal or replacement. This study describes a modified simple method of anchoring a PD catheter on the anterior peritoneal wall without using a laparoscopic system (peritoneal wall anchor technique, PWAT). ♦ METHODS: We performed a retrospective cohort study of consecutive PD catheter insertions, and compared the catheter survival rate between the traditional method and the modified simple PWAT. The traditional method was used in 54 cases and the modified simple PWAT was used in 17 cases. The primary endpoint was the occurrence of surgical catheter repair because of outflow obstruction by day 365. The secondary endpoint was the occurrence of catheter migration with obstruction requiring any interventions, including the alpha-replacement method by day 365. Catheter survival was analyzed by Kaplan-Meier survival curves. ♦ RESULTS: Migration-free catheter survival was significantly (p = 0.02) higher in the PWAT group (100%, 17/17) than in the traditional group (72.2%, 39/54). Catheter survival without surgical repair or cessation of PD was also significantly (p = 0.04) higher in the PWAT group (100%, 17/17) than in the traditional group (77.8%, 42/54). Similarly, migration-free and surgery-free catheter survival rates in cases with a straight-type catheter in the PWAT group were significantly higher than those in cases with a straight-type catheter in the traditional group. ♦ CONCLUSIONS: Our results suggest that the modified simple PWAT provides a better catheter survival rate than the traditional method by preventing catheter migration with obstruction in PD. Copyright © 2017 International Society for Peritoneal Dialysis.

  14. Video Observations of Anchor Ice/Sediment Interactions in Two Rocky Mountain Streams

    NASA Astrophysics Data System (ADS)

    Kempema, E. W.; McGee, B. W.; Pueblitz, L.

    2004-12-01

    Anchor ice is submerged ice that is attached or anchored to the bottom of rivers, lakes, and shallow seas. It forms in turbulent, supercooled water. In rivers, anchor ice usually forms at night and is released from the riverbed in the morning as the sun warms the water column. Released anchor ice often contains significant amounts of coarse-grained sediment. This entrained sediment can be transported (ice rafted) long distances downstream before it is released from the buoyant, floating ice back to the riverbed. Understanding sediment entrainment into anchor ice requires knowledge of the processes occurring at the sediment/ice interface. Viewing this interface through a freezing water column is a difficult task. Removing anchor ice from the water usually destroys the fragile contact between the ice and entrained sediment. In this study, we used an underwater video system to document anchor ice/sediment interactions in two small Rocky Mountain rivers. The video system allowed us to view: (1) the contact between attached anchor ice and the streambed; (2) the distribution of sediment in attached and released anchor ice masses; (3) the release of anchor ice from the bed; and (4) ice rafting of sediment in released anchor ice masses. One of the greatest benefits of the video system is the ability to see the in situ sediment distribution in attached and recently released anchor ice. Analysis of the video reveals that gravel concentrations are high on the top and bottom surfaces of anchor ice masses with relatively little gravel in the interior. The video system is not perfect for viewing ice underwater, however. The small difference between the refractive indices of ice and water makes it very difficult to discern ice crystal shapes and the exact nature of ice/sediment contacts. We are continuing to develop ways to enhance the image quality of ice crystals in water. Even with this shortcoming, the underwater video system is a very useful tool for observing in situ anchor

  15. BEHAVIOR OF POST-INSTALLED ANCHORS TESTED BY STEPWISE INCREASING CYCLIC LOAD PROTOCOLS

    PubMed Central

    Mahrenholtz, Philipp; Eligehausen, Rolf; Hutchinson, Tara C.; Hoehler, Matthew S.

    2016-01-01

    Cyclic loads are a characteristic feature of actions acting on structures and anchorages during earthquakes. For this reason, seismic qualification of post-installed concrete anchors according to the internationally recognized American Concrete Institute (ACI) standard ACI 355 is based on cyclic load tests. The protocols for these tests, however, have limited scientific basis. Therefore, in the present paper newly-developed test protocols with stepwise-increasing load amplitudes are utilized to more realistically evaluate anchor seismic performance. The study focuses on the load-displacement behavior of common anchor types installed in cracked concrete and subjected to both cyclic tension and cyclic shear actions. The results confirmed robust behavior for anchors loaded in cyclic tension even in the presence of crack widths in the anchorage material larger than currently required by ACI 355. In addition, the critical influence of low cycle fatigue on the performance of anchors loaded in cyclic shear is demonstrated. PMID:27890969

  16. Development and validation of European guidelines for seismic qualification of post-installed anchors.

    PubMed

    Mahrenholtz, Philipp; Wood, Richard L; Eligehausen, Rolf; Hutchinson, Tara C; Hoehler, Matthew S

    2017-10-01

    This paper presents the technical background for the seismic qualification procedures for post-installed anchors in the European Technical Approval Guideline (ETAG 001) seismic annex issued in 2013. We discuss requirements for a comprehensive guideline and reference supporting documentation. Numerical studies to generate new simulated seismic protocols for anchors are summarized with focus on their application to Europe. To reduce the time and cost of anchor product qualification testing while fulfilling the requirement of European building codes to assess two performance categories, we combine the results of our numerical studies to generate novel testing protocols that allow for the assessment of anchor behavior at multiple levels in a unified protocol. Validation tests demonstrate that the unified protocol results in anchor performance comparable with that achieved in multiple, single-performance-level tests.

  17. Treatment of photoaged skin with topical tretinoin increases epidermal-dermal anchoring fibrils

    SciTech Connect

    Woodley, D.T.; Briggaman, R.A.; Zelickson, A.S.

    1990-06-13

    Topical 0.1% tretinoin or vehicle control was applied daily to the forearm skin of six caucasian adults for 4 months. Two-millimeter punch biopsy specimens were obtained from treatment sites at the beginning and end of the study period for electron microscopy. Anchoring fibrils within the epidermal-dermal junction of skin treatment sites were quantitated by blinded, standardized, computer-assisted morphometry. After 4 months of continual daily treatment, skin sites that received topical tretinoin showed double the anchoring fibril density compared with vehicle control sites. The possible mechanism by which topical tretinoin increases anchoring fibrils in skin include the drug's property of inhibitingmore » collagenase, a dermal enzyme that degrades anchoring fibril collagen. The authors speculate that increased numbers of collagenous anchoring fibrils within the papillary dermis of human skin is one of the connective-tissue correlates of the clinical improvement observed in photoaged skin after treatment with topical tretinoin.« less

  18. The detrimental consequences for seagrass of ineffective marine park management related to boat anchoring.

    PubMed

    La Manna, G; Donno, Y; Sarà, G; Ceccherelli, G

    2015-01-15

    Posidonia oceanica (L.) Delile meadows are recognized as priority habitat for conservation by the EU Habitats Directive. The La Maddalena Archipelago National Park (Mediterranean Sea) P. oceanica meadow, the dominant coastal habitat of the area, is mostly threatened by boat anchoring. 12 years after the establishment of mooring fields and anchoring restrictions, a study was conducted to measure their effectiveness on the conservation of seagrass and the mitigation of anchoring damage. We found that: (i) the condition of P. oceanica was disturbed, both in the mooring fields and in control locations; (ii) mooring fields and anchoring restrictions did not show to be an efficient system for the protection of seagrass, in fact anchor scars increased after the tourist season; (iii) the mooring systems had an impact on the surrounding area of the meadow, probably due to their misuse. On the basis of these results, management recommendations for marine parks are proposed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Activation of Duck RIG-I by TRIM25 Is Independent of Anchored Ubiquitin

    PubMed Central

    Miranzo-Navarro, Domingo; Magor, Katharine E.

    2014-01-01

    Retinoic acid inducible gene I (RIG-I) is a viral RNA sensor crucial in defense against several viruses including measles, influenza A and hepatitis C. RIG-I activates type-I interferon signalling through the adaptor for mitochondrial antiviral signaling (MAVS). The E3 ubiquitin ligase, tripartite motif containing protein 25 (TRIM25), activates human RIG-I through generation of anchored K63-linked polyubiquitin chains attached to lysine 172, or alternatively, through the generation of unanchored K63-linked polyubiquitin chains that interact non-covalently with RIG-I CARD domains. Previously, we identified RIG-I of ducks, of interest because ducks are the host and natural reservoir of influenza viruses, and showed it initiates innate immune signaling leading to production of interferon-beta (IFN-β). We noted that K172 is not conserved in RIG-I of ducks and other avian species, or mouse. Because K172 is important for both mechanisms of activation of human RIG-I, we investigated whether duck RIG-I was activated by TRIM25, and if other residues were the sites for attachment of ubiquitin. Here we show duck RIG-I CARD domains are ubiquitinated for activation, and ubiquitination depends on interaction with TRIM25, as a splice variant that cannot interact with TRIM25 is not ubiquitinated, and cannot be activated. We expressed GST-fusion proteins of duck CARD domains and characterized TRIM25 modifications of CARD domains by mass spectrometry. We identified two sites that are ubiquitinated in duck CARD domains, K167 and K193, and detected K63 linked polyubiquitin chains. Site directed mutagenesis of each site alone, does not alter the ubiquitination profile of the duck CARD domains. However, mutation of both sites resulted in loss of all attached ubiquitin and polyubiquitin chains. Remarkably, the double mutant duck RIG-I CARD still interacts with TRIM25, and can still be activated. Our results demonstrate that anchored ubiquitin chains are not necessary for TRIM25

  20. Protein Kinase A, Ca2+/Calmodulin-Dependent Kinase II, and Calcineurin Regulate the Intracellular Trafficking of Myopodin between the Z-Disc and the Nucleus of Cardiac Myocytes▿

    PubMed Central

    Faul, Christian; Dhume, Ashwini; Schecter, Alison D.; Mundel, Peter

    2007-01-01

    Spatial and temporal resolution of intracellular signaling can be achieved by compartmentalizing transduction units. Myopodin is a dual-compartment, actin-bundling protein that shuttles between the nucleus and the Z-disc of myocytes in a differentiation- and stress-dependent fashion. Importin α binding and nuclear import of myopodin are regulated by serine/threonine phosphorylation-dependent binding of myopodin to 14-3-3. Here we show that in the heart myopodin forms a Z-disc signaling complex with α-actinin, calcineurin, Ca2+/calmodulin-dependent kinase II (CaMKII), muscle-specific A-kinase anchoring protein, and myomegalin. Phosphorylation of myopodin by protein kinase A (PKA) or CaMKII mediates 14-3-3 binding and nuclear import in myoblasts. Dephosphorylation of myopodin by calcineurin abrogates 14-3-3β binding. Activation of PKA or inhibition of calcineurin in adult cardiac myocytes releases myopodin from the Z-disc and induces its nuclear import. The identification of myopodin as a direct target of PKA, CaMKII, and calcineurin defines a novel intracellular signaling pathway whereby changes in Z-disc dynamics may translate into compartmentalized signal transduction in the heart. PMID:17923693

  1. Are Rib Versus Spine Anchors Protective Against Breakage of Growing Rods?

    PubMed

    Yamaguchi, Kent T; Skaggs, David L; Mansour, Shaun; Myung, Karen S; Yazici, Muharram; Johnston, Charles; Thompson, George; Sponseller, Paul; Akbarnia, Behrooz A; Vitale, Michael G

    2014-11-01

    Retrospective multicenter, case-control study. To compare the risks of rod breakage and anchor complications between distraction-based growing rods with proximal spine versus rib anchors. Rod breakage is a known complication of distraction-based growing rod instrumentation. A total of 176 patients met inclusion criteria: minimum 2-year follow-up, younger than age 9 years at index surgery, non-Vertical Expandable Prosthetic Titanium Rib distraction-based growing rods, and known anchor locations. Mean follow-up was 56 months (range, 24-152 months). Survival analyses using Cox proportional hazards model (accounting for varying lengths of follow-up) of rod breakage, anchor complications, preoperative Cobb angle, number of growing rods, age, and number of levels instrumented were performed using a significance level of p < .05. Thirty-four patients had rib-anchored growing rods and 142 had spine-anchored growing rods. This analysis found that proximal rib-anchored growing rods have a 23% risk of lifetime rod breakage compared with spine-anchored growing rods (6% vs. 29%) (p = .041) without a significant increase in risk of anchor complications (38% vs. 33%) (p = .117). The number of implanted rods (p = .839), age (p = .649), and number of instrumented levels (p = .447) were not statistically significant regarding rod breakage risk, although higher preoperative Cobb angles were significant (p = .014). Preoperative Cobb angle appears to be the most influential factor in determining whether growing rods break (p = .014). Univariate analysis found that rib anchors were associated with less than one-fourth the risk of rod breakage than spine anchors (p = .04) but multivariate analysis found no significant association between anchors and rod breakage (p = .07). This trend suggests that rib-anchored growing rod systems may be associated with less rod breakage because the system is less rigid as a result of some "slop" at the hook-rib interface, as well as the normal motion

  2. Attaching transmitters to waterbirds using one versus two subcutaneous anchors: Retention and survival trade-offs

    USGS Publications Warehouse

    Lewis, Tyler; Esler, Daniel N.; Uher-Koch, Brian D.; Dickson, Rian D.; Anderson, Eric M.; Evenson, Joseph R.; Hupp, Jerry; Flint, Paul L.

    2017-01-01

    A major challenge of wildlife telemetry is choosing an attachment technique that maximizes transmitter retention while minimizing negative side effects. For waterbirds, attachment of transmitters with subcutaneous anchors has been an effective and well-established technique, having been used on >40 species. This method was recently modified to include a second subcutaneous anchor, presumably increasing transmitter retention beyond that of single-anchor attachments. This putative benefit may be offset, however, by increased health risks related to additional incisions and subcutaneous protrusions. To test this potential trade-off, we attached radiotransmitters to molting and wintering surf (Melanitta perspicillata) and white-winged scoters (M. fusca) during 2008 and 2009 in Washington State and southeast Alaska, USA, using single- (121 scoters) and double-anchor (128 scoters) attachment techniques. We estimated daily probabilities of survival and radio retention for each group, this being apparent retention for wintering scoters because we could not differentiate shed transmitters from flighted emigration. For scoters during the flightless remigial molt, we found that addition of a second anchor increased cumulative retention probability (±SE) over a 49-day period from 0.69 ± 0.11 for single-anchor to 0.88 ± 0.07 for double-anchor attachments, while having no effect on survival. However, during winter, scoters with double-anchor attachments experienced no improvement in apparent retention, while having significantly lower survival during their first 14 days following transmitter attachment; of 15 mortalities during this period, 11 had 2 subcutaneous anchors. From day 15 onward, winter survival rates were nearly identical for single- versus double-anchor attachments, indicating that adverse effects of subcutaneous anchors were mainly limited to the 14-day postattachment period. Overall, given that the survival cost of adding a second subcutaneous anchor

  3. Examining the Impact of Covariates on Anchor Tests to Ascertain Quality over Time in a College Admissions Test

    ERIC Educational Resources Information Center

    Wiberg, Marie; von Davier, Alina A.

    2017-01-01

    We propose a comprehensive procedure for the implementation of a quality control process of anchor tests for a college admissions test with multiple consecutive administrations. We propose to examine the anchor tests and their items in connection with covariates to investigate if there was any unusual behavior in the anchor test results over time…

  4. Linking Composite Scores: Effects of Anchor Test Length and Content Representativeness. Research Report. ETS RR-16-36

    ERIC Educational Resources Information Center

    Lin, Peng; Dorans, Neil; Weeks, Jonathan

    2016-01-01

    The nonequivalent groups with anchor test (NEAT) design is frequently used in test score equating or linking. One important assumption of the NEAT design is that the anchor test is a miniversion of the 2 tests to be equated/linked. When the content of the 2 tests is different, it is not possible for the anchor test to be adequately representative…

  5. Career Anchors and the Effects of Downsizing: Implications for Generations and Cultures at Work. A Preliminary Investigation.

    ERIC Educational Resources Information Center

    Marshall, Verena; Bonner, Dede

    2003-01-01

    The relationships among career anchors, age, culture, gender, employment experience, and the impact of career planning on downsizing were examined with data from 423 management students (49% had been downsized). Lifestyle was the most valued anchor across age groups, stability/security the least; compared with Schein's earlier anchors research,…

  6. The Place of White in a World of Grays: A Double-Anchoring Theory of Lightness Perception

    ERIC Educational Resources Information Center

    Bressan, Paola

    2006-01-01

    The specific gray shades in a visual scene can be derived from relative luminance values only when an anchoring rule is followed. The double-anchoring theory I propose in this article, as a development of the anchoring theory of Gilchrist et al. (1999), assumes that any given region (a) belongs to one or more frameworks, created by Gestalt…

  7. Hierarchical assembly of centriole subdistal appendages via centrosome binding proteins CCDC120 and CCDC68.

    PubMed

    Huang, Ning; Xia, Yuqing; Zhang, Donghui; Wang, Song; Bao, Yitian; He, Runsheng; Teng, Junlin; Chen, Jianguo

    2017-04-19

    In animal cells, the centrosome is the main microtubule-organizing centre where microtubules are nucleated and anchored. The centriole subdistal appendages (SDAs) are the key structures that anchor microtubules in interphase cells, but the composition and assembly mechanisms of SDAs are not well understood. Here, we reveal that centrosome-binding proteins, coiled-coil domain containing (CCDC) 120 and CCDC68 are two novel SDA components required for hierarchical SDA assembly in human cells. CCDC120 is anchored to SDAs by ODF2 and recruits CEP170 and Ninein to the centrosome through different coiled-coil domains at its N terminus. CCDC68 is a CEP170-interacting protein that competes with CCDC120 in recruiting CEP170 to SDAs. Furthermore, CCDC120 and CCDC68 are required for centrosome microtubule anchoring. Our findings elucidate the molecular basis for centriole SDA hierarchical assembly and microtubule anchoring in human interphase cells.

  8. Determining the mechanical properties of electrospun poly-ε-caprolactone (PCL) nanofibers using AFM and a novel fiber anchoring technique.

    PubMed

    Baker, Stephen R; Banerjee, Soham; Bonin, Keith; Guthold, Martin

    2016-02-01

    Due to its low cost, biocompatibility and slow bioresorption, poly-ε-caprolactone (PCL) continues to be a suitable material for select biomedical engineering applications. We used a combined atomic force microscopy (AFM)/optical microscopy technique to determine key mechanical properties of individual electrospun PCL nanofibers with diameters between 440-1040nm. Compared to protein nanofibers, PCL nanofibers showed much lower adhesion, as they slipped on the substrate when mechanically manipulated. We, therefore, first developed a novel technique to anchor individual PCL nanofibers to micrometer-sized ridges on a substrate, and then mechanically tested anchored nanofibers. When held at constant strain, tensile stress relaxed with fast and slow relaxation times of 1.0±0.3s and 8.8±3.1s, respectively. The total tensile modulus was 62±26MPa, the elastic (non-relaxing) component of the tensile modulus was 53±36MPa. Individual PCL fibers could be stretched elastically (without permanent deformation) to strains of 19-23%. PCL nanofibers are rather extensible; they could be stretched to a strain of at least 98%, and a tensile strength of at least 12MPa, before they slipped off the AFM tip. PCL nanofibers that had aged for over a month at ambient conditions became stiffer and less elastic. Our technique provides accurate nanofiber mechanical data, which are needed to guide construction of scaffolds for cells and other biomedical devices. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Modulation of non steroidal anti-inflammatory drug induced membrane fusion by copper coordination of these drugs: anchoring effect.

    PubMed

    Majumdar, Anupa; Chakraborty, Sreeja; Sarkar, Munna

    2014-12-04

    Membrane fusion, an integral event in several biological processes, is characterized by several intermediate steps guided by specific energy barriers. Hence, it requires the aid of fusogens to complete the process. Common fusogens, such as proteins/peptides, have the ability to overcome theses barriers by their conformational reorganization, an advantage not shared by small drug molecules. Hence, drug induced fusion at physiologically relevant drug concentrations is rare and occurs only in the case of the oxicam group of non steroidal anti-inflammatory drugs (NSAIDs). To use drugs to induce and control membrane fusion in various biochemical processes requires the understanding of how different parameters modulate fusion. Also, fusion efficacy needs to be enhanced. Here we have synthesized and used Cu(II) complexes of fusogenic oxicam NSAIDs, Meloxicam and Piroxicam, to induce fusion in model membranes monitored by using DSC, TEM, steady-state, and time-resolved spectroscopy. The ability of the complexes to anchor apposing model membranes to initiate/facilitate fusion has been demonstrated. This results in better fusion efficacy compared to the bare drugs. These complexes can take the fusion to its final step. Unlike other designed membrane anchors, the role of molecular recognition and strength of interaction between molecular partners is obliterated for these preformed Cu(II)-NSAIDs.

  10. Surface engineered and ligand anchored nanobioconjugate: an effective therapeutic approach for oral insulin delivery in experimental diabetic rats.

    PubMed

    Sharma, Rajeev; Gupta, Umesh; Garg, Neeraj K; Tyagi, Rajeev K; Jain, N K

    2015-03-01

    The present study was designed to enhance intestinal absorption of insulin by nanobioconjugate formulated with PEGylation and Concanavalin A based targeted synergistic approach. The attempts were aimed at maximizing bioavailability and therapeutic efficacy of insulin by incorporating it in Concanavalin A anchored PEGylated nanoconstructs. The Con A anchored PEGylated PLGA diblock copolymer was synthesized by modified surface functionalization method, and was then characterized by FTIR and 1H NMR spectrum analysis. The nanoparticles from synthesized polymers were prepared and characterized for mean size and distribution by laser diffraction spectroscopy. The physicochemically characterized (by SEM and TEM) formulations were evaluated for optimum particle size, polydispersity index, zeta potential and entrapment efficiency 196.3±4.5 nm, 0.15±0.04, -25.6±1.68 and 44.6±3.5% respectively. The insulin encapsulation efficiency and in vitro release were assessed by bicinchoninic protein assay (BCA). The in vitro results corroborated in vivo studies carried out in experimentally created diabetic albino rats. The nano-encapsulated insulin was discovered to meet the requirements by achieving better stability, improved absorption and enhanced oral bioavailability elucidated by in vivo and in vitro bioassays. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Basic Values, Career Orientations, and Career Anchors: Empirical Investigation of Relationships

    PubMed Central

    Abessolo, Marc; Rossier, Jérôme; Hirschi, Andreas

    2017-01-01

    In today's dynamic and uncertain career context, values play an important role for career choice and lifelong career self-management. Values are desirable goals that are sought by individuals to satisfy their needs and are important for understanding career orientations in terms of protean and boundaryless career orientations and career anchors. However, how career orientations or career anchors fit into a well-established and supported model and into the structure of basic human values remains an important and under-investigated question. The aim of this study was to use Schwartz's model of structural values to empirically explore the relationships and structural correspondences among basic values, career orientations, and career anchors. A heterogeneous sample of 238 employees from French-speaking Switzerland (Mage = 35.60, SD = 13.03) completed the Portrait Values Questionnaire (PVQ5X), the Protean and Boundaryless Career Attitudes Scales (PCAS, BCAS), and the Career Orientation Inventory (COI) via an anonymous and confidential survey questionnaire. The results showed that it was possible to meaningfully position both career orientations and career anchors in Schwartz's values structure. The protean and boundaryless career orientations were positively related to Schwartz's basic values that emphasized openness to change and career anchors meaningfully followed the motivational continuum of these basic values. Overall, the overlap among the basic values, career orientations, and career anchors appeared relatively important, suggesting that these basic values, orientations, and anchors should be considered simultaneously to understand and address the factors and processes underlying individuals' career choices and paths. PMID:28955275

  12. Modified method for external attachment of transmitters to birds using two subcutaneous anchors

    USGS Publications Warehouse

    Lewis, T.L.; Flint, P.L.

    2008-01-01

    Of the transmitter attachment techniques for birds, the subcutaneous anchor provides a secure attachment that yields relatively few secondary effects. However, the use of subcutaneous anchors has been limited by transmitter size and retention time. Using a modified method of attachment that utilized two subcutaneous anchors, we deployed 69 GPS transmitters, plus 13 VHF transmitters that were similar in size and weight to GPS models, on Pacific Black Brant (Branta bernicla nigricans). Prior to our study, only harnesses were used for attaching GPS transmitters on birds, mainly because GPS transmitters are too large for other external attachment techniques and implantation in the body cavity attenuates the GPS signal. Thus, to increase the size capacity of anchor attachment and to avoid the well-documented negative effects of harnesses on behavior and survival, we added a second anchor at the transmitter's posterior end. The double-anchor attachment technique was quickly and easily accomplished in the field, requiring bird handling times of <10 min. Incidental recoveries of tagged Brant indicate a high degree of transmitter retention. Five recaptured birds (4-6 weeks after deployment) and eight killed by hunters (3-6 mo after deployment) retained their GPS transmitters. For studies involving the use of relatively large transmitters, the double-anchor method appears to provide a viable alternative for external attachment. ?? 2008 Association of Field Ornithologists.

  13. Basic Values, Career Orientations, and Career Anchors: Empirical Investigation of Relationships.

    PubMed

    Abessolo, Marc; Rossier, Jérôme; Hirschi, Andreas

    2017-01-01

    In today's dynamic and uncertain career context, values play an important role for career choice and lifelong career self-management. Values are desirable goals that are sought by individuals to satisfy their needs and are important for understanding career orientations in terms of protean and boundaryless career orientations and career anchors. However, how career orientations or career anchors fit into a well-established and supported model and into the structure of basic human values remains an important and under-investigated question. The aim of this study was to use Schwartz's model of structural values to empirically explore the relationships and structural correspondences among basic values, career orientations, and career anchors. A heterogeneous sample of 238 employees from French-speaking Switzerland (Mage = 35.60, SD = 13.03) completed the Portrait Values Questionnaire (PVQ5X), the Protean and Boundaryless Career Attitudes Scales (PCAS, BCAS), and the Career Orientation Inventory (COI) via an anonymous and confidential survey questionnaire. The results showed that it was possible to meaningfully position both career orientations and career anchors in Schwartz's values structure. The protean and boundaryless career orientations were positively related to Schwartz's basic values that emphasized openness to change and career anchors meaningfully followed the motivational continuum of these basic values. Overall, the overlap among the basic values, career orientations, and career anchors appeared relatively important, suggesting that these basic values, orientations, and anchors should be considered simultaneously to understand and address the factors and processes underlying individuals' career choices and paths.

  14. Follow Your Heart: How Is Willingness to Pay Formed under Multiple Anchors?

    PubMed Central

    Lin, Chien-Huang; Chen, Ming

    2017-01-01

    In sales, a common promotional tactic is to supplement a required purchase (i.e., a focal product) by offering a free or discounted product (i.e., a supplementary product). The present research examines the contextual factors driving consumer evaluations of the supplementary product after the promotion has been terminated. Two experiments are used to demonstrate that consumers use multiple anchors to determine the value of a supplementary product. Consumers use other types of price information, such as the internal reference price (IRP), promotional price, and original price of the supplementary product, as anchors to adjust their willingness to pay. Among the multiple anchors, the consumer’s IRP is not only the crucial anchor to estimate the willingness to pay but also the criterion to determine whether other price information can serve as anchors. Price information, such as the promotional and original price of the supplementary product, which is higher (lower) than the IRP, will increase (decrease) the willingness to pay. However, these anchors are only employed when the price information is considered to be plausible. Assimilation and contrast effects occur when the IRP is used by consumers as a criterion to judge the reasonableness of other anchors. When the external price information belongs (does not belong) to consumers’ distribution of IRP, assimilation (contrast) effects occur, and consumers will regard the external reference price (ERP) to be a plausible (implausible) price. Limitations and future avenues for research are also discussed. PMID:29312098

  15. Wireless and acoustic hearing with bone-anchored hearing devices

    PubMed Central

    Bosman, Arjan J.; Mylanus, Emmanuel A.M.; Hol, Myrthe K.S.; Snik, Ad F.M.

    2016-01-01

    Abstract Objective: The efficacy of wireless connectivity in bone-anchored hearing was studied by comparing the wireless and acoustic performance of the Ponto Plus sound processor from Oticon Medical relative to the acoustic performance of its predecessor, the Ponto Pro. Study sample: Nineteen subjects with more than two years' experience with a bone-anchored hearing device were included. Thirteen subjects were fitted unilaterally and six bilaterally. Design: Subjects served as their own control. First, subjects were tested with the Ponto Pro processor. After a four-week acclimatization period performance the Ponto Plus processor was measured. In the laboratory wireless and acoustic input levels were made equal. In daily life equal settings of wireless and acoustic input were used when watching TV, however when using the telephone the acoustic input was reduced by 9 dB relative to the wireless input. Results: Speech scores for microphone with Ponto Pro and for both input modes of the Ponto Plus processor were essentially equal when equal input levels of wireless and microphone inputs were used. Only the TV-condition showed a statistically significant (p <5%) lower speech reception threshold for wireless relative to microphone input. In real life, evaluation of speech quality, speech intelligibility in quiet and noise, and annoyance by ambient noise, when using landline phone, mobile telephone, and watching TV showed a clear preference (p <1%) for the Ponto Plus system with streamer over the microphone input. Due to the small number of respondents with landline phone (N = 7) the result for noise annoyance was only significant at the 5% level. Conclusion: Equal input levels for acoustic and wireless inputs results in equal speech scores, showing a (near) equivalence for acoustic and wireless sound transmission with Ponto Pro and Ponto Plus. The default 9-dB difference between microphone and wireless input when using the telephone results in a substantial

  16. Wireless and acoustic hearing with bone-anchored hearing devices.

    PubMed

    Bosman, Arjan J; Mylanus, Emmanuel A M; Hol, Myrthe K S; Snik, Ad F M

    2015-07-01

    The efficacy of wireless connectivity in bone-anchored hearing was studied by comparing the wireless and acoustic performance of the Ponto Plus sound processor from Oticon Medical relative to the acoustic performance of its predecessor, the Ponto Pro. Nineteen subjects with more than two years' experience with a bone-anchored hearing device were included. Thirteen subjects were fitted unilaterally and six bilaterally. Subjects served as their own control. First, subjects were tested with the Ponto Pro processor. After a four-week acclimatization period performance the Ponto Plus processor was measured. In the laboratory wireless and acoustic input levels were made equal. In daily life equal settings of wireless and acoustic input were used when watching TV, however when using the telephone the acoustic input was reduced by 9 dB relative to the wireless input. Speech scores for microphone with Ponto Pro and for both input modes of the Ponto Plus processor were essentially equal when equal input levels of wireless and microphone inputs were used. Only the TV-condition showed a statistically significant (p <5%) lower speech reception threshold for wireless relative to microphone input. In real life, evaluation of speech quality, speech intelligibility in quiet and noise, and annoyance by ambient noise, when using landline phone, mobile telephone, and watching TV showed a clear preference (p <1%) for the Ponto Plus system with streamer over the microphone input. Due to the small number of respondents with landline phone (N = 7) the result for noise annoyance was only significant at the 5% level. Equal input levels for acoustic and wireless inputs results in equal speech scores, showing a (near) equivalence for acoustic and wireless sound transmission with Ponto Pro and Ponto Plus. The default 9-dB difference between microphone and wireless input when using the telephone results in a substantial wireless benefit when using the telephone. The preference of

  17. Study of crack patterns in beam column joint due to upwards anchoring beam effect

    NASA Astrophysics Data System (ADS)

    Sabariman, Bambang; Sofianto, Mochamad Firmansyah

    2017-06-01

    Beam-column joint (BCJ) of reinforced concrete is an area of critical which must be carefully designed to absorb the seismic energy. Inability to absorb seismic energy will cause serious damage to structural failure. It will lead to shear failure, i.e. brittle and endanger users of the building if the BCJ area is not designed properly. In order to get a building structure is safe and resistant to disasters, mainly due to the earthquake, the structure must be designed in accordance to the rules of standard construction (eligible detailing). In general, the anchorages (detailing) reinforcement beams to the column is pointing downwards, however, what if the steel anchoring beams are pointing upwards. Different anchoring will result in the behavior of different structures, such as fracturing pattern. This study observes two anchoring model, by making two test specimens of BCJ. One set of reinforcement beams anchored BCJ upward and downward respectively, which are applied with monotonic static load. The study investigated the pattern of cracks and ductility of the beam displacement on BCJ. The results showed that the downward anchoring in the fracturing pattern out of the area BCJ was anchoring upward pattern rift goes deep into the area BCJ, thus anchoring above makes BCJ unsafe. Downward anchoring are more ductile than the upward anchoring, however the deflection of all BJC specimens was higher than the boundaries maximum deflection prediction (Δprediction = 10.45 mm < eksp Δexp-min = 13.15 mm), which can be assumed that all BCJ specimens were ductile, but it still in ductile limited due to displacement ductility μΔ<3,5.

  18. SYP73 Anchors the ER to the Actin Cytoskeleton for Maintenance of ER Integrity and Streaming in Arabidopsis.

    PubMed

    Cao, Pengfei; Renna, Luciana; Stefano, Giovanni; Brandizzi, Federica

    2016-12-05

    The endoplasmic reticulum (ER) is an essential organelle that spreads throughout the cytoplasm as one interconnected network of narrow tubules and dilated cisternae that enclose a single lumen. The ER network undergoes extensive remodeling, which critically depends on membrane-cytoskeleton interactions [1]. In plants, the ER is also highly mobile, and its streaming contributes significantly to the movement of other organelles [2, 3]. The remodeling and motility of the plant ER rely mainly on actin [4] and to a minor extent on microtubules [5]. Although a three-way interaction between the ER, cytosolic myosin-XI, and F-actin mediates the plant ER streaming [6], the mechanisms underlying stable interaction of the ER membrane with actin are unknown. Early electron microscopy studies suggested a direct attachment of the plant ER with actin filaments [7, 8], but it is plausible that yet-unknown proteins facilitate anchoring of the ER membrane with the cytoskeleton. We demonstrate here that SYP73, a member of the plant Syp7 subgroup of SNARE proteins [9] containing actin-binding domains, is a novel ER membrane-associated actin-binding protein. We show that overexpression of SYP73 causes a striking rearrangement of the ER over actin and that, similar to mutations of myosin-XI [4, 10, 11], loss of SYP73 reduces ER streaming and affects overall ER network morphology and plant growth. We propose a model for plant ER remodeling whereby the dynamic rearrangement and streaming of the ER network depend on the propelling action of myosin-XI over actin coupled with a SYP73-mediated bridging, which dynamically anchors the ER membrane with actin filaments. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Structure of the Membrane Anchor of Pestivirus Glycoprotein Erns, a Long Tilted Amphipathic Helix

    PubMed Central

    Aberle, Daniel; Muhle-Goll, Claudia; Bürck, Jochen; Wolf, Moritz; Reißer, Sabine; Luy, Burkhard; Wenzel, Wolfgang; Ulrich, Anne S.; Meyers, Gregor

    2014-01-01

    Erns is an essential virion glycoprotein with RNase activity that suppresses host cellular innate immune responses upon being partially secreted from the infected cells. Its unusual C-terminus plays multiple roles, as the amphiphilic helix acts as a membrane anchor, as a signal peptidase cleavage site, and as a retention/secretion signal. We analyzed the structure and membrane binding properties of this sequence to gain a better understanding of the underlying mechanisms. CD spectroscopy in different setups, as well as Monte Carlo and molecular dynamics simulations confirmed the helical folding and showed that the helix is accommodated in the amphiphilic region of the lipid bilayer with a slight tilt rather than lying parallel to the surface. This model was confirmed by NMR analyses that also identified a central stretch of 15 residues within the helix that is fully shielded from the aqueous layer, which is C-terminally followed by a putative hairpin structure. These findings explain the strong membrane binding of the protein and provide clues to establishing the Erns membrane contact, processing and secretion. PMID:24586172

  20. Structure of the membrane anchor of pestivirus glycoprotein E(rns), a long tilted amphipathic helix.

    PubMed

    Aberle, Daniel; Muhle-Goll, Claudia; Bürck, Jochen; Wolf, Moritz; Reißer, Sabine; Luy, Burkhard; Wenzel, Wolfgang; Ulrich, Anne S; Meyers, Gregor

    2014-02-01

    E(rns) is an essential virion glycoprotein with RNase activity that suppresses host cellular innate immune responses upon being partially secreted from the infected cells. Its unusual C-terminus plays multiple roles, as the amphiphilic helix acts as a membrane anchor, as a signal peptidase cleavage site, and as a retention/secretion signal. We analyzed the structure and membrane binding properties of this sequence to gain a better understanding of the underlying mechanisms. CD spectroscopy in different setups, as well as Monte Carlo and molecular dynamics simulations confirmed the helical folding and showed that the helix is accommodated in the amphiphilic region of the lipid bilayer with a slight tilt rather than lying parallel to the surface. This model was confirmed by NMR analyses that also identified a central stretch of 15 residues within the helix that is fully shielded from the aqueous layer, which is C-terminally followed by a putative hairpin structure. These findings explain the strong membrane binding of the protein and provide clues to establishing the E(rns) membrane contact, processing and secretion.

  1. Dynamic Rewiring of Promoter-Anchored Chromatin Loops during Adipocyte Differentiation.

    PubMed

    Siersbæk, Rasmus; Madsen, Jesper Grud Skat; Javierre, Biola Maria; Nielsen, Ronni; Bagge, Emilie Kristine; Cairns, Jonathan; Wingett, Steven William; Traynor, Sofie; Spivakov, Mikhail; Fraser, Peter; Mandrup, Susanne

    2017-05-04

    Interactions between transcriptional promoters and their distal regulatory elements play an important role in transcriptional regulation; however, the extent to which these interactions are subject to rapid modulations in response to signals is unknown. Here, we use promoter capture Hi-C to demonstrate a rapid reorganization of promoter-anchored chromatin loops within 4 hr after inducing differentiation of 3T3-L1 preadipocytes. The establishment of new promoter-enhancer loops is tightly coupled to activation of poised (histone H3 lysine 4 mono- and dimethylated) enhancers, as evidenced by the acquisition of histone H3 lysine 27 acetylation and the binding of MED1, SMC1, and P300 proteins to these regions, as well as to activation of target genes. Intriguingly, formation of loops connecting activated enhancers and promoters is also associated with extensive recruitment of corepressors such as NCoR and HDACs, indicating that this class of coregulators may play a previously unrecognized role during enhancer activation. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. New OprM structure highlighting the nature of the N-terminal anchor.

    PubMed

    Monlezun, Laura; Phan, Gilles; Benabdelhak, Houssain; Lascombe, Marie-Bernard; Enguéné, Véronique Y N; Picard, Martin; Broutin, Isabelle

    2015-01-01

    Among the different mechanisms used by bacteria to resist antibiotics, active efflux plays a major role. In Gram-negative bacteria, active efflux is carried out by tripartite efflux pumps that form a macromolecular assembly spanning both membranes of the cellular wall. At the outer membrane level, a well-conserved outer membrane factor (OMF) protein acts as an exit duct, but its sequence varies greatly among different species. The OMFs share a similar tri-dimensional structure that includes a beta-barrel pore domain that stabilizes the channel within the membrane. In addition, OMFs are often subjected to different N-terminal post-translational modifications (PTMs), such as an acylation with a lipid. The role of additional N-terminal anchors is all the more intriguing since it is not always required among the OMFs family. Understanding this optional PTM could open new research lines in the field of antibiotics resistance. In Escherichia coli, it has been shown that CusC is modified with a tri-acylated lipid, whereas TolC does not show any modification. In the case of OprM from Pseudomonas aeruginosa, the N-terminal modification remains a matter of debate, therefore, we used several approaches to investigate this issue. As definitive evidence, we present a new X-ray structure at 3.8 Å resolution that was solved in a new space group, making it possible to model the N-terminal residue as a palmitoylated cysteine.

  3. Membrane-anchored human Rab GTPases directly mediate membrane tethering in vitro.

    PubMed

    Tamura, Naoki; Mima, Joji

    2014-10-31

    Rab GTPases are master regulators of eukaryotic endomembrane systems, particularly functioning in membrane tethering to confer the directionality of intracellular membrane trafficking. However, how exactly Rab GTPases themselves act upon membrane tethering processes has remained enigmatic. Here, we thoroughly tested seven purified Rab GTPases in human, which localize at the various representative organelles, for their capacity to support membrane tethering in vitro. Strikingly, we found that three specific human Rabs (endoplasmic reticulum/Golgi Rab2a, early endosomal Rab5a, and late endosomal/lysosomal Rab7a) strongly accelerated membrane aggregation of synthetic liposomes even in the absence of any additional components, such as classical tethers, tethering factors, and Rab effectors. This Rab-induced membrane aggregation was a reversible membrane tethering reaction that can be strictly controlled by the membrane recruitment of Rab proteins on both apposing membranes. Thus, our current reconstitution studies establish that membrane-anchored human Rab GTPases are an essential tethering factor to directly mediate membrane tethering events. © 2014. Published by The Company of Biologists Ltd.

  4. Membrane-anchored human Rab GTPases directly mediate membrane tethering in vitro

    PubMed Central

    Tamura, Naoki; Mima, Joji

    2014-01-01

    ABSTRACT Rab GTPases are master regulators of eukaryotic endomembrane systems, particularly functioning in membrane tethering to confer the directionality of intracellular membrane trafficking. However, how exactly Rab GTPases themselves act upon membrane tethering processes has remained enigmatic. Here, we thoroughly tested seven purified Rab GTPases in human, which localize at the various representative organelles, for their capacity to support membrane tethering in vitro. Strikingly, we found that three specific human Rabs (endoplasmic reticulum/Golgi Rab2a, early endosomal Rab5a, and late endosomal/lysosomal Rab7a) strongly accelerated membrane aggregation of synthetic liposomes even in the absence of any additional components, such as classical tethers, tethering factors, and Rab effectors. This Rab-induced membrane aggregation was a reversible membrane tethering reaction that can be strictly controlled by the membrane recruitment of Rab proteins on both apposing membranes. Thus, our current reconstitution studies establish that membrane-anchored human Rab GTPases are an essential tethering factor to directly mediate membrane tethering events. PMID:25361579

  5. Structural Basis for a Safety-Belt Mechanism That Anchors Condensin to Chromosomes.

    PubMed

    Kschonsak, Marc; Merkel, Fabian; Bisht, Shveta; Metz, Jutta; Rybin, Vladimir; Hassler, Markus; Haering, Christian H

    2017-10-19

    Condensin protein complexes coordinate the formation of mitotic chromosomes and thereby ensure the successful segregation of replicated genomes. Insights into how condensin complexes bind to chromosomes and alter their topology are essential for understanding the molecular principles behind the large-scale chromatin rearrangements that take place during cell divisions. Here, we identify a direct DNA-binding site in the eukaryotic condensin complex, which is formed by its Ycg1 Cnd3 HEAT-repeat and Brn1 Cnd2 kleisin subunits. DNA co-crystal structures reveal a conserved, positively charged groove that accommodates the DNA double helix. A peptide loop of the kleisin subunit encircles the bound DNA and, like a safety belt, prevents its dissociation. Firm closure of the kleisin loop around DNA is essential for the association of condensin complexes with chromosomes and their DNA-stimulated ATPase activity. Our data suggest a sophisticated molecular basis for anchoring condensin complexes to chromosomes that enables the formation of large-sized chromatin loops. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. Suture locking of isolated internal locking knotless suture anchors is not affected by bone quality

    PubMed Central

    Woodmass, Jarret M; Matthewson, Graeme; Ono, Yohei; Bois, Aaron J; Boorman, Richard S; Lo, Ian KY; Thornton, Gail M

    2015-01-01

    Purpose The purpose of this study was to evaluate the mechanical performance of different suture locking mechanisms including: i) interference fit between the anchor and the bone (eg, 4.5 mm PushLock, 5.5 mm SwiveLock), ii) internal locking mechanism within the anchor itself (eg, 5.5 mm SpeedScrew), or iii) a combination of interference fit and internal locking (eg, 4.5 mm MultiFIX P, 5.5 mm MultiFIX S). Methods Anchors were tested in foam blocks representing normal (20/8 foam) or osteopenic (8/8 foam) bone, using standard suture loops pulled in-line with the anchor to isolate suture locking. Mechanical testing included cyclic testing for 500 cycles from 10 N to 60 N at 60 mm/min, followed by failure testing at 60 mm/min. Displacement after 500 cycles at 60 N, number of cycles at 3 mm displacement, load at 3 mm displacement, and maximum load were evaluated. Results Comparing 8/8 foam to 20/8 foam, load at 3 mm displacement and maximum load were significantly decreased (P<0.05) with decreased bone quality for anchors that, even in part, relied on an interference fit suture locking mechanism (ie, 4.5 mm PushLock, 5.5 mm SwiveLock, 4.5 mm MultiFIX P, 5.5 mm MultiFIX S). Bone quality did not affect the mechanical performance of 5.5 mm SpeedScrew anchors which have an isolated internal locking mechanism. Conclusion The mechanical performance of anchors that relied, even in part, on interference fit were affected by bone quality. Isolated internal locking knotless suture anchors functioned independently of bone quality. Anchors with a combined type (interference fit and internal locking) suture locking mechanism demonstrated similar mechanical performance to isolated internal locking anchors in osteopenic foam comparing similar sized anchors. Clinical relevance In osteopenic bone, knotless suture anchors that have an internal locking mechanism (isolated or combined type) may be advantageous for secure tendon fixation to bone. PMID:26124683

  7. Autonomous Landing and Smart Anchoring for In-Situ Exploration of Small Bodies

    NASA Technical Reports Server (NTRS)

    Ghavimi, Ali R.; Serricchio, Frederick; Hadaegh, Fred Y.; Dolgin, Ben

    2000-01-01

    Future NASA missions include in-situ scientific explorations of small interplanetary objects like comets and asteroids. Sample acquisition systems are envisioned to operate directly from the landers that are anchored to the surface. Landing and anchoring proves to be challenging in the absence of an attitude control system and in the presence of nearly zero-gravity environments with uncertain surface terrain and unknown mechanical properties. This paper presents recent advancements in developing a novel landing and anchoring control system for the exploration of small bodies.

  8. Collagenous microstructure of the glenoid labrum and biceps anchor

    PubMed Central

    Hill, A M; Hoerning, E J; Brook, K; Smith, C D; Moss, J; Ryder, T; Wallace, A L; Bull, A M J

    2008-01-01

    The glenoid labrum is a significant passive stabilizer of the shoulder joint. However, its microstructural form remains largely unappreciated, particularly in the context of its variety of functions. The focus of labral microscopy has often been histology and, as such, there is very little appreciation of collagen composition and arrangement of the labrum, and hence the micromechanics of the structure. On transmission electron microscopy, significant differences in diameter, area and perimeter were noted in the two gross histological groups of collagen fibril visualized; this suggests a heterogeneous collagenous composition with potentially distinct mechanical function. Scanning electron microscopy demonstrated three distinct zones of interest: a superficial mesh, a dense circumferential braided core potentially able to accommodate hoop stresses, and a loosely packed peri-core zone. Confocal microscopy revealed an articular surface fine fibrillar mesh potentially able to reduce surface friction, bundles of circumferential encapsulated fibres in the bulk of the tissue, and bone anchoring fibres at the osseous interface. Varying microstructure throughout the depth of the labrum suggests a role in accommodating different types of loading. An understanding of the labral microstructure can lead to development of hypotheses based upon an appreciation of this component of material property. This may aid an educated approach to surgical timing and repair. PMID:18429974

  9. Corrected high-frame rate anchored ultrasound with software alignment.

    PubMed

    Miller, Amanda L; Finch, Kenneth B

    2011-04-01

    To improve lingual ultrasound imaging with the corrected high frame rate anchored ultrasound with software alignment (CHAUSA; Miller, 2008) 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 data collection paths, using dominant ultrasound technology and the CHAUSA method, are compared. The probe stabilization and head movement correction paradigm is demonstrated. The CHAUSA method increases the frame rate from the standard National Television System Committee (NTSC) video rate (29.97) to the ultrasound internal machine rate--in this case, 124 frames per second (fps)--by using Digital Imaging and Communications in Medicine (DICOM; National Electrical Manufacturers Association, 2008) data transfer. DICOM avoids spatiotemporal inaccuracies introduced by dominant ultrasound export techniques. The data display alignment of the acoustic and articulatory signals to the correct high-frame rate (FR) frame (± 4 ms at 124 fps). CHAUSA produces high-FR, high-spatial-quality ultrasound images, which are head corrected to 1 mm. The method reveals tongue dorsum retraction during the posterior release of the alveolar click and tongue tip recoil following the anterior release of the alveolar click, both of which were previously undetectable. CHAUSA visualizes most of the tongue in studies of dynamic consonants with a major reduction in field problems, opening up important areas of speech research.

  10. Financial fluctuations anchored to economic fundamentals: A mesoscopic network approach.

    PubMed

    Sharma, Kiran; Gopalakrishnan, Balagopal; Chakrabarti, Anindya S; Chakraborti, Anirban

    2017-08-14

    We demonstrate the existence of an empirical linkage between nominal financial networks and the underlying economic fundamentals, across countries. We construct the nominal return correlation networks from daily data to encapsulate sector-level dynamics and infer the relative importance of the sectors in the nominal network through measures of centrality and clustering algorithms. Eigenvector centrality robustly identifies the backbone of the minimum spanning tree defined on the return networks as well as the primary cluster in the multidimensional scaling map. We show that the sectors that are relatively large in size, defined with three metrics, viz., market capitalization, revenue and number of employees, constitute the core of the return networks, whereas the periphery is mostly populated by relatively smaller sectors. Therefore, sector-level nominal return dynamics are anchored to the real size effect, which ultimately shapes the optimal portfolios for risk management. Our results are reasonably robust across 27 countries of varying degrees of prosperity and across periods of market turbulence (2008-09) as well as periods of relative calmness (2012-13 and 2015-16).

  11. Magnetic resonance imaging and bone anchored hearing implants: pediatric considerations.

    PubMed

    Doshi, Jayesh; Schneiders, Sara; Foster, Katherine; Reid, Andrew; McDermott, Ann Louise

    2014-02-01

    Recent developments in bone conduction hearing systems have seen the introduction of transcutaneous devices comprising of magnetic components. Our aim was to identify the number of children implanted with a traditional, non-magnetic percutaneous bone anchored hearing implant (BAHI) who would not have been eligible for a transcutaneous implant based on magnetic resonance imaging (MRI) need. A retrospective case review of 206 children who had a percutaneous BAHI at the Birmingham Children's Hospital (January 2009-October 2012) for auditory rehabilitation. Twenty-eight percent (56/206) of children required at least one MRI scan after receiving a BAHI and 10 percent of patients (20/206) required two or more MRI scans. The main indication for MRI scanning was for neurological co-morbidities; a MRI brain was the most common scan performed. Although transcutaneous hearing devices/middle ear implants have their clear benefits, it may be argued that these relatively more invasive surgical procedures may not be the best option for the child who will require MRI scanning at some point in the future. Clinicians should be mindful of any need for MRI scanning when considering implant choices in the pediatric population. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  12. Gold nanoparticles assembled with dithiocarbamate-anchored molecular wires

    PubMed Central

    Reeler, Nini E. A.; Lerstrup, Knud A.; Somerville, Walter; Speder, Jozsef; Petersen, Søren V.; Laursen, Bo W.; Arenz, Matthias; Qiu, Xiaohui; Vosch, Tom; Nørgaard, Kasper

    2015-01-01

    A protocol for the bottom-up self-assembly of nanogaps is developed through molecular linking of gold nanoparticles (AuNPs). Two π-conjugated oligo(phenylene ethynylene) molecules (OPE) with dithiocarbamate anchoring groups are used as ligands for the AuNPs. OPE-4S with a dithiocarbamate in each end of the molecule and a reference molecule OPE-2S with only a single dithiocarbamate end group. The linking mechanism of OPE-4S is investigated by using a combination of TEM, UV-Vis absorption and surface enhanced Raman spectroscopy (SERS) as well as studying the effect of varying the OPE-4S to AuNP concentration ratio. UV-Vis absorption confirms the formation of AuNP aggregates by the appearance of an extended plasmon band (EPB) for which the red shift and intensity depend on the OPE-4S:AuNP ratio. SERS confirms the presence of OPE-4S and shows a gradual increase of the signal intensity with increasing OPE-4S:AuNP ratios up to a ratio of about 4000, after which the SERS intensity does not increase significantly. For OPE-2S, no linking is observed below full coverage of the AuNPs indicating that the observed aggregate formation at high OPE-2S:AuNP ratios, above full AuNP coverage, is most likely of a physical nature (van der Waals forces or π-π interactions). PMID:26471461

  13. Dynamics of anchor last deployment of submersible buoy system

    NASA Astrophysics Data System (ADS)

    Zheng, Zhongqiang; Xu, Jianpeng; Huang, Peng; Wang, Lei; Yang, Xiaoguang; Chang, Zongyu

    2016-02-01

    Submersible buoy systems are widely used for oceanographic research, ocean engineering and coastal defense. Severe sea environment has obvious effects on the dynamics of submersible buoy systems. Huge tension can occur and may cause the snap of cables, especially during the deployment period. This paper studies the deployment dynamics of submersible buoy systems with numerical and experimental methods. By applying the lumped mass approach, a three-dimensional multi-body model of submersible buoy system is developed considering the hydrodynamic force, tension force and impact force between components of submersible buoy system and seabed. Numerical integration method is used to solve the differential equations. The simulation output includes tension force, trajectory, profile and dropping location and impact force of submersible buoys. In addition, the deployment experiment of a simplified submersible buoy model was carried out. The profile and different nodes' velocities of the submersible buoy are obtained. By comparing the results of the two methods, it is found that the numerical model well simulates the actual process and conditions of the experiment. The simulation results agree well with the results of the experiment such as gravity anchor's location and velocities of different nodes of the submersible buoy. The study results will help to understand the conditions of submersible buoy's deployment, operation and recovery, and can be used to guide the design and optimization of the system.

  14. Ultrasonically implanted PLA suture anchors are stable in osteopenic bone.

    PubMed

    Meyer, D C; Mayer, J; Weber, U; Mueller, A; Koch, P P; Gerber, C

    2006-01-01

    We compared the in vitro pullout strengths of a metallic screw-type suture anchor (5-mm Corkscrew) and a new ultrasonically implanted absorbable pin from cadaveric humeral heads with different bone densities. They were assessed quantitatively using microcomputed tomography. Pullout tests were done at four standardized sites and then correlated with the local bone density. The mean pullout strengths for the 5-mm Corkscrew and the 3.5-mm polylactic pin were similar in weak bone (76 +/- 24 N versus 104 +/- 46 N), intermediate quality bone (194 +/- 81 N versus 218 +/- 76 N), and strong bone (349 +/- 127 N versus 325 +/- 100 N). Pullout strength correlated with bone density for both implants (r = 0.76 and r = 0.86 for the Corkscrew and polylactic pin, respectively). The ultrasonically implanted absorbable 3.5-mm polylactic pin achieved equal pullout strength in weak bone as the larger 5-mm titanium Corkscrew. Bone density is highly variable in humeral heads and influenced the pullout strength of both implants.

  15. Collagenous microstructure of the glenoid labrum and biceps anchor.

    PubMed

    Hill, A M; Hoerning, E J; Brook, K; Smith, C D; Moss, J; Ryder, T; Wallace, A L; Bull, A M J

    2008-06-01

    The glenoid labrum is a significant passive stabilizer of the shoulder joint. However, its microstructural form remains largely unappreciated, particularly in the context of its variety of functions. The focus of labral microscopy has often been histology and, as such, there is very little appreciation of collagen composition and arrangement of the labrum, and hence the micromechanics of the structure. On transmission electron microscopy, significant differences in diameter, area and perimeter were noted in the two gross histological groups of collagen fibril visualized; this suggests a heterogeneous collagenous composition with potentially distinct mechanical function. Scanning electron microscopy demonstrated three distinct zones of interest: a superficial mesh, a dense circumferential braided core potentially able to accommodate hoop stresses, and a loosely packed peri-core zone. Confocal microscopy revealed an articular surface fine fibrillar mesh potentially able to reduce surface friction, bundles of circumferential encapsulated fibres in the bulk of the tissue, and bone anchoring fibres at the osseous interface. Varying microstructure throughout the depth of the labrum suggests a role in accommodating different types of loading. An understanding of the labral microstructure can lead to development of hypotheses based upon an appreciation of this component of material property. This may aid an educated approach to surgical timing and repair.

  16. Defining a Key Receptor-CheA Kinase Contact and Elucidating Its Function in the Membrane-Bound Bacterial Chemosensory Array: A Disulfide Mapping and TAM-IDS Study

    PubMed Central

    Piasta, Kene N.; Ulliman, Caleb J.; Slivka, Peter F.; Crane, Brian R.; Falke, Joseph J.

    2013-01-01

    The three core components of the ubiquitous bacterial chemosensory array – the transmembrane chemoreceptor, the histidine kinase CheA and the adaptor protein CheW – assemble to form a membrane-bound, hexagonal lattice in which receptor transmembrane signals regulate kinase activity. Both the regulatory domain of the kinase and the adaptor protein bind to overlapping sites on the cytoplasmic tip of the receptor (termed the protein interaction region). Notably, the kinase regulatory domain (P5) and the adaptor protein share the same fold constructed of two SH3-like domains. The present study focuses on the structural interface between the receptor and the kinase regulatory domain. Two models have been proposed for this interface: Model 1 is based on the crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheW adaptor protein. This model has been used in current models of chemosensory array architecture to build the receptor-CheA kinase interface. Model 2 is based on a newly determined crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheA kinase regulatory domain. Both models present unique strengths and weaknesses, and current evidence is unable to resolve which model best describes contacts in the native chemosensory arrays of Escherichia coli, Salmonella typhimurium and other bacteria. Here we employ disulfide mapping and TAM-IDS (Tryptophan and Alanine Mutation to Identify Docking Sites) to test Models 1 and 2 in well-characterized membrane-bound arrays formed from E. coli and S. typhimurium components. The results reveal that the native array interface between the receptor protein interaction region and the kinase regulatory domain is accurately described by Model 2, but not by Model 1. In addition, the results show that the interface possesses both a structural function that contributes to stable CheA kinase binding in the array, and a regulatory function central to transmission of

  17. Enhancement of polar anchoring strength in a graphene-nematic suspension and its effect on nematic electro-optic switching

    NASA Astrophysics Data System (ADS)

    Basu, Rajratan

    2017-07-01

    A small quantity of monolayer graphene flakes is doped in a nematic liquid crystal (LC), and the effective polar anchoring strength coefficient between the LC and the alignment substrate is found to increase by an order of magnitude. The hexagonal pattern of graphene can interact with the LC's benzene rings via π -π electron stacking, enabling the LC to anchor to the graphene surface homogeneously (i.e., planar anchoring). When the LC cell is filled with the graphene-doped LC, some graphene flakes are preferentially attached to the alignment layer and modify the substrate's anchoring property. These spontaneously deposited graphene flakes promote planar anchoring at the substrate and the polar anchoring energy at alignment layer is enhanced significantly. The enhanced anchoring energy is found to impact favorably on the electro-optic response of the LC. Additional studies reveal that the nematic electro-optic switching is significantly faster in the LC-graphene hybrid than that of the pure LC.

  18. Glycosyl Phosphatidylinositol Anchor Biosynthesis Is Essential for Maintaining Epithelial Integrity during Caenorhabditis elegans Embryogenesis

    PubMed Central

    Zaidel-Bar, Ronen

    2015-01-01

    Glycosylphosphatidylinositol (GPI) is a post-translational modification resulting in the attachment of modified proteins to the outer leaflet of the plasma membrane. Tissue culture experiments have shown GPI-anchored proteins (GPI-APs) to be targeted to the apical membrane of epithelial cells. However, the in vivo importance of this targeting has not been investigated since null mutations in GPI biosynthesis enzymes in mice result in very early embryonic lethality. Missense mutations in the human GPI biosynthesis enzyme pigv are associated with a multiple congenital malformation syndrome with a high frequency of Hirschsprung disease and renal anomalies. However, it is currently unknown how these phenotypes are linked to PIGV function. Here, we identify a temperature-sensitive hypomorphic allele of PIGV in Caenorhabditis elegans, pigv-1(qm34), enabling us to study the role of GPI-APs in development. At the restrictive temperature we found a 75% reduction in GPI-APs at the surface of embryonic cells. Consequently, ~80% of pigv-1(qm34) embryos arrested development during the elongation phase of morphogenesis, exhibiting internal cysts and/or surface ruptures. Closer examination of the defects revealed them all to be the result of breaches in epithelial tissues: cysts formed in the intestine and excretory canal, and ruptures occurred through epidermal cells, suggesting weakening of the epithelial membrane or membrane-cortex connection. Knockdown of piga-1, another GPI biosynthesis enzymes resulted in similar phenotypes. Importantly, fortifying the link between the apical membrane and actin cortex by overexpression of the ezrin/radixin/moesin ortholog ERM-1, significantly rescued cyst formation and ruptures in the pigv-1(qm34) mutant. In conclusion, we discovered GPI-APs play a critical role in maintaining the integrity of the epithelial tissues, allowing them to withstand the pressure and stresses of morphogenesis. Our findings may help to explain some of the

  19. Electron cryomicroscopy structure of a membrane-anchored mitochondrial AAA protease.

    PubMed

    Lee, Sukyeong; Augustin, Steffen; Tatsuta, Takashi; Gerdes, Florian; Langer, Thomas; Tsai, Francis T F

    2011-02-11

    FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo- and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo- and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides. These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases.

  20. Dynamic Effects of Anchor Positional Tolerance on Tension Moored Floating Wind Turbine

    NASA Astrophysics Data System (ADS)

    Wright, Christopher; Pakrashi, Vikram; Murphy, Jimmy

    2016-09-01

    For water depths greater than 60m floating wind turbines will become the most economical option for generating offshore wind energy. Tension mooring stabilised units are one type of platform being considered by the offshore wind energy industry. The complex mooring arrangement used by this type of platform means that the dynamics are greatly effected by offsets in the positioning of the anchors. This paper examines the issue of tendon anchor position tolerances. The dynamic effects of three positional tolerances are analysed in survival state using the time domain FASTLink. The severe impact of worst case anchor positional offsets on platform and turbine survivability is shown. The worst anchor misposition combinations are highlighted and should be strongly avoided. Novel methods to mitigate this issue are presented.

  1. PROBLEMS ABOUT THE DESIGN STAN DARD OF THE EXTERNAL FRICTION TYPE ANCHOR AND THE IMPROVEMENT PLAN

    NASA Astrophysics Data System (ADS)

    Sesaki, Sigeru; Hamano, Hiroki

    It has been about 50 years since ground anchorages were introduced to Japan. The number of permanent anchors constructed has already reached ab out 100,000 a year. Howeve r, an increasing number of corrupted anchors have been reported recently. If th ere is a problem in the design of anchorages, one of its possible causes is the yet-to-b e-clarified mechanism of the anchors' adhesion to the bedrock, in particular. In this study, we conducted a detailed examination on the behavior of the anchor body in relation to the pseudo bedrock and examined support mechanism of anchorages. Based on its results, we suggest improvements for the designing standard of the anchorages.

  2. Compartment A125, view of after end of anchor windlass. Windlass ...

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

    Compartment A-125, view of after end of anchor windlass. Windlass is steam powered; note cylinder at center right of photograph. (031) - USS Olympia, Penn's Landing, 211 South Columbus Boulevard, Philadelphia, Philadelphia County, PA

  3. Two novel self-assemblies of supramolecular solar cells using N-heterocyclic-anchoring porphyrins

    NASA Astrophysics Data System (ADS)

    Zhang, Qian; Wu, Fang-Yuan; Liu, Jia-Cheng; Li, Ren-Zhi; Jin, Neng-Zhi

    2018-02-01

    Two novel N-substituted anchoring porphyrins (ZnPAtz and ZnPAim) have been devised and synthesized. Moreover, these two anchoring porphyrins were linked to the TiO2 semiconductor through carboxyl groups and then a zinc porphyrin ZnP was bound to the anchoring porphyrin using a zinc-to-ligand axial coordination approach. The different performances of these assemblies were compared with single anchoring porphyrin devices ZnPAtz and ZnPAim. The photoelectric conversion efficiency of the new supramolecular solar cells sensitized by ZnP-ZnPAx (x = tz, im) has been improved. The ZnP-ZnPAtz-based DSSCs provided the highest photovoltaic efficiency (1.86%). Fundamental studies showed that incorporation of these assemblies promote light-harvesting efficiency.

  4. Suture slippage in knotless suture anchors resulting in subacromial-subdeltoid bursitis.

    PubMed

    Hayeri, Mohammad Reza; Keefe, Daniel T; Chang, Eric Y

    2016-05-01

    Rotator cuff repair using a suture bridge and knotless suture anchors is a relatively new, but increasingly used technique. The suture bridge technique creates an anatomically similar and more secure rotator cuff repair compared with conventional arthroscopic techniques and the use of knotless anchors eliminates the challenges associated with knot tying during arthroscopic surgery. However, previous in vitro biomechanical tests have shown that the hold of the suture in a knotless suture anchor is far lower than the pullout strength of the anchor from bone. Up until now slippage has been a theoretical concern. We present a prospectively diagnosed case of in vivo suture loosening after rotator cuff repair using a knotless bridge technique resulting in subacromial-subdeltoid bursitis.

  5. Targeting of liposomes to cells expressing CD4 using glycosylphosphatidylinositol-anchored gp120. Influence of liposome composition on intracellular trafficking.

    PubMed

    Schreier, H; Moran, P; Caras, I W

    1994-03-25

    To test the concept that glycosylphosphatidylinositol (GPI)-anchored proteins might be useful as targeting molecules for liposomes, we engineered a GPI-anchored form of gp120 from human immunodeficiency virus type 1 (termed gp120DAF) using the GPI signal of decay-accelerating factor (DAF). We show that (i) purified gp120DAF spontaneously inserts into liposome membranes via the GPI anchor; (ii) liposomes bearing gp120DAF bind specifically to cells expressing CD4, the cellular receptor for gp120; and (iii) the receptor-bound liposomes are internalized and recycle in Chinese hamster ovary cells. To test whether the lipid composition of the liposome affects any of these processes, we compared small unilamellar liposomes containing only phosphatidylcholine and cholesterol in a 7:1 molar ratio with artificial viral envelopes that mimic the lipid composition of human immunodeficiency virus type 1. We show that when tagged with gp120DAF, both liposome preparations bind specifically to cells expressing CD4, and both are endocytosed. However, artificial viral envelope liposomes are transported to late endosomes or lysosomes in the cell interior, whereas phosphatidylcholine:cholesterol liposomes are confined to a population of vesicles that remain close to the plasma membrane. Since the binding and internalization of both liposome preparations are mediated by the same receptor, we conclude that the lipid composition of the liposome profoundly influences the subsequent intracellular trafficking of the liposome-receptor complex.

  6. New benzimidazoles and their antitumor effects with Aurora A kinase and KSP inhibitory activities.

    PubMed

    Abd El-All, Amira S; Magd-El-Din, Asmaa A; Ragab, Fatma A F; ElHefnawi, Mahmoud; Abdalla, Mohamed M; Galal, Shadia A; El-Rashedy, Ahmed A

    2015-07-01

    A newly synthesized series of anticancer compounds comprising thiazolo[3,2-a]pyrimidine derivatives 6a-q bearing a benzimidazole moiety was produced via a one-pot reaction of N-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-2-cyanoacetamide 5 with 2-aminothiazole and an appropriate aromatic aldehyde. Compound 7 was obtained via the reaction of 4-(1H-benzo[d]imidazol-2yl)benzenamide 1 with carbon disulphide and methyl iodide in the presence of concentrated aqueous solution of NaOH, then treated with o-phenylenediamine to give N-(4-1H-benzo[d]imidazol-2-yl)phenyl)-1H-benzo[d]imidazol-2-amine 8. The structures of the newly synthesized compounds were confirmed by analytical and spectroscopic measurements (IR, MS, and (1) H NMR). The synthesized products were screened and studied for their in vitro antitumor activity against three human cancer cell lines (namely colorectal cancer cell line HCT116, human liver cancer cell line HepG2, and human ovarian cancer cell line A2780) and their Aurora A kinase and KSP inhibitory activities. All newly synthesized compounds revealed marked results comparable with the standard drug CK0106023. The compounds 6e and 6k of the thiazolopyrimidine derivatives were the most active compounds when tested against the three cell lines in comparison with the standard drug CK0106023, and showed potent dual KSP and Aurora A kinase inhibition. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Archaeometallurgical investigation of the iron anchor from the Tantura F shipwreck

    SciTech Connect

    Aronson, A.; Ashkenazi, D., E-mail: dana@eng.tau.ac.il; Barkai, O.

    2013-04-15

    The Tantura F shipwreck was a coaster or a fishing vessel about 15.7 m long, discovered in the Dor/Tantura lagoon, Israel in 1995. It was dated to between the mid-7th and the end of the 8th centuries CE. Among the finds excavated were two T-shaped type iron anchors. Of the two anchors, one (anchor A) was thoroughly studied by archaeometallurgical methods in order to identify forge-welding lines, to determine the welding quality and to understand the manufacturing technology. The examinations included X-ray radiography, XRF analysis, optical microscopy, SEM/EDS observation and analysis, OES analysis and microhardness tests. The investigation included characterizationmore » of the composition, microstructure, thermal treatments, forge-welding junctions and slag analysis. The results revealed a heterogeneous microstructure, rich in glassy, fayalite and wüstite slag. Iron based phases included ferrite, pearlite, cementite and Widmanstätten plates, all typical to wrought iron. The forge-welds of Anchor A were located. Each arm was made of one piece, weighing about 2.5–3 kg and the shank was made of a few 1.5–2 kg pieces. The second anchor (anchor B) was only briefly examined visually and with a few radiographs, which support the results from anchor A. The research results revealed significant information about T-shaped anchors and their manufacturing process, including hot-working processes without any additional heat treatments, and folding techniques. The microstructure was similar to other ancient simple tools such as saws, sickles, axes and mortise chisels, and though the technology to make complicated structures and objects, such as swords, existed at that time, the anchors did not require this sophistication; thus simpler techniques were used, presumably because they were more cost-effective. - Highlights: ► Tantura F was a coaster dated to mid-7th–end-8th centuries. ► Two iron anchors were discovered at the Tantura F shipwreck-site. ► Anchor A was

  8. Contact area and pressure in suture bridge rotator cuff repair using knotless lateral anchors.

    PubMed

    Tompkins, Marc; Monchik, Keith O; Plante, Matthew J; Fleming, Braden C; Fadale, Paul D

    2011-10-01

    To evaluate whether the use of knotless lateral anchors in a suture bridge construct produces better contact area and pressure parameters than a suture bridge construct with standard lateral anchors that require knots or a double-row repair. The hypothesis was that knotless lateral anchors would produce better contact area and pressure parameters than the other two constructs. A total of fifteen matched pairs of cadaveric shoulders were divided into three groups. In Group 1, a suture bridge using knotless anchors for the lateral row was performed on five shoulders. A suture bridge using standard lateral row anchors that require knots was performed on the contralateral shoulders. In Group 2, suture bridge with knotless lateral row anchors was compared with double-row repair. In Group 3, suture bridge using standard lateral row anchors was compared with double-row repair. The contact conditions of the rotator cuff footprint were measured using pressure-sensitive film. There were no statistically significant differences between any of the techniques regarding contact area F(2, 15.7) = 3.09, P = 0.07 or mean contact pressure F(2, 15.1) = 2.35, P = 0.12. A post hoc power analysis suggests differences between techniques are likely less than 91-113 mm(2) for area and 0.071-0.089 N for pressure. The use of knotless anchors in the lateral row of a suture bridge repair did not increase the footprint contact area or contact pressure when compared to a suture bridge repair requiring knots laterally or to a double-row repair.

  9. Fragile X gene instability: anchoring AGGs and linked microsatellites.

    PubMed Central

    Zhong, N; Yang, W; Dobkin, C; Brown, W T

    1995-01-01

    Interspersed AGGs within the FMR1 gene CGG repeat region may anchor the sequence and prevent slippage during replication. In order to detect the AGG position variations, we developed a method employing partial MnlI restriction analysis and analyzed X chromosomes from 187 males, including 133 normal controls (117 with 20-34 and 16 with 35-52 repeats), plus 54 fragile X premutations with 56-180 repeats. Among controls, the interspersed AGG positions were highly polymorphic, with a heterozygosity of 91%. Among the control samples, 1.5% had no AGG positions, 25% had one, 71% had two, and 3% had three. Among the fragile X premutation samples, 63% had no AGG, while 37% had only one AGG. Analysis of premutation samples within fragile X families showed that variation occurred only within the 3' end of the region. Thus, the instability was polar. Controls with > or = 15 pure CGG repeats were associated with the longest alleles of two nearby microsatellites, FRAXAC1 with 20-21 repeats and DXS548 with 202-206 bp and with increased microsatellite heterozygosity. The association of long pure CGG regions, as with fragile X chromosomes, with the longer and more heterozygous microsatellite alleles suggests they may be related mechanistically. Further, our results do not support a recent suggestion that the frequency of fragile X alleles may be increasing. Finally, analysis of a set of nonhuman primate samples showed that long pure CGG tracks are variable in size and are located within the 3' region, which suggests that polar instability within FMR1 is evolutionarily quite old. Images Figure 1 PMID:7668261

  10. Effect of Class III bone anchor treatment on airway.

    PubMed

    Nguyen, Tung; De Clerck, Hugo; Wilson, Michael; Golden, Brent

    2015-07-01

    To compare airway volumes and minimum cross-section area changes of Class III patients treated with bone-anchored maxillary protraction (BAMP) versus untreated Class III controls. Twenty-eight consecutive skeletal Class III patients between the ages of 10 and 14 years (mean age, 11.9 years) were treated using Class III intermaxillary elastics and bilateral miniplates (two in the infra-zygomatic crests of the maxilla and two in the anterior mandible). The subjects had cone beam computed tomographs (CBCTs) taken before initial loading (T1) and 1 year out (T2). Twenty-eight untreated Class III patients (mean age, 12.4 years) had CBCTs taken and cephalograms generated. The airway volumes and minimum cross-sectional area measurements were performed using Dolphin Imaging 11.7 3D software. The superior border of the airway was defined by a plane that passes through the posterior nasal spine and basion, while the inferior border included the base of the epiglottis to the lower border of C3. From T1 to T2, airway volume from BAMP-treated subjects showed a statistically significant increase (1499.64 mm(3)). The area in the most constricted section of the airway (choke point) increased slightly (15.44 mm(2)). The airway volume of BAMP patients at T2 was 14136.61 mm(3), compared with 14432.98 mm(3) in untreated Class III subjects. Intraexaminer correlation coefficients values and 95% confidence interval values were all greater than .90, showing a high degree of reliability of the measurements. BAMP treatment did not hinder the development of the oropharynx.

  11. Molecular beacon anchored onto a graphene oxide substrate

    NASA Astrophysics Data System (ADS)

    Darbandi, Arash; Datta, Debopam; Patel, Krunal; Lin, Gary; Stroscio, Michael A.; Dutta, Mitra

    2017-09-01

    In this article, we report a graphene oxide-based nanosensor incorporating semiconductor quantum dots linked to DNA-aptamers that functions as a ‘turn-off’ fluorescent nanosensor for detection of low concentrations of analytes. A specific demonstration of this turn-off aptasensor is presented for the case of the detection of mercury (II) ions. In this system, ensembles of aptamer-based quantum-dot sensors are anchored onto graphene oxide (GO) flakes which provide a platform for analyte detection in the vicinity of GO. Herein, the operation of this ensemble-based nanosensor is demonstrated for mercury ions, which upon addition of mercury, quenching of the emission intensity from the quantum dots is observed due to resonance energy transfer between quantum dots and the gold nanoparticle connected via a mercury target aptamer. A key result is that the usually dominant effect of quenching of the quantum dot due to close proximity to the GO can be reduced to negligible levels by using a linker molecule in conjunctions with the aptamer-based nanosensor. The effect of ionic concentration of the background matrix on the emission intensity was also investigated. The sensor system is found to be highly selective towards mercury and exhibits a linear behavior (r 2 > 0.99) in the nanomolar concentration range. The detection limit of the sensor towards mercury with no GO present was found to be 16.5 nM. With GO attached to molecular beacon via 14 base, 35 base, and 51 base long linker DNA, the detection limit was found to be 38.4 nM, 9.45 nM, and 11.38 nM; respectively.

  12. Universal microfluidic platform for bioassays in anchored droplets.

    PubMed

    Amselem, Gabriel; Guermonprez, Cyprien; Drogue, Benoît; Michelin, Sébastien; Baroud, Charles N

    2016-10-18

    In spite of the large number of droplet-based microfluidic tools that have appeared in recent years, their penetration into non-specialist labs remains limited to a small number of applications. This is partly due to the lack of a generic platform that integrates all of the necessary operations for end-users, and partly to the increasing complexity that emerges as several operations are combined together. Here we report the development of a platform that provides the capabilities of multiwell plates in a two-dimensional array of nanoliter droplets: encapsulation, time-resolved monitoring and variation of well contents, as well as the ability to selectively extract the contents of any of the wells. We demonstrate these capabilities by encapsulating thousands of individual bacterial cells in droplets that are stored on a two-dimensional array of surface-energy anchors. Bacterial culture can be performed either in liquid or hydrogel droplets, both of which allow precise quantification using either standard measurements or digital enumeration. Using hydrogels allows the removal of the external oil that surrounds the aqueous drops, for instance in order to apply a gradient of antibiotics across the droplet population. This defines a protocol to obtain an antibiogram in a single experiment. Finally, the liquid to gel transition provides a robust way to selectively extract any droplet from the array, by melting it with a focused laser. When combined with further off-chip culture or genotyping, this platform provides a unique culturing environment to relate phenotype and genotype measurements on monoclonal colonies.

  13. Evolutionary modularity and morphological integration in the haptoral anchor structures of Ligophorus spp. (Monogenea: Dactylogyridae).

    PubMed

    Rodríguez-González, A; Míguez-Lozano, R; Sarabeev, V; Balbuena, J A

    2016-09-01

    An important question in the study of phenotypic evolution is whether characters are independent of each other or behave and evolve as integrated modules. Morphological integration and modularity provide a powerful framework for the analysis of the evolution of morphological traits. We used geometric morphometrics and phylogenetically independent contrasts (PIC) to test four different modularity hypotheses in the haptoral anchors of 14 monogenean species of Ligophorus. Integration between the modular units identified was further evaluated with two-block partial least squares analysis. Roots and points represented two modules in the dorsal and ventral anchors, but modularity was not statistically supported when parasite phylogeny was accounted for, which may indicate convergent evolution related to host characteristics and gill morphology. In contrast, PIC revealed medial and lateral modules in ventral anchors only. Moreover, we found evidence for ventral and dorsal anchor pairs forming two modules, supporting the notion that they play different functional roles. Integration between all identified modules was strong. We conclude that there is modular structure in the anchors of Ligophorus spp., accounted by adaptive and phylogenetic factors acting at different levels, and ventral and dorsal anchors evolve as integrated modules with specific roles in attachment.

  14. Study on interaction between soil and anchor chain with finite element method

    NASA Astrophysics Data System (ADS)

    Li, Sa; Xu, Bao-zhao; Wu, Yun-zhou; Li, Zhong-gang

    2016-12-01

    With the development of offshore engineering, deeply embedded anchors are needed to be penetrated to appreciable depth and attached at the pad-eye. The interaction between anchor chain and soil is a very complex process and has not been thoroughly understood yet. In this paper, the finite element method (FEM) was used to study the interaction of soil-chain system. Results of the analysis show that when the attachment point is at a shallow depth, the load-development characteristics of the chain from FEM are in good agreement with that from the model tests and theoretical analysis. But with the depth increment, the results are different obviously in different methods. This phenomenon is resulted from a variety of reasons, and the plastic zone around the chain was studied to try finding the mechanism behind it. It could be seen that the plastic zone extended in different modes at different depths of attachment points. The interaction between the soil and anchor chain makes the load acting on the anchor decrease, but the soil disturbed surrounding the chain increases the anchor failure possibility. When the anchor bearing capacity is evaluated, these two factors should be considered properly at the same time.

  15. Caisson foundations as alternative anchors for permanent mooring of a process barge offshore Congo

    SciTech Connect

    Colliat, J.L.; Boisard, P.; Andersen, K.

    1995-12-01

    Shallow caisson foundations are considered as alternative anchors for permanent mooring of a process barge at an oil and gas field developed by ELF Congo in the Gulf of Guinea. The 12 mooring lines are scheduled to be installed on site in July 1995 and the process barge will be connected in 1996 for start of production. With a water depth of about 170m, this field will be the deepest one offshore Western Africa to date. The soils at the site consist of soft normally consolidated clays. The paper presents the results of a comparative study of potential anchor solutionsmore » for this production barge, including high capacity drag anchors, driven piles, and shallow caisson foundations installed by underpressure. This comparative study, including design, construction, and installation of the mooring system, has shown that the caisson foundations represent the best suited solution, technically as well as on an economical point of view, because of the following advantages: (1) vertical load capability, thus allowing to reduce the anchoring pattern, (2) once positioned on the seabed, the location of each anchor is fixed and known with accuracy, and (3) more simple and shorter installation procedure, since caisson anchors, as piles, do not require to be proof-loaded on site.« less

  16. Bone-anchored hearing aids in children and young adults: the Freeman Hospital experience.

    PubMed

    Ramakrishnan, Y; Marley, S; Leese, D; Davison, T; Johnson, I J M

    2011-02-01

    To investigate the utilisation of bone-anchored hearing aids and Softband, as well as the effects on quality of life, amongst the paediatric and young adult population of Freeman Hospital, Newcastle Upon Tyne, UK. Retrospective, anonymised, cross-sectional survey using the Glasgow Benefit Inventory and Listening Situation Questionnaire (parent version), administered at least three months following the start of bone-anchored hearing aid or Softband use. One hundred and nine patients were included, of whom syndromic children made up a significant proportion (22 of 109). Patients using bone-anchored hearing aids obtained significant educational and social benefit from their aids. The mean Listening Situation Questionnaire difficulty score was 17 (15 patients), which is below the trigger score of 22+ at which further reassessment and rehabilitation is required. 87% (of 15 patients) did not require further intervention. The overall mean GBI score for the 22 patients (syndromic and non-syndromic) was +29. The use of bone-anchored hearing aids and Softband results in significant improvements in quality of life for children and young adults with hearing impairment. There is significant under-utilisation of bone-anchored hearing aids in children with skull and congenital abnormalities, and we would advocate bone-anchored hearing aid implantation for these patients.

  17. The late endosome/lysosome-anchored p18-mTORC1 pathway controls terminal maturation of lysosomes

    SciTech Connect

    Takahashi, Yusuke; Nada, Shigeyuki; Mori, Shunsuke

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer p18 is a membrane adaptor that anchors mTORC1 to late endosomes/lysosomes. Black-Right-Pointing-Pointer We examine the role of the p18-mTORC1 pathway in lysosome biogenesis. Black-Right-Pointing-Pointer The loss of p18 causes accumulation of intact late endosomes by arresting lysosome maturation. Black-Right-Pointing-Pointer Inhibition of mTORC1 activity with rapamycin phenocopies the defects of p18 loss. Black-Right-Pointing-Pointer The p18-mTORC1 pathway plays crucial roles in the terminal maturation of lysosomes. -- Abstract: The late endosome/lysosome membrane adaptor p18 (or LAMTOR1) serves as an anchor for the mammalian target of rapamycin complex 1 (mTORC1) and is required for its activation on lysosomes. The loss ofmore » p18 causes severe defects in cell growth as well as endosome dynamics, including membrane protein transport and lysosome biogenesis. However, the mechanisms underlying these effects on lysosome biogenesis remain unknown. Here, we show that the p18-mTORC1 pathway is crucial for terminal maturation of lysosomes. The loss of p18 causes aberrant intracellular distribution and abnormal sizes of late endosomes/lysosomes and an accumulation of late endosome specific components, including Rab7, RagC, and LAMP1; this suggests that intact late endosomes accumulate in the absence of p18. These defects are phenocopied by inhibiting mTORC1 activity with rapamycin. Loss of p18 also suppresses the integration of late endosomes and lysosomes, resulting in the defective degradation of tracer proteins. These results suggest that the p18-mTORC1 pathway plays crucial roles in the late stages of lysosomal maturation, potentially in late endosome-lysosome fusion, which is required for processing of various macromolecules.« less

  18. Use of carbon fiber reinforced polymer (CFRP) with CFRP anchors for shear-strengthening and design recommendations/quality control procedures for CFRP anchors.

    DOT National Transportation Integrated Search

    2017-03-01

    The objective of the study was to demonstrate the feasibility of using bi-directional layouts of CFRP for shear : strengthening of bridge girders. In a previous project (0-6306), it was demonstrated that uni-directional CFRP : strips and CFRP anchors...

  19. LRD-22, a novel dual dithiocarbamatic acid ester, inhibits Aurora-A kinase and induces apoptosis and cell cycle arrest in HepG2 cells

    SciTech Connect

    Wang, Huiling; Li, Ridong; Li, Li

    2015-02-27

    In this study we investigated the antitumor activity of the novel dual dithiocarbamatic acid ester LRD-22 in vitro and in vivo. Several cancer cell lines were employed to determine the effect of LRD-22 on cell growth, and the MTT assay showed there was a significant decrease in viable tumor cell numbers in the presence of LRD-22, especially in the HepG2 cell line. Colony formation assay also showed LRD-22 strongly inhibits HepG2 cell growth. Evaluation of the mechanism involved showed that inhibitory effects of LRD-22 on cell growth are due to induction of apoptosis and G2/M arrest. LRD-22 inhibited Aurora-A phosphorylation at Thr{submore » 288} and subsequently impaired p53 phosphorylation at Ser{sub 315} which was associated with the proteasome degradation pathway. Tumor suppressor protein p53 is stabilized by this mechanism and accumulates through inhibition of Aurora-A kinase activity via treatment with LRD-22. In vivo study of HepG2 xenograft in nude mice also shows LRD-22 suppresses tumor growth at a concentration of 5 mg/kg without animals suffering loss of body weight. In conclusion, our results demonstrate LRD-22 acts as an Aurora-A kinase inhibitor to induce apoptosis and inhibit proliferation in HepG2 cells, and should be considered as a promising targeting agent for HCC therapy. - Highlights: • LRD-22 significantly inhibits cancer cell growth, especially in the HepG2 cell line. • The inhibitory effect of LRD-22 is due to induction of apoptosis and cell cycle arrest. • LRD-22 inhibits Aurora-A phosphorylation which results in subsequent impairment of the p53 pathway. • LRD-22 suppresses tumor growth in xenograft mice without body weight loss.« less

  20. Restoring the Labral Height for Treatment of Bankart Lesions: A Comparison of Suture Anchor Constructs

    PubMed Central

    Slabaugh, Mark A.; Friel, Nicole A.; Wang, Vincent M.; Cole, Brian J.

    2013-01-01

    Purpose The purpose of this study was to evaluate glenoid labral heights before injury and after repair with 2 suture anchors: (1) traditional suture anchor secured with knots and (2) knotless suture anchor. Methods Ten matched pairs of human cadaveric glenoids were examined. In each specimen the labrum was detached from the 3-o’clock position to the 6:30 clock position on the anteroinferior glenoid, and labral repair was performed with either (1) traditional Bio-SutureTak suture anchors (n = 10) (Arthrex, Naples, FL) or (2) knotless PushLock suture anchors (n = 10, contralateral side) (Arthrex). By use of a 3-dimensional digitizer, the labral height, measured from the deepest point of the glenoid articular surface to the highest tip of the labrum, was measured in all specimens before injury and after repair at the 3:30, 4:30, and 5:30 clock positions. The degree of labral height increase was computed as a percent increase in labral height from before injury to after repair. Results Labral height increased significantly for all specimens from before injury (5.35 mm) to after repair (8.05 mm) (159.1% ± 13.7%, P < .0001). Increases in labral height from before injury to after repair were similar (P > .05) for Bio-SutureTak suture anchors (164.6% ± 18.7%, P < .0001) and PushLock suture anchors (153.6% ± 5.8%, P < .0001). The amount of labral height increase did not vary by anatomic location (157.0% ± 50.2%, 168.9% ± 51.0%, and 150.4% ± 35.2% at 3:30, 4:30, and 5:30, respectively; P = .46). Conclusions An increase in labral height can be achieved to create a significant height increase from before injury to after labral repair. The difference in labral height afforded by a traditional suture anchor and a knotless anchor is not statistically significant. Clinical Relevance Both traditional and knotless suture anchor constructs provide a reliable restoration of labral height in an acute Bankart model. PMID:20434654