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Sample records for midgut gpi-anchored proteins

  1. Proteomic analysis of GPI-anchored membrane proteins

    DEFF Research Database (Denmark)

    Jung, Hye Ryung; Jensen, Ole Nørregaard

    2006-01-01

    Glycosyl-phosphatidyl-inositol-anchored proteins (GPI-APs) represent a subset of post-translationally modified proteins that are tethered to the outer leaflet of the plasma membrane via a C-terminal GPI anchor. GPI-APs are found in a variety of eukaryote species, from pathogenic microorganisms...... to humans. GPI-APs confer important cellular functions as receptors, enzymes and scaffolding molecules. Specific enzymes and detergent extraction methods combined with separation technologies and mass spectrometry permit proteomic analysis of GPI-APs from plasma membrane preparations to reveal cell...

  2. Plant glycosylphosphatidylinositol (GPI) anchored proteins at the plasma membrane-cell wall nexus.

    Science.gov (United States)

    Yeats, Trevor H; Bacic, Antony; Johnson, Kim L

    2018-04-18

    Approximately 1% of plant proteins are predicted to be post-translationally modified with a glycosylphosphatidylinositol (GPI) anchor that tethers the polypeptide to the outer leaflet of the plasma membrane. While the synthesis and structure of GPI anchors is largely conserved across eukaryotes, the repertoire of functional domains present in the GPI-anchored proteome has diverged substantially. In plants, this includes a large fraction of the GPI-anchored proteome being further modified with plant-specific arabinogalactan (AG) O-glycans. The importance of the GPI-anchored proteome to plant development is underscored by the fact that GPI biosynthetic null mutants exhibit embryo lethality. Mutations in genes encoding specific GPI-anchored proteins (GAPs) further supports their contribution to diverse biological processes occurring at the interface of the plasma membrane and cell wall, including signaling, cell wall metabolism, cell wall polymer cross-linking, and plasmodesmatal transport. Here, we review the literature concerning plant GPI-anchored proteins in the context of their potential to act as molecular hubs that mediate interactions between the plasma membrane and the cell wall and their potential to transduce the signal into the protoplast and thereby activate signal transduction pathways. This article is protected by copyright. All rights reserved.

  3. Mutational analysis of the glycosylphosphatidylinositol (GPI) anchor pathway demonstrates that GPI-anchored proteins are required for cell wall biogenesis and normal hyphal growth in Neurospora crassa.

    Science.gov (United States)

    Bowman, Shaun M; Piwowar, Amy; Al Dabbous, Mash'el; Vierula, John; Free, Stephen J

    2006-03-01

    Using mutational and proteomic approaches, we have demonstrated the importance of the glycosylphosphatidylinositol (GPI) anchor pathway for cell wall synthesis and integrity and for the overall morphology of the filamentous fungus Neurospora crassa. Mutants affected in the gpig-1, gpip-1, gpip-2, gpip-3, and gpit-1 genes, which encode components of the N. crassa GPI anchor biosynthetic pathway, have been characterized. GPI anchor mutants exhibit colonial morphologies, significantly reduced rates of growth, altered hyphal growth patterns, considerable cellular lysis, and an abnormal "cell-within-a-cell" phenotype. The mutants are deficient in the production of GPI-anchored proteins, verifying the requirement of each altered gene for the process of GPI-anchoring. The mutant cell walls are abnormally weak, contain reduced amounts of protein, and have an altered carbohydrate composition. The mutant cell walls lack a number of GPI-anchored proteins, putatively involved in cell wall biogenesis and remodeling. From these studies, we conclude that the GPI anchor pathway is critical for proper cell wall structure and function in N. crassa.

  4. GPI-anchored protein organization and dynamics at the cell surface.

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    Saha, Suvrajit; Anilkumar, Anupama Ambika; Mayor, Satyajit

    2016-02-01

    The surface of eukaryotic cells is a multi-component fluid bilayer in which glycosylphosphatidylinositol (GPI)-anchored proteins are an abundant constituent. In this review, we discuss the complex nature of the organization and dynamics of GPI-anchored proteins at multiple spatial and temporal scales. Different biophysical techniques have been utilized for understanding this organization, including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, single particle tracking, and a number of super resolution methods. Major insights into the organization and dynamics have also come from exploring the short-range interactions of GPI-anchored proteins by fluorescence (or Förster) resonance energy transfer microscopy. Based on the nanometer to micron scale organization, at the microsecond to the second time scale dynamics, a picture of the membrane bilayer emerges where the lipid bilayer appears inextricably intertwined with the underlying dynamic cytoskeleton. These observations have prompted a revision of the current models of plasma membrane organization, and suggest an active actin-membrane composite. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

  5. Hypomorphic mutations in PGAP2, encoding a GPI-anchor-remodeling protein, cause autosomal-recessive intellectual disability

    DEFF Research Database (Denmark)

    Hansen, Lars; Tawamie, Hasan; Murakami, Yoshiko

    2013-01-01

    PGAP2 encodes a protein involved in remodeling the glycosylphosphatidylinositol (GPI) anchor in the Golgi apparatus. After synthesis in the endoplasmic reticulum (ER), GPI anchors are transferred to the proteins and are remodeled while transported through the Golgi to the cell membrane. Germline...... mutations in six genes (PIGA, PIGL, PIGM, PIGV, PIGN, and PIGO) in the ER-located part of the GPI-anchor-biosynthesis pathway have been reported, and all are associated with phenotypes extending from malformation and lethality to severe intellectual disability, epilepsy, minor dysmorphisms, and elevated...... alkaline phosphatase (ALP). We performed autozygosity mapping and ultra-deep sequencing followed by stringent filtering and identified two homozygous PGAP2 alterations, p.Tyr99Cys and p.Arg177Pro, in seven offspring with nonspecific autosomal-recessive intellectual disability from two consanguineous...

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

    Directory of Open Access Journals (Sweden)

    Yusuke Masuishi

    2016-06-01

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

  7. Spontaneous insertion of GPI anchors into cholesterol-rich membrane domains

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    Jing Li

    2018-05-01

    Full Text Available GPI-Anchored proteins (GPI-APs can be exogenously transferred onto bilayer membranes both in vivo and in vitro, while the mechanism by which this transfer process occurs is unknown. In this work, we used atomistic molecular dynamics simulations and free energy calculations to characterize the essential influence of cholesterol on insertion of the GPI anchors into plasma membranes. We demonstrate, both dynamically and energetically, that in the presence of cholesterol, the tails of GPI anchors are able to penetrate inside the core of the lipid membrane spontaneously with a three-step mechanism, while in the absence of cholesterol no spontaneous insertion was observed. We ascribe the failure of insertion to the strong thermal fluctuation of lipid molecules in cholesterol-free bilayer, which generates a repulsive force in entropic origin. In the presence of cholesterol, however, the fluctuation of lipids is strongly reduced, thus decreasing the barrier for the anchor insertion. Based on this observation, we propose a hypothesis that addition of cholesterol creates vertical creases in membranes for the insertion of acyl chains. Moreover, we find that the GPI anchor could also spontaneously inserted into the boundary between cholesterol-rich and cholesterol-depleted domains. Our results shed light on the mechanism of cholesterol-mediated interaction between membrane proteins with acyl chain and plasma membranes in living cells.

  8. Spontaneous insertion of GPI anchors into cholesterol-rich membrane domains

    Science.gov (United States)

    Li, Jing; Liu, Xiuhua; Tian, Falin; Yue, Tongtao; Zhang, Xianren; Cao, Dapeng

    2018-05-01

    GPI-Anchored proteins (GPI-APs) can be exogenously transferred onto bilayer membranes both in vivo and in vitro, while the mechanism by which this transfer process occurs is unknown. In this work, we used atomistic molecular dynamics simulations and free energy calculations to characterize the essential influence of cholesterol on insertion of the GPI anchors into plasma membranes. We demonstrate, both dynamically and energetically, that in the presence of cholesterol, the tails of GPI anchors are able to penetrate inside the core of the lipid membrane spontaneously with a three-step mechanism, while in the absence of cholesterol no spontaneous insertion was observed. We ascribe the failure of insertion to the strong thermal fluctuation of lipid molecules in cholesterol-free bilayer, which generates a repulsive force in entropic origin. In the presence of cholesterol, however, the fluctuation of lipids is strongly reduced, thus decreasing the barrier for the anchor insertion. Based on this observation, we propose a hypothesis that addition of cholesterol creates vertical creases in membranes for the insertion of acyl chains. Moreover, we find that the GPI anchor could also spontaneously inserted into the boundary between cholesterol-rich and cholesterol-depleted domains. Our results shed light on the mechanism of cholesterol-mediated interaction between membrane proteins with acyl chain and plasma membranes in living cells.

  9. Use of green fluorescent protein fusions to analyse the N- and C-terminal signal peptides of GPI-anchored cell wall proteins in Candida albicans.

    Science.gov (United States)

    Mao, Yuxin; Zhang, Zimei; Wong, Brian

    2003-12-01

    Glycophosphatidylinositol (GPI)-anchored proteins account for 26-35% of the Candida albicans cell wall. To understand the signals that regulate these proteins' cell surface localization, green fluorescent protein (GFP) was fused to the N- and C-termini of the C. albicans cell wall proteins (CWPs) Hwp1p, Als3p and Rbt5p. C. albicans expressing all three fusion proteins were fluorescent at the cell surface. GFP was released from membrane fractions by PI-PLC and from cell walls by beta-glucanase, which implied that GFP was GPI-anchored to the plasma membrane and then covalently attached to cell wall glucans. Twenty and 25 amino acids, respectively, from the N- and C-termini of Hwp1p were sufficient to target GFP to the cell surface. C-terminal substitutions that are permitted by the omega rules (G613D, G613N, G613S, G613A, G615S) did not interfere with GFP localization, whereas some non-permitted substitutions (G613E, G613Q, G613R, G613T and G615Q) caused GFP to accumulate in intracellular ER-like structures and others (G615C, G613N/G615C and G613D/G615C) did not. These results imply that (i) GFP fusions can be used to analyse the N- and C-terminal signal peptides of GPI-anchored CWPs, (ii) the omega amino acid in Hwp1p is G613, and (iii) C can function at the omega+2 position in C. albicans GPI-anchored proteins.

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

    Science.gov (United States)

    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. Novel applications for glycosylphosphatidylinositol-anchored proteins in pharmaceutical and industrial biotechnology.

    Science.gov (United States)

    Müller, Günter

    2011-04-01

    Glycosylphosphatidylinositol (GPI)-anchored proteins have been regarded as typical cell surface proteins found in most eukaryotic cells from yeast to man. They are embedded in the outer plasma membrane leaflet via a carboxy-terminally linked complex glycolipid GPI structure. The amphiphilic nature of the GPI anchor, its compatibility with the function of the attached protein moiety and the capability of GPI-anchored proteins for spontaneous insertion into and transfer between artificial and cellular membranes initially suggested their potential for biotechnological applications. However, these expectations have been hardly fulfilled so far. Recent developments fuel novel hopes with regard to: (i) Automated online expression, extraction and purification of therapeutic proteins as GPI-anchored proteins based on their preferred accumulation in plasma membrane lipid rafts, (ii) multiplex custom-made protein chips based on GPI-anchored cell wall proteins in yeast, (iii) biomaterials and biosensors with films consisting of sets of distinct GPI-anchored binding-proteins or enzymes for sequential or combinatorial catalysis, and (iv) transport of therapeutic proteins across or into relevant tissue cells, e.g., enterocytes or adipocytes. Latter expectations are based on the demonstrated translocation of GPI-anchored proteins from plasma membrane lipid rafts to cytoplasmic lipid droplets and eventually further into microvesicles which upon release from donor cells transfer their GPI-anchored proteins to acceptor cells. The value of these technologies, which are all based on the interaction of GPI-anchored proteins with membranes and surfaces, for the engineering, production and targeted delivery of biomolecules for a huge variety of therapeutic and biotechnological purposes should become apparent in the near future.

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

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2013-06-01

    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.

  14. GPI-anchored proteins are confined in subdiffraction clusters at the apical surface of polarized epithelial cells.

    Science.gov (United States)

    Paladino, Simona; Lebreton, Stéphanie; Lelek, Mickaël; Riccio, Patrizia; De Nicola, Sergio; Zimmer, Christophe; Zurzolo, Chiara

    2017-12-01

    Spatio-temporal compartmentalization of membrane proteins is critical for the regulation of diverse vital functions in eukaryotic cells. It was previously shown that, at the apical surface of polarized MDCK cells, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are organized in small cholesterol-independent clusters of single GPI-AP species (homoclusters), which are required for the formation of larger cholesterol-dependent clusters formed by multiple GPI-AP species (heteroclusters). This clustered organization is crucial for the biological activities of GPI-APs; hence, understanding the spatio-temporal properties of their membrane organization is of fundamental importance. Here, by using direct stochastic optical reconstruction microscopy coupled to pair correlation analysis (pc-STORM), we were able to visualize and measure the size of these clusters. Specifically, we show that they are non-randomly distributed and have an average size of 67 nm. We also demonstrated that polarized MDCK and non-polarized CHO cells have similar cluster distribution and size, but different sensitivity to cholesterol depletion. Finally, we derived a model that allowed a quantitative characterization of the cluster organization of GPI-APs at the apical surface of polarized MDCK cells for the first time. Experimental FRET (fluorescence resonance energy transfer)/FLIM (fluorescence-lifetime imaging microscopy) data were correlated to the theoretical predictions of the model. © 2017 The Author(s).

  15. Proteomic analysis of glycosylphosphatidylinositol-anchored membrane proteins

    DEFF Research Database (Denmark)

    Elortza, Felix; Nühse, Thomas S; Foster, Leonard J

    2003-01-01

    Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a functionally and structurally diverse family of post-translationally modified membrane proteins found mostly in the outer leaflet of the plasma membrane in a variety of eukaryotic cells. Although the general role of GPI-APs remains...... unclear, they have attracted attention because they act as enzymes and receptors in cell adhesion, differentiation, and host-pathogen interactions. GPI-APs may represent potential diagnostic and therapeutic targets in humans and are interesting in plant biotechnology because of their key role in root...... and 44 GPI-APs in an Arabidopsis thaliana membrane preparation, representing the largest experimental dataset of GPI-anchored proteins to date....

  16. Two endoplasmic reticulum (ER) membrane proteins that facilitate ER-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins.

    Science.gov (United States)

    Barz, W P; Walter, P

    1999-04-01

    Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes in Saccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for "delayed GPI-anchored protein transport"), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Delta dgt1Delta cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting that LAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non-GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Delta dgt1Delta cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Delta dgt1Delta cells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.

  17. Targeting the GPI biosynthetic pathway.

    Science.gov (United States)

    Yadav, Usha; Khan, Mohd Ashraf

    2018-02-27

    The GPI (Glycosylphosphatidylinositol) biosynthetic pathway is a multistep conserved pathway in eukaryotes that culminates in the generation of GPI glycolipid which in turn anchors many proteins (GPI-APs) to the cell surface. In spite of the overall conservation of the pathway, there still exist subtle differences in the GPI pathway of mammals and other eukaryotes which holds a great promise so far as the development of drugs/inhibitors against specific targets in the GPI pathway of pathogens is concerned. Many of the GPI structures and their anchored proteins in pathogenic protozoans and fungi act as pathogenicity factors. Notable examples include GPI-anchored variant surface glycoprotein (VSG) in Trypanosoma brucei, GPI-anchored merozoite surface protein 1 (MSP1) and MSP2 in Plasmodium falciparum, protein-free GPI related molecules like lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) in Leishmania spp., GPI-anchored Gal/GalNAc lectin and proteophosphoglycans in Entamoeba histolytica or the GPI-anchored mannoproteins in pathogenic fungi like Candida albicans. Research in this active area has already yielded encouraging results in Trypanosoma brucei by the development of parasite-specific inhibitors of GlcNCONH 2 -β-PI, GlcNCONH 2 -(2-O-octyl)-PI and salicylic hydroxamic acid (SHAM) targeting trypanosomal GlcNAc-PI de-N-acetylase as well as the development of antifungal inhibitors like BIQ/E1210/gepinacin/G365/G884 and YW3548/M743/M720 targeting the GPI specific fungal inositol acyltransferase (Gwt1) and the phosphoethanolamine transferase-I (Mcd4), respectively. These confirm the fact that the GPI pathway continues to be the focus of researchers, given its implications for the betterment of human life.

  18. Chemical biology of Glycosylphosphatidylinositol (GPI) anchors

    Indian Academy of Sciences (India)

    Admin

    CSIR-IIIM. Chemical biology of. Glycosylphosphatidylinositol (GPI) anchors. Ram Vishwakarma. CSIR-Indian Institute of Integrative Medicine, Jammu. N ti l I tit t f I l. N. D lhi. National Institute of Immunology, New Delhi. Piramal Life Sciences Ltd, Mumbai ...

  19. Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting

    Directory of Open Access Journals (Sweden)

    Sander A. A. Kooijmans

    2016-03-01

    Full Text Available Background: Extracellular vesicles (EVs are attractive candidate drug delivery systems due to their ability to functionally transport biological cargo to recipient cells. However, the apparent lack of target cell specificity of exogenously administered EVs limits their therapeutic applicability. In this study, we propose a novel method to equip EVs with targeting properties, in order to improve their interaction with tumour cells. Methods: EV producing cells were transfected with vectors encoding for anti-epidermal growth factor receptor (EGFR nanobodies, which served as targeting ligands for tumour cells, fused to glycosylphosphatidylinositol (GPI anchor signal peptides derived from decay-accelerating factor (DAF. EVs were isolated using ultrafiltration/size-exclusion liquid chromatography and characterized using western blotting, Nanoparticle Tracking Analysis, and electron microscopy. EV–tumour cell interactions were analyzed under static conditions using flow cytometry and under flow conditions using a live-cell fluorescence microscopy-coupled perfusion system. Results: V analysis showed that GPI-linked nanobodies were successfully displayed on EV surfaces and were highly enriched in EVs compared with parent cells. Display of GPI-linked nanobodies on EVs did not alter general EV characteristics (i.e. morphology, size distribution and protein marker expression, but greatly improved EV binding to tumour cells dependent on EGFR density under static conditions. Moreover, nanobody-displaying EVs showed a significantly improved cell association to EGFR-expressing tumour cells under flow conditions. Conclusions: We show that nanobodies can be anchored on the surface of EVs via GPI, which alters their cell targeting behaviour. Furthermore, this study highlights GPI-anchoring as a new tool in the EV toolbox, which may be applied for EV display of a variety of proteins, such as antibodies, reporter proteins and signaling molecules.

  20. GPI anchor biosynthesis in yeast : phosphoethanolamine is attached to the alpha1,4-linked mannose of the complete precursor glycophospholipid

    NARCIS (Netherlands)

    Canivenc-Gansel, E; Imhof, I; Reggiori, F; Burda, P; Conzelmann, A; Benachour, A; Reggiori, Fulvio

    Cells synthesize the GPI anchor carbohydrate core by successively adding N-acetylglucosamine, three mannoses, and phosphoethanolamine (EtN-P) onto phosphatidylinositol, thus forming the complete GPI precursor lipid which is then added to proteins. Previously, we isolated a GPI deficient yeast mutant

  1. Diffusion of lipids and GPI-anchored proteins in actin-free plasma membrane vesicles measured by STED-FCS

    DEFF Research Database (Denmark)

    Schneider, Falk; Waithe, Dominic; Clausen, Mathias P

    2017-01-01

    (STED-FCS) to access and compare the diffusion characteristics of fluorescent lipid analogues and GPI-anchored proteins (GPI-APs) in the live cell plasma membrane and in actin cytoskeleton-free cell-derived giant plasma membrane vesicles (GPMVs). Hindered diffusion of phospholipids and sphingolipids......Diffusion and interaction dynamics of molecules at the plasma membrane play an important role in cellular signalling, and they are suggested to be strongly associated with the actin cytoskeleton. Here, we utilise super-resolution STED microscopy combined with fluorescence correlation spectroscopy...... forming immobile clusters, both of which disappear in GPMVs. Our data underline the crucial role of the actin cortex in maintaining hindered diffusion modes of many but not all of the membrane molecules, and highlight a powerful experimental approach to decipher specific influences on molecular plasma...

  2. Proteome analysis of Aspergillus fumigatus identifies glycosylphosphatidylinositol-anchored proteins associated to the cell wall biosynthesis.

    Science.gov (United States)

    Bruneau, J M; Magnin, T; Tagat, E; Legrand, R; Bernard, M; Diaquin, M; Fudali, C; Latgé, J P

    2001-08-01

    Previous studies in Aspergillus fumigatus (Mouyna I., Fontaine T., Vai M., Monod M., Fonzi W. A., Diaquin M., Popolo L., Hartland R. P., Latgé J.-P, J. Biol. Chem. 2000, 275, 14882-14889) have shown that a glucanosyltransferase playing an important role in fungal cell wall biosynthesis is glycosylphosphatidylinositol (GPI) anchored to the membrane. To identify other GPI-anchored proteins putatively involved in cell wall biogenesis, a proteomic analysis has been undertaken in A. fumigatus and the protein data were matched with the yeast genomic data. GPI-anchored proteins of A. fumigatus were released from membrane preparation by an endogenous GPI-phospholipase C, purified by liquid chromatography and separated by two-dimensional electrophoresis. They were characterized by their peptide mass fingerprint through matrix-assisted laser desorption/ionization-time of flight-(MALDI-TOF)-mass spectrometry and by internal amino acid sequencing. Nine GPI-anchored proteins were identified in A. fumigatus. Five of them were homologs of putatively GPI-anchored yeast proteins (Csa1p, Crh1p, Crh2p, Ecm33p, Gas1p) of unknown function but shown by gene disruption analysis to play a role in cell wall morphogenesis. In addition, a comparative study performed with chitin synthase and glucanosyl transferase mutants of A. fumigatus showed that a modification of the growth phenotype seen in these mutants was associated to an alteration of the pattern of GPI-anchored proteins. These results suggest that GPI-anchored proteins identified in this study are involved in A. fumigatus cell wall organization.

  3. Two homologous genes, DCW1 (YKL046c) and DFG5, are essential for cell growth and encode glycosylphosphatidylinositol (GPI)-anchored membrane proteins required for cell wall biogenesis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kitagaki, Hiroshi; Wu, Hong; Shimoi, Hitoshi; Ito, Kiyoshi

    2002-11-01

    The cell wall of Saccharomyces cerevisiae consists of glucan, chitin and various kinds of mannoproteins. Major parts of mannoproteins are synthesized as glycosylphosphatidylinositol (GPI)-anchored proteins and are then transferred to cell wall beta-1,6-glucan. A glycosyltransferase has been hypothesized to catalyse this transfer reaction. A database search revealed that the products of YKL046c and DFG5 are homologous to bacterial mannosidase. These genes are homologous to each other and have primary structures characteristic of GPI-anchored proteins. Although single disruptants of ykl046c and dfg5 were viable, ykl046cDelta was hypersensitive to a cell wall-digesting enzyme (zymolyase), suggesting that this gene is involved in cell wall biosynthesis. We therefore designated this gene as DCW1 (defective cell wall). A double disruptant of dcw1 and dfg5 was synthetically lethal, indicating that the functions of these gene products are redundant, and at least one of them is required for cell growth. Cells deficient in both Dcw1p and Dfg5p were round and large, had cell walls that contained an increased amount of chitin and secreted a major cell wall protein, Cwp1p, into the medium. Biochemical analyses showed that epitope-tagged Dcw1p is an N-glycosylated, GPI-anchored membrane protein and is localized in the membrane fraction including the cell surface. These results suggest that both Dcw1p and Dfg5p are GPI-anchored membrane proteins and are required for normal biosynthesis of the cell wall.

  4. Midgut GPI-anchored proteins with alkaline phosphatase activity from the cotton boll weevil (Anthonomus grandis) are putative receptors for the Cry1B protein of Bacillus thuringiensis.

    Science.gov (United States)

    Martins, Erica Soares; Monnerat, Rose Gomes; Queiroz, Paulo Roberto; Dumas, Vinicius Fiuza; Braz, Shélida Vasconcelos; de Souza Aguiar, Raimundo Wagner; Gomes, Ana Cristina Menezes Mendes; Sánchez, Jorge; Bravo, Alejandra; Ribeiro, Bergmann Morais

    2010-02-01

    Cry toxins from Bacillus thuringiensis (Bt) are used for insect control. They interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding resulting in midgut epithelial cells lysis. In this work we had cloned, sequenced and expressed a cry1Ba toxin gene from the B thuringiensis S601 strain which was previously shown to be toxic to Anthonomus grandis, a cotton pest. The Cry1Ba6 protein expressed in an acrystaliferous B. thuringiensis strain was toxic to A. grandis in bioassays. The binding of Cry1Ba6 toxin to proteins located in the midgut brush border membrane of A. grandis was analyzed and we found that Cry1Ba6 binds to two proteins (62 and 65kDa) that showed alkaline phosphatase (ALP) activity. This work is the first report that shows the localization of Cry toxin receptors in the midgut cells of A. grandis. 2009. Published by Elsevier Ltd.

  5. Defects in GPI biosynthesis perturb Cripto signaling during forebrain development in two new mouse models of holoprosencephaly

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    David M. McKean

    2012-07-01

    Holoprosencephaly is the most common forebrain defect in humans. We describe two novel mouse mutants that display a holoprosencephaly-like phenotype. Both mutations disrupt genes in the glycerophosphatidyl inositol (GPI biosynthesis pathway: gonzo disrupts Pign and beaker disrupts Pgap1. GPI anchors normally target and anchor a diverse group of proteins to lipid raft domains. Mechanistically we show that GPI anchored proteins are mislocalized in GPI biosynthesis mutants. Disruption of the GPI-anchored protein Cripto (mouse and TDGF1 (human ortholog have been shown to result in holoprosencephaly, leading to our hypothesis that Cripto is the key GPI anchored protein whose altered function results in an HPE-like phenotype. Cripto is an obligate Nodal co-factor involved in TGFβ signaling, and we show that TGFβ signaling is reduced both in vitro and in vivo. This work demonstrates the importance of the GPI anchor in normal forebrain development and suggests that GPI biosynthesis genes should be screened for association with human holoprosencephaly.

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

    Science.gov (United States)

    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.

  7. Glycosylphosphatidylinositol-anchored proteins are required for cell wall synthesis and morphogenesis in Arabidopsis.

    Science.gov (United States)

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

    2005-04-01

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

  8. Deletion of Smgpi1 encoding a GPI-anchored protein suppresses sterility of the STRIPAK mutant ΔSmmob3 in the filamentous ascomycete Sordaria macrospora.

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    Frey, Stefan; Lahmann, Yasmine; Hartmann, Thomas; Seiler, Stephan; Pöggeler, Stefanie

    2015-08-01

    The striatin interacting phosphatase and kinase (STRIPAK) complex, which is composed of striatin, protein phosphatase PP2A and kinases, is required for fruiting-body development and cell fusion in the filamentous ascomycete Sordaria macrospora. Here, we report on the interplay of the glycosylphosphatidylinositol (GPI)-anchored protein SmGPI1 with the kinase activator SmMOB3, a core component of human and fungal STRIPAK complexes. SmGPI1 is conserved among filamentous ascomycetes and was first identified in a yeast two-hybrid screen using SmMOB3 as bait. The physical interaction of SmMOB3 and SmGPI1 was verified by co-immunoprecipitation. In vivo localization and differential centrifugation revealed that SmGPI1 is predominantly secreted and attached to the cell wall but is also associated with mitochondria and appears to be a dual-targeted protein. Deletion of Smgpi1 led to an increased number of fruiting bodies that were normally shaped but reduced in size. In addition, Smmob3 and Smgpi1 genetically interact. In the sterile ΔSmmob3 background deletion of Smgpi1 restores fertility, vegetative growth as well as hyphal-fusion defects. The suppression effect was specific for the ΔSmmob3 mutant as deletion of Smgpi1 in other STRIPAK mutants does not restore fertility. © 2015 John Wiley & Sons Ltd.

  9. Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts

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    Du, Yijun [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan (China); Pattnaik, Asit K. [School of Veterinary Medicine and Biomedical Sciences and the Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583-0900 (United States); Song, Cheng [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Yoo, Dongwan, E-mail: dyoo@illinois.edu [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Li, Gang, E-mail: dyoo@illinois.edu [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences, Beijing (China)

    2012-03-01

    The porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 4 (GP4) resembles a typical type I membrane protein in its structure but lacks a hydrophilic tail at the C-terminus, suggesting that GP4 may be a lipid-anchored membrane protein. Using the human decay-accelerating factor (DAF; CD55), a known glycosyl-phosphatidylinositol (GPI) lipid-anchored protein, chimeric constructs were made to substitute the GPI-anchor domain of DAF with the putative lipid-anchor domain of GP4, and their membrane association and lipase cleavage were determined in cells. The DAF-GP4 fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC), indicating that the C-terminal domain of GP4 functions as a GPI anchor. Mutational studies for residues adjacent to the GPI modification site and characterization of respective mutant viruses generated from infectious cDNA clones show that the ability of GP4 for membrane association corresponded to virus viability and growth characteristics. The residues T158 ({omega} - 2, where {omega} is the GPI moiety at E160), P159 ({omega} - 1), and M162 ({omega} + 2) of GP4 were determined to be important for virus replication, with M162 being of particular importance for virus infectivity. The complete removal of the peptide-anchor domain in GP4 resulted in a complete loss of virus infectivity. The depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in PRRSV infection. Remarkably, GP4 was found to co-localize with CD163 in the lipid rafts on the plasma membrane. Since CD163 has been reported as a cellular receptor for PRRSV and GP4 has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus.

  10. Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts

    International Nuclear Information System (INIS)

    Du, Yijun; Pattnaik, Asit K.; Song, Cheng; Yoo, Dongwan; Li, Gang

    2012-01-01

    The porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 4 (GP4) resembles a typical type I membrane protein in its structure but lacks a hydrophilic tail at the C-terminus, suggesting that GP4 may be a lipid-anchored membrane protein. Using the human decay-accelerating factor (DAF; CD55), a known glycosyl-phosphatidylinositol (GPI) lipid-anchored protein, chimeric constructs were made to substitute the GPI-anchor domain of DAF with the putative lipid-anchor domain of GP4, and their membrane association and lipase cleavage were determined in cells. The DAF-GP4 fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC), indicating that the C-terminal domain of GP4 functions as a GPI anchor. Mutational studies for residues adjacent to the GPI modification site and characterization of respective mutant viruses generated from infectious cDNA clones show that the ability of GP4 for membrane association corresponded to virus viability and growth characteristics. The residues T158 (ω − 2, where ω is the GPI moiety at E160), P159 (ω − 1), and M162 (ω + 2) of GP4 were determined to be important for virus replication, with M162 being of particular importance for virus infectivity. The complete removal of the peptide–anchor domain in GP4 resulted in a complete loss of virus infectivity. The depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in PRRSV infection. Remarkably, GP4 was found to co-localize with CD163 in the lipid rafts on the plasma membrane. Since CD163 has been reported as a cellular receptor for PRRSV and GP4 has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus.

  11. Glycosylphosphatidylinositol-Anchored Proteins in Fusarium graminearum: Inventory, Variability, and Virulence

    Science.gov (United States)

    Rittenour, William R.; Harris, Steven D.

    2013-01-01

    The contribution of cell surface proteins to plant pathogenicity of fungi is not well understood. As such, the objective of this study was to investigate the functions and importance of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the wheat pathogen F. graminearum. GPI-APs are surface proteins that are attached to either the membrane or cell wall. In order to simultaneously disrupt several GPI-APs, a phosphoethanolamine transferase-encoding gene gpi7 was deleted and the resultant mutant characterized in terms of growth, development, and virulence. The Δgpi7 mutants exhibited slower radial growth rates and aberrantly shaped macroconidia. Furthermore, virulence tests and microscopic analyses indicated that Gpi7 is required for ramification of the fungus throughout the rachis of wheat heads. In parallel, bioinformatics tools were utilized to predict and inventory GPI-APs within the proteome of F. graminearum. Two of the genes identified in this screen (FGSG_01588 and FGSG_08844) displayed isolate-specific length variability as observed for other fungal cell wall adhesion genes. Nevertheless, deletion of these genes failed to reveal obvious defects in growth, development, or virulence. This research demonstrates the global importance of GPI-APs to in planta proliferation in F. graminearum, and also highlights the potential of individual GPI-APs as diagnostic markers. PMID:24312325

  12. COBRA encodes a putative GPI-anchored protein, which is polarly localized and necessary for oriented cell expansion in Arabidopsis.

    Science.gov (United States)

    Schindelman, G; Morikami, A; Jung, J; Baskin, T I; Carpita, N C; Derbyshire, P; McCann, M C; Benfey, P N

    2001-05-01

    To control organ shape, plant cells expand differentially. The organization of the cellulose microfibrils in the cell wall is a key determinant of differential expansion. Mutations in the COBRA (COB) gene of Arabidopsis, known to affect the orientation of cell expansion in the root, are reported here to reduce the amount of crystalline cellulose in cell walls in the root growth zone. The COB gene, identified by map-based cloning, contains a sequence motif found in proteins that are anchored to the extracellular surface of the plasma membrane through a glycosylphosphatidylinositol (GPI) linkage. In animal cells, this lipid linkage is known to confer polar localization to proteins. The COB protein was detected predominately on the longitudinal sides of root cells in the zone of rapid elongation. Moreover, COB RNA levels are dramatically upregulated in cells entering the zone of rapid elongation. Based on these results, models are proposed for the role of COB as a regulator of oriented cell expansion.

  13. Complementation of essential yeast GPI mannosyltransferase mutations suggests a novel specificity for certain Trypanosoma and Plasmodium PigB proteins.

    Directory of Open Access Journals (Sweden)

    Leslie K Cortes

    Full Text Available The glycosylphosphatidylinositol (GPI anchor is an essential glycolipid that tethers certain eukaryotic proteins to the cell surface. The core structure of the GPI anchor is remarkably well conserved across evolution and consists of NH2-CH2-CH2-PO4-6Manα1,2Manα1,6Manα1,4-GlcNα1,6-myo-inositol-PO4-lipid. The glycan portion of this structure may be modified with various side-branching sugars or other compounds that are heterogeneous and differ from organism to organism. One such modification is an α(1,2-linked fourth mannose (Man-IV that is side-branched to the third mannose (Man-III of the trimannosyl core. In fungi and mammals, addition of Man-III and Man-IV occurs by two distinct Family 22 α(1,2-mannosyltransferases, Gpi10/PigB and Smp3/PigZ, respectively. However, in the five protozoan parasite genomes we examined, no genes encoding Smp3/PigZ proteins were observed, despite reports of tetramannosyl-GPI structures (Man4-GPIs being produced by some parasites. In this study, we tested the hypothesis that the Gpi10/PigB proteins produced by protozoan parasites have the ability to add both Man-III and Man-IV to GPI precursors. We used yeast genetics to test the in vivo specificity of Gpi10/PigB proteins from several Plasmodium and Trypanosoma species by examining their ability to restore viability to Saccharomyces cerevisiae strains harboring lethal defects in Man-III (gpi10Δ or Man-IV (smp3Δ addition to GPI precursor lipids. We demonstrate that genes encoding PigB enzymes from T. cruzi, T. congolense and P. falciparum are each capable of separately complementing essential gpi10Δ and smp3Δ mutations, while PIGB genes from T. vivax and T. brucei only complement gpi10Δ. Additionally, we show the ability of T. cruzi PIGB to robustly complement a gpi10Δ/smp3Δ double mutant. Our data suggest that certain Plasmodium and Trypanosoma PigB mannosyltransferases can transfer more than one mannose to GPI precursors in vivo, and suggest a novel

  14. BcCFEM1, a CFEM Domain-Containing Protein with Putative GPI-Anchored Site, Is Involved in Pathogenicity, Conidial Production, and Stress Tolerance in Botrytis cinerea

    Directory of Open Access Journals (Sweden)

    Wenjun Zhu

    2017-09-01

    Full Text Available We experimentally isolated and characterized a CFEM protein with putative GPI-anchored site BcCFEM1 in Botrytis cinerea. BcCFEM1 contains a CFEM (common in several fungal extracellular membrane proteins domain with the characteristic eight cysteine residues at N terminus, and a predicted GPI modification site at C terminus. BcCFEM1 was significantly up-regulated during early stage of infection on bean leaves and induced chlorosis in Nicotiana benthamiana leaves using Agrobacterium infiltration method. Targeted deletion of BcCFEM1 in B. cinerea affected virulence, conidial production and stress tolerance, but not growth rate, conidial germination, colony morphology, and sclerotial formation. However, over expression of BcCFEM1 did not make any observable phenotype change. Therefore, our data suggested that BcCFEM1 contributes to virulence, conidial production, and stress tolerance. These findings further enhance our understanding on the sophisticated pathogenicity of B. cinerea beyond necrotrophic stage, highlighting the importance of CFEM protein to B. cinerea and other broad-host-range necrotrophic pathogens.

  15. The GPI-anchored protein Ecm33 is vital for conidiation, cell wall integrity, and multi-stress tolerance of two filamentous entomopathogens but not for virulence.

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    Chen, Ying; Zhu, Jing; Ying, Sheng-Hua; Feng, Ming-Guang

    2014-06-01

    Ecm33 is one of several glycosylphosphatidylinositol (GPI)-anchored proteins. This protein is known to be involved in fungal cell wall integrity, but its contribution to multi-stress tolerance is largely unknown. Here we characterized the functions of two Ecm33 orthologues, i.e., Bbecm33 in Beauveria bassiana and Mrecm33 in Metarhizium robertsii. Bbecm33 and Mrecm33 were both confirmed as GPI-anchored cell wall proteins in immunogold localization. Single-gene disruptions of Bbecm33 and Mrecm33 caused slight growth defects, but conidial yield decreased much more in ΔBbecm33 (76 %) than in ΔMrecm33 (42 %), accompanied with significant reductions of intracellular mannitol and trehalose contents in both mutants and weakened cell walls in ΔBbecm33 only. Consequently, ΔBbecm33 was far more sensitive to the cell wall-perturbating agents Congo red and sodium dodecyl sulfate (SDS) than ΔMrecm33, which showed null response to SDS. Both deletion mutants became significantly more sensitive to two oxidants (menadione and H2O2), two fungicides (carbendazim and ethirimol), osmotic salt NaCl, and Ca(2+) during growth despite some degrees of differences in their sensitivities to the chemical stressors. Strikingly, conidial UV-B resistance decreased by 55 % in ΔBbecm33 but was unaffected in ΔMrecm33, unlike a similar decrease (25-28 %) of conidial thermotolerance in both. All the changes were restored to wild-type levels by gene complementation through ectopic gene integration in each fungus. However, neither ΔBbecm33 nor ΔMrecm33 showed a significant change in virulence to a susceptible insect host. Our results indicate that Bbecm33 and Mrecm33 contribute differentially to the conidiation and multi-stress tolerance of B. bassiana and M. robertsii.

  16. Extraneural manifestations of prion infection in GPI-anchorless transgenic mice

    International Nuclear Information System (INIS)

    Lee, Andrew M.; Paulsson, Johan F.; Cruite, Justin; Andaya, Abegail A.; Trifilo, Matthew J.; Oldstone, Michael B.A.

    2011-01-01

    Earlier studies indicated that transgenic (tg) mice engineered to express prion protein (PrP) lacking the glycophosphatidylinositol (GPI -/- ) membrane anchor formed abnormal proteinase-resistant prion (PrPsc) amyloid deposits in their brains and hearts when infected with the RML strain of murine scrapie. In contrast, RML scrapie infection of normal mice with a GPI-anchored PrP did not deposit amyloid with PrPsc in the brain or the heart. Here we report that scrapie-infected GPI -/- PrP tg mice also deposit PrP and transmissible infectious material in the gut, kidneys, and islets of Langerhans. Similar to previously reported amyloid deposits in the brain and heart, amyloid deposits were found in the gut; however, no amyloid deposited in the islets. By high-resolution electron microscopy, we show PrP is located primarily in α cells and also β cells. Islets contain abundant insulin and there is no abnormality in glucose metabolism in infected GPI -/- PrP tg mice.

  17. Cytosolically expressed PrP GPI-signal peptide interacts with mitochondria.

    Science.gov (United States)

    Guizzunti, Gianni; Zurzolo, Chiara

    2015-01-01

    We previously reported that PrP GPI-anchor signal peptide (GPI-SP) is specifically degraded by the proteasome. Additionally, we showed that the point mutation P238S, responsible for a genetic form of prion diseases, while not affecting the GPI-anchoring process, results in the accumulation of PrP GPI-SP, suggesting the possibility that PrP GPI-anchor signal peptide could play a role in neurodegenerative prion diseases. We now show that PrP GPI-SP, when expressed as a cytosolic peptide, is able to localize to the mitochondria and to induce mitochondrial fragmentation and vacuolarization, followed by loss in mitochondrial membrane potential, ultimately resulting in apoptosis. Our results identify the GPI-SP of PrP as a novel candidate responsible for the impairment in mitochondrial function involved in the synaptic pathology observed in prion diseases, establishing a link between PrP GPI-SP accumulation and neuronal death.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-02-19

    The normal cellular prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein. However, in pancreatic ductal adenocarcinoma cell lines, such as BxPC-3, PrP exists as a pro-PrP retaining its glycosylphosphatidylinositol (GPI) peptide signaling sequence. Here, we report the identification of another pancreatic ductal adenocarcinoma cell line, AsPC-1, which expresses a mature GPI-anchored PrP. Comparison of the 24 genes involved in the GPI anchor modification pathway between AsPC-1 and BxPC-3 revealed 15 of the 24 genes, including PGAP1 and PIG-F, were down-regulated in the latter cells. We also identified six missense mutations in DPM2, PIG-C, PIG-N, and PIG-P alongside eight silent mutations. When BxPC-3 cells were fused with Chinese hamster ovary (CHO) cells, which lack endogenous PrP, pro-PrP was successfully converted into mature GPI-anchored PrP. Expression of the individual gene, such as PGAP1, PIG-F, or PIG-C, into BxPC-3 cells does not result in phosphoinositide-specific phospholipase C sensitivity of PrP. However, when PIG-F but not PIG-P is expressed in PGAP1-expressing BxPC-3 cells, PrP on the surface of the cells becomes phosphoinositide-specific phospholipase C-sensitive. Thus, low expression of PIG-F and PGAP1 is the major factor contributing to the accumulation of pro-PrP. More importantly, BxPC-3 cells expressing GPI-anchored PrP migrate much slower than BxPC-3 cells bearing pro-PrP. In addition, GPI-anchored PrP-bearing AsPC-1 cells also migrate slower than pro-PrP bearing BxPC-3 cells, although both cells express filamin A. "Knocking out" PRNP in BxPC-3 cell drastically reduces its migration. Collectively, these results show that multiple gene irregularity in BxPC-3 cells is responsible for the formation of pro-PrP, and binding of pro-PrP to filamin A contributes to enhanced tumor cell motility. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Dynamic partitioning of a glycosyl-phosphatidylinositol-anchored protein in glycosphingolipid-rich microdomains imaged by single-quantum dot tracking.

    Science.gov (United States)

    Pinaud, Fabien; Michalet, Xavier; Iyer, Gopal; Margeat, Emmanuel; Moore, Hsiao-Ping; Weiss, Shimon

    2009-06-01

    Recent experimental developments have led to a revision of the classical fluid mosaic model proposed by Singer and Nicholson more than 35 years ago. In particular, it is now well established that lipids and proteins diffuse heterogeneously in cell plasma membranes. Their complex motion patterns reflect the dynamic structure and composition of the membrane itself, as well as the presence of the underlying cytoskeleton scaffold and that of the extracellular matrix. How the structural organization of plasma membranes influences the diffusion of individual proteins remains a challenging, yet central, question for cell signaling and its regulation. Here we have developed a raft-associated glycosyl-phosphatidyl-inositol-anchored avidin test probe (Av-GPI), whose diffusion patterns indirectly report on the structure and dynamics of putative raft microdomains in the membrane of HeLa cells. Labeling with quantum dots (qdots) allowed high-resolution and long-term tracking of individual Av-GPI and the classification of their various diffusive behaviors. Using dual-color total internal reflection fluorescence (TIRF) microscopy, we studied the correlation between the diffusion of individual Av-GPI and the location of glycosphingolipid GM1-rich microdomains and caveolae. We show that Av-GPI exhibit a fast and a slow diffusion regime in different membrane regions, and that slowing down of their diffusion is correlated with entry in GM1-rich microdomains located in close proximity to, but distinct, from caveolae. We further show that Av-GPI dynamically partition in and out of these microdomains in a cholesterol-dependent manner. Our results provide direct evidence that cholesterol-/sphingolipid-rich microdomains can compartmentalize the diffusion of GPI-anchored proteins in living cells and that the dynamic partitioning raft model appropriately describes the diffusive behavior of some raft-associated proteins across the plasma membrane.

  1. Grasshopper Lazarillo, a GPI-anchored Lipocalin, increases Drosophila longevity and stress resistance, and functionally replaces its secreted homolog NLaz.

    Science.gov (United States)

    Ruiz, Mario; Wicker-Thomas, Claude; Sanchez, Diego; Ganfornina, Maria D

    2012-10-01

    Lazarillo (Laz) is a glycosyl-phosphatidylinositol (GPI)-linked glycoprotein first characterized in the developing nervous system of the grasshopper Schistocerca americana. It belongs to the Lipocalins, a functionally diverse family of mostly secreted proteins. In this work we test whether the protective capacity known for Laz homologs in flies and vertebrates (NLaz, GLaz and ApoD) is evolutionarily conserved in grasshopper Laz, and can be exerted from the plasma membrane in a cell-autonomous manner. First we demonstrate that extracellular forms of Laz have autocrine and paracrine protecting effects for oxidative stress-challenged Drosophila S2 cells. Then we assay the effects of overexpressing GPI-linked Laz in adult Drosophila and whether it rescues both known and novel phenotypes of NLaz null mutants. Local effects of GPI-linked Laz inside and outside the nervous system promote survival upon different stress forms, and extend lifespan and healthspan of the flies in a cell-type dependent manner. Outside the nervous system, expression in fat body cells but not in hemocytes results in protection. Within the nervous system, glial cell expression is more effective than neuronal expression. Laz actions are sexually dimorphic in some expression domains. Fat storage promotion and not modifications in hydrocarbon profiles or quantities explain the starvation-desiccation resistance caused by Laz overexpression. This effect is exerted when Laz is expressed ubiquitously or in dopaminergic cells, but not in hemocytes. Grasshopper Laz functionally restores the loss of NLaz, rescuing stress-sensitivity as well as premature accumulation of aging-related damage, monitored by advanced glycation end products (AGEs). However Laz does not rescue NLaz courtship behavioral defects. Finally, the presence of two new Lipocalins with predicted GPI-anchors in mosquitoes shows that the functional advantages of GPI-linkage have been commonly exploited by Lipocalins in the arthropodan lineage

  2. Glycolipid precursors for the membrane anchor of Trypanosoma brucei variant surface glycoproteins. II. Lipid structures of phosphatidylinositol-specific phospholipase C sensitive and resistant glycolipids

    International Nuclear Information System (INIS)

    Mayor, S.; Menon, A.K.; Cross, G.A.

    1990-01-01

    A common diagnostic feature of glycosylinositol phospholipid (GPI)-anchored proteins is their release from the membrane by a phosphatidylinositol-specific phospholipase C (PI-PLC). However, some GPI-anchored proteins are resistant to this enzyme. The best characterized example of this subclass is the human erythrocyte acetylcholinesterase, where the structural basis of PI-PLC resistance has been shown to be the acylation of an inositol hydroxyl group(s). Both PI-PLC-sensitive and resistant GPI-anchor precursors (P2 and P3, respectively) have been found in Trypanosoma brucei, where the major surface glycoprotein is anchored by a PI-PLC-sensitive glycolipid anchor. The accompanying paper shows that P2 and P3 have identical glycans, indistinguishable from the common core glycan found on all the characterized GPI protein anchors. This paper shows that the single difference between P2 and P3, and the basis for the PI-PLC insusceptibility of P3, is a fatty acid, ester-linked to the inositol residue in P3. The inositol-linked fatty acid can be removed by treatment with mild base to restore PI-PLC sensitivity. Biosynthetic labeling experiments with [3H]palmitic acid and [3H]myristic acid show that [3H]palmitic acid specifically labels the inositol residue in P3 while [3H]myristic acid labels the diacylglycerol portion. Possible models to account for the simultaneous presence of PI-PLC-resistant and sensitive glycolipids are discussed in the context of available information on the biosynthesis of GPI-anchors

  3. Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

    Science.gov (United States)

    Zhang, Li; Liang, Shuli; Zhou, Xinying; Jin, Zi; Jiang, Fengchun; Han, Shuangyan; Zheng, Suiping

    2013-01-01

    Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro. In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface. PMID:23835174

  4. N-Glycosylation instead of cholesterol mediates oligomerization and apical sorting of GPI-APs in FRT cells.

    Science.gov (United States)

    Imjeti, Naga Salaija; Lebreton, Stéphanie; Paladino, Simona; de la Fuente, Erwin; Gonzalez, Alfonso; Zurzolo, Chiara

    2011-12-01

    Sorting of glycosylphosphatidyl-inositol--anchored proteins (GPI-APs) in polarized epithelial cells is not fully understood. Oligomerization in the Golgi complex has emerged as the crucial event driving apical segregation of GPI-APs in two different kind of epithelial cells, Madin-Darby canine kidney (MDCK) and Fisher rat thyroid (FRT) cells, but whether the mechanism is conserved is unknown. In MDCK cells cholesterol promotes GPI-AP oligomerization, as well as apical sorting of GPI-APs. Here we show that FRT cells lack this cholesterol-driven oligomerization as apical sorting mechanism. In these cells both apical and basolateral GPI-APs display restricted diffusion in the Golgi likely due to a cholesterol-enriched membrane environment. It is striking that N-glycosylation is the critical event for oligomerization and apical sorting of GPI-APs in FRT cells but not in MDCK cells. Our data indicate that at least two mechanisms exist to determine oligomerization in the Golgi leading to apical sorting of GPI-APs. One depends on cholesterol, and the other depends on N-glycosylation and is insensitive to cholesterol addition or depletion.

  5. Bst1 is required for Candida albicans infecting host via facilitating cell wall anchorage of Glycosylphosphatidyl inositol anchored proteins

    Science.gov (United States)

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

    2016-01-01

    Glycosylphosphatidyl inositol anchored proteins (GPI-APs) on fungal cell wall are essential for invasive infections. While the function of inositol deacylation of GPI-APs in mammalian cells has been previously characterized the impact of inositol deacylation in fungi and implications to host infection remains largely unexplored. Herein we describe our identification of BST1, an inositol deacylase of GPI-Aps in Candida albicans, was critical for GPI-APs cell wall attachment and host infection. BST1-deficient C. albicans (bst1Δ/Δ) was associated with severely impaired cell wall anchorage of GPI-APs and subsequen unmasked β-(1,3)-glucan. Consistent with the aberrant cell wall structures, bst1Δ/Δ strain did not display an invasive ability and could be recognized more efficiently by host immune systems. Moreover, BST1 null mutants or those expressing Bst1 variants did not display inositol deacylation activity and exhibited severely attenuated virulence and reduced organic colonization in a murine systemic candidiasis model. Thus, Bst1 can facilitate cell wall anchorage of GPI-APs in C. albicans by inositol deacylation, and is critical for host invasion and immune escape. PMID:27708385

  6. Increased infectivity of anchorless mouse scrapie prions in transgenic mice overexpressing human prion protein.

    Science.gov (United States)

    Race, Brent; Phillips, Katie; Meade-White, Kimberly; Striebel, James; Chesebro, Bruce

    2015-06-01

    Prion protein (PrP) is found in all mammals, mostly as a glycoprotein anchored to the plasma membrane by a C-terminal glycosylphosphatidylinositol (GPI) linkage. Following prion infection, host protease-sensitive prion protein (PrPsen or PrPC) is converted into an abnormal, disease-associated, protease-resistant form (PrPres). Biochemical characteristics, such as the PrP amino acid sequence, and posttranslational modifications, such as glycosylation and GPI anchoring, can affect the transmissibility of prions as well as the biochemical properties of the PrPres generated. Previous in vivo studies on the effects of GPI anchoring on prion infectivity have not examined cross-species transmission. In this study, we tested the effect of lack of GPI anchoring on a species barrier model using mice expressing human PrP. In this model, anchorless 22L prions derived from tg44 mice were more infectious than 22L prions derived from C57BL/10 mice when tested in tg66 transgenic mice, which expressed wild-type anchored human PrP at 8- to 16-fold above normal. Thus, the lack of the GPI anchor on the PrPres from tg44 mice appeared to reduce the effect of the mouse-human PrP species barrier. In contrast, neither source of prions induced disease in tgRM transgenic mice, which expressed human PrP at 2- to 4-fold above normal. Prion protein (PrP) is found in all mammals, usually attached to cells by an anchor molecule called GPI. Following prion infection, PrP is converted into a disease-associated form (PrPres). While most prion diseases are species specific, this finding is not consistent, and species barriers differ in strength. The amino acid sequence of PrP varies among species, and this variability affects prion species barriers. However, other PrP modifications, including glycosylation and GPI anchoring, may also influence cross-species infectivity. We studied the effect of PrP GPI anchoring using a mouse-to-human species barrier model. Experiments showed that prions produced by

  7. Homozygous missense mutation (G56R in glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPI-HBP1 in two siblings with fasting chylomicronemia (MIM 144650

    Directory of Open Access Journals (Sweden)

    Hegele Robert A

    2007-09-01

    Full Text Available Abstract Background Mice with a deleted Gpihbp1 gene encoding glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPI-HBP1 develop severe chylomicronemia. We screened the coding regions of the human homologue – GPIHBP1 – from the genomic DNA of 160 unrelated adults with fasting chylomicronemia and plasma triglycerides >10 mmol/L, each of whom had normal sequence of the LPL and APOC2 genes. Results One patient with severe type 5 hyperlipoproteinemia (MIM 144650, fasting chylomicronemia and relapsing pancreatitis resistant to standard therapy was found to be homozygous for a novel GPIHBP1 missense variant, namely G56R. This mutation was absent from the genomes of 600 control subjects and 610 patients with hyperlipidemia. The GPIHBP1 G56 residue has been conserved throughout evolution and the G56R mutation was predicted to have compromised function. Her homozygous brother also had refractory chylomicronemia and relapsing pancreatitis together with early coronary heart disease. G56R heterozygotes in the family had fasting mild hypertriglyceridemia. Conclusion Thus, a very rare GPIHBP1 missense mutation appears to be associated with severe hypertriglyceridemia and chylomicronemia.

  8. Sex-specific and blood meal-induced proteins of Anopheles gambiae midguts: analysis by two-dimensional gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Laurent-Winter C

    2003-02-01

    Full Text Available Abstract Background Anopheles gambiae is the main vector of Plasmodium falciparum in Africa. The mosquito midgut constitutes a barrier that the parasite must cross if it is to develop and be transmitted. Despite the central role of the mosquito midgut in the host/parasite interaction, little is known about its protein composition. Characterisation of An. gambiae midgut proteins may identify the proteins that render An. gambiae receptive to the malaria parasite. Methods We carried out two-dimensional gel electrophoresis of An. gambiae midgut proteins and compared protein profiles for midguts from males, sugar-fed females and females fed on human blood. Results Very few differences were detected between male and female mosquitoes for the approximately 375 silver-stained proteins. Male midguts contained ten proteins not detected in sugar-fed or blood-fed females, which are therefore probably involved in male-specific functions; conversely, female midguts contained twenty-three proteins absent from male midguts. Eight of these proteins were specific to sugar-fed females, and another ten, to blood-fed females. Conclusion Mass spectrometry analysis of the proteins found only in blood-fed female midguts, together with data from the recent sequencing of the An. gambiae genome, should make it possible to determine the role of these proteins in blood digestion or parasite receptivity.

  9. A glycosylphosphatidylinositol anchor is required for membrane localization but dispensable for cell wall association of chitin deacetylase 2 in Cryptococcus neoformans.

    Science.gov (United States)

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

    2012-01-01

    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. The surface of a pathogenic microbe is a major interface with its host. In fungi, the outer surface consists of a complex matrix known as the cell wall, which includes polysaccharides, proteins, and other molecules. The mammalian host recognizes many of these surface molecules and mounts appropriate responses to combat the microbial infection. Cryptococcus neoformans is a

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

    Science.gov (United States)

    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.

  11. Proteomics-based identification of midgut proteins correlated with Cry1Ac resistance in Plutella xylostella (L.).

    Science.gov (United States)

    Xia, Jixing; Guo, Zhaojiang; Yang, Zezhong; Zhu, Xun; Kang, Shi; Yang, Xin; Yang, Fengshan; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Xu, Weijun; Zhang, Youjun

    2016-09-01

    The diamondback moth, Plutella xylostella (L.), is a worldwide pest of cruciferous crops and can rapidly develop resistance to many chemical insecticides. Although insecticidal crystal proteins (i.e., Cry and Cyt toxins) derived from Bacillus thuringiensis (Bt) have been useful alternatives to chemical insecticides for the control of P. xylostella, resistance to Bt in field populations of P. xylostella has already been reported. A better understanding of the resistance mechanisms to Bt should be valuable in delaying resistance development. In this study, the mechanisms underlying P. xylostella resistance to Bt Cry1Ac toxin were investigated using two-dimensional differential in-gel electrophoresis (2D-DIGE) and ligand blotting for the first time. Comparative analyses of the constitutive expression of midgut proteins in Cry1Ac-susceptible and -resistant P. xylostella larvae revealed 31 differentially expressed proteins, 21 of which were identified by mass spectrometry. Of these identified proteins, the following fell into diverse eukaryotic orthologous group (KOG) subcategories may be involved in Cry1Ac resistance in P. xylostella: ATP-binding cassette (ABC) transporter subfamily G member 4 (ABCG4), trypsin, heat shock protein 70 (HSP70), vacuolar H(+)-ATPase, actin, glycosylphosphatidylinositol anchor attachment 1 protein (GAA1) and solute carrier family 30 member 1 (SLC30A1). Additionally, ligand blotting identified the following midgut proteins as Cry1Ac-binding proteins in Cry1Ac-susceptible P. xylostella larvae: ABC transporter subfamily C member 1 (ABCC1), solute carrier family 36 member 1 (SLC36A1), NADH dehydrogenase iron-sulfur protein 3 (NDUFS3), prohibitin and Rap1 GTPase-activating protein 1. Collectively, these proteomic results increase our understanding of the molecular resistance mechanisms to Bt Cry1Ac toxin in P. xylostella and also demonstrate that resistance to Bt Cry1Ac toxin is complex and multifaceted. Copyright © 2016 Elsevier B.V. All

  12. Plasmodium falciparum merozoite surface protein 1 - Glycosylation and localization to low-density, detergent-resistant membranes in the parasitized erythrocyte

    DEFF Research Database (Denmark)

    Hoessli, D.C.; Poincelet, M.; Gupta, Ramneek

    2003-01-01

    In addition to the major carbohydrate moieties of the glycosylphosphatidylinositol (GPI) anchor, we report that Plasmodium falciparum merozoite surface protein 1 (MSP-1) bears O-GlcNAc modifications predominantly in beta-anomeric configuration, in both the C- and N-terminal portions of the protein....... Subcellular fractionation of parasitized erythrocytes in the late trophozoite/schizont stage reveals that GPI-anchored C-terminal fragments of MSP-1 are recovered in Triton X-100 resistant, low-density membrane fractions. Our results suggest that O -GlcNAc-modified MSP-1 N-terminal fragments tend to localize...... within the parasitophorous vacuolar membrane while GPI-anchored MSP-1 C-terminal fragments associate with low-density, Triton X-100 resistant membrane domains (rafts), redistribute in the parasitized erythrocyte and are eventually shed as membrane vesicles that also contain the endogenous, GPI-linked CD...

  13. Protein expression in the midgut of sugar-fed Aedes albopictus females

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    Saboia-Vahia Leonardo

    2012-12-01

    Full Text Available Abstract Background Aedes albopictus is a vector for several fatal arboviruses in tropical and sub-tropical regions of the world. The midgut of the mosquito is the first barrier that pathogens must overcome to establish infection and represents one of the main immunologically active sites of the insect. Nevertheless, little is known about the proteins involved in the defense against pathogens, and even in the processing of food, and the detoxification of metabolites. The identification of proteins exclusively expressed in the midgut is the first step in understanding the complex physiology of this tissue and can provide insight into the mechanisms of pathogen-vector interaction. However, identification of the locally expressed proteins presents a challenge because the Ae. albopictus genome has not been sequenced. Methods In this study, two-dimensional electrophoresis (2DE was combined with liquid chromatography in line with tandem mass spectrometry (LC-MS/MS and data mining to identify the major proteins in the midgut of sugar-fed Ae. albopictus females. Results Fifty-six proteins were identified by sequence similarity to entries from the Ae. aegypti genome. In addition, two hypothetical proteins were experimentally confirmed. According to the gene ontology analysis, the identified proteins were classified into 16 clusters of biological processes. Use of the STRING database to investigate protein functional associations revealed five functional networks among the identified proteins, including a network for carbohydrate and amino acid metabolism, a group associated with ATP production and a network of proteins that interact during detoxification of toxic free radicals, among others. This analysis allowed the assignment of a potential role for proteins with unknown function based on their functional association with other characterized proteins. Conclusion Our findings represent the first proteome map of the Ae. albopictus midgut and denotes the

  14. A lepidopteran-specific gene family encoding valine-rich midgut proteins.

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    Jothini Odman-Naresh

    Full Text Available Many lepidopteran larvae are serious agricultural pests due to their feeding activity. Digestion of the plant diet occurs mainly in the midgut and is facilitated by the peritrophic matrix (PM, an extracellular sac-like structure, which lines the midgut epithelium and creates different digestive compartments. The PM is attracting increasing attention to control lepidopteran pests by interfering with this vital function. To identify novel PM components and thus potential targets for insecticides, we performed an immunoscreening with anti-PM antibodies using an expression library representing the larval midgut transcriptome of the tobacco hornworm, Manduca sexta. We identified three cDNAs encoding valine-rich midgut proteins of M. sexta (MsVmps, which appear to be loosely associated with the PM. They are members of a lepidopteran-specific family of nine VMP genes, which are exclusively expressed in larval stages in M. sexta. Most of the MsVMP transcripts are detected in the posterior midgut, with the highest levels observed for MsVMP1. To obtain further insight into Vmp function, we expressed MsVMP1 in insect cells and purified the recombinant protein. Lectin staining and glycosidase treatment indicated that MsVmp1 is highly O-glycosylated. In line with results from qPCR, immunoblots revealed that MsVmp1 amounts are highest in feeding larvae, while MsVmp1 is undetectable in starving and molting larvae. Finally using immunocytochemistry, we demonstrated that MsVmp1 localizes to the cytosol of columnar cells, which secrete MsVmp1 into the ectoperitrophic space in feeding larvae. In starving and molting larvae, MsVmp1 is found in the gut lumen, suggesting that the PM has increased its permeability. The present study demonstrates that lepidopteran species including many agricultural pests have evolved a set of unique proteins that are not found in any other taxon and thus may reflect an important adaptation in the highly specialized lepidopteran

  15. Differential protein modulation in midguts of Aedes aegypti infected with chikungunya and dengue 2 viruses.

    Science.gov (United States)

    Tchankouo-Nguetcheu, Stéphane; Khun, Huot; Pincet, Laurence; Roux, Pascal; Bahut, Muriel; Huerre, Michel; Guette, Catherine; Choumet, Valérie

    2010-10-05

    Arthropod borne virus infections cause several emerging and resurgent infectious diseases. Among the diseases caused by arboviruses, dengue and chikungunya are responsible for a high rate of severe human diseases worldwide. The midgut of mosquitoes is the first barrier for pathogen transmission and is a target organ where arboviruses must replicate prior to infecting other organs. A proteomic approach was undertaken to characterize the key virus/vector interactions and host protein modifications that happen in the midgut for viral transmission to eventually take place. Using a proteomics differential approach with two-Dimensional Differential in-Gel Electrophoresis (2D-DIGE), we defined the protein modulations in the midgut of Aedes aegypti that were triggered seven days after an oral infection (7 DPI) with dengue 2 (DENV-2) and chikungunya (CHIKV) viruses. Gel profile comparisons showed that the level of 18 proteins was modulated by DENV-2 only and 12 proteins were modulated by CHIKV only. Twenty proteins were regulated by both viruses in either similar or different ways. Both viruses caused an increase of proteins involved in the generation of reactive oxygen species, energy production, and carbohydrate and lipid metabolism. Midgut infection by DENV-2 and CHIKV triggered an antioxidant response. CHIKV infection produced an increase of proteins involved in detoxification. Our study constitutes the first analysis of the protein response of Aedes aegypti's midgut infected with viruses belonging to different families. It shows that the differentially regulated proteins in response to viral infection include structural, redox, regulatory proteins, and enzymes for several metabolic pathways. Some of these proteins like antioxidant are probably involved in cell protection. On the other hand, we propose that the modulation of other proteins like transferrin, hsp60 and alpha glucosidase, may favour virus survival, replication and transmission, suggesting a subversion of

  16. Differential protein modulation in midguts of Aedes aegypti infected with chikungunya and dengue 2 viruses.

    Directory of Open Access Journals (Sweden)

    Stéphane Tchankouo-Nguetcheu

    Full Text Available BACKGROUND: Arthropod borne virus infections cause several emerging and resurgent infectious diseases. Among the diseases caused by arboviruses, dengue and chikungunya are responsible for a high rate of severe human diseases worldwide. The midgut of mosquitoes is the first barrier for pathogen transmission and is a target organ where arboviruses must replicate prior to infecting other organs. A proteomic approach was undertaken to characterize the key virus/vector interactions and host protein modifications that happen in the midgut for viral transmission to eventually take place. METHODOLOGY AND PRINCIPAL FINDINGS: Using a proteomics differential approach with two-Dimensional Differential in-Gel Electrophoresis (2D-DIGE, we defined the protein modulations in the midgut of Aedes aegypti that were triggered seven days after an oral infection (7 DPI with dengue 2 (DENV-2 and chikungunya (CHIKV viruses. Gel profile comparisons showed that the level of 18 proteins was modulated by DENV-2 only and 12 proteins were modulated by CHIKV only. Twenty proteins were regulated by both viruses in either similar or different ways. Both viruses caused an increase of proteins involved in the generation of reactive oxygen species, energy production, and carbohydrate and lipid metabolism. Midgut infection by DENV-2 and CHIKV triggered an antioxidant response. CHIKV infection produced an increase of proteins involved in detoxification. CONCLUSION/SIGNIFICANCE: Our study constitutes the first analysis of the protein response of Aedes aegypti's midgut infected with viruses belonging to different families. It shows that the differentially regulated proteins in response to viral infection include structural, redox, regulatory proteins, and enzymes for several metabolic pathways. Some of these proteins like antioxidant are probably involved in cell protection. On the other hand, we propose that the modulation of other proteins like transferrin, hsp60 and alpha

  17. Possible Insecticidal Mechanisms Mediated by Immune-Response-Related Cry-Binding Proteins in the Midgut Juice of Plutella xylostella and Spodoptera exigua.

    Science.gov (United States)

    Lu, Keyu; Gu, Yuqing; Liu, Xiaoping; Lin, Yi; Yu, Xiao-Qiang

    2017-03-15

    Cry toxins are insecticidal toxin proteins produced by a spore-forming Gram-positive bacterium Bacillus thuringiensis. Interactions between the Cry toxins and the receptors from midgut brush border membrane vesicles (BBMVs), such as cadherin, alkaline phosphatase, and aminopeptidase, are key steps for the specificity and insecticidal activity of Cry proteins. However, little is known about the midgut juice proteins that may interfere with Cry binding to the receptors. To validate the hypothesis that there exist Cry-binding proteins that can interfere with the insecticidal process of Cry toxins, we applied Cry1Ab1-coupled Sepharose beads to isolate Cry-binding proteins form midgut juice of Plutella xylostella and Spodoptera exigua. Trypsin-like serine proteases and Dorsal were found to be Cry1Ab1-binding proteins in the midgut juice of P. xylostella. Peroxidase-C (POX-C) was found to be the Cry1Ab1-binding protein in the midgut juice of S. exigua. We proposed possible insecticidal mechanisms of Cry1Ab1 mediated by the two immune-related proteins: Dorsal and POX-C. Our results suggested that there exist, in the midgut juice, Cry-binding proteins, which are different from BBMV-specific receptors.

  18. Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions.

    Science.gov (United States)

    Tavares, Evandro; Macedo, Joana A; Paulo, Pedro M R; Tavares, Catarina; Lopes, Carlos; Melo, Eduardo P

    2014-07-01

    Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10±2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17±2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4±1% to 7±1% in the stable clone and from 10±2% to 16±1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Anchoring Proteins as Regulators of Signaling Pathways

    Science.gov (United States)

    Perino, Alessia; Ghigo, Alessandra; Scott, John D.; Hirsch, Emilio

    2012-01-01

    Spatial and temporal organization of signal transduction is coordinated through the segregation of signaling enzymes in selected cellular compartments. This highly evolved regulatory mechanism ensures the activation of selected enzymes only in the vicinity of their target proteins. In this context, cAMP-responsive triggering of protein kinase A is modulated by a family of scaffold proteins referred to as A-kinase anchoring proteins. A-kinase anchoring proteins form the core of multiprotein complexes and enable simultaneous but segregated cAMP signaling events to occur in defined cellular compartments. In this review we will focus on the description of A-kinase anchoring protein function in the regulation of cardiac physiopathology. PMID:22859670

  20. The Glycosylphosphatidylinositol-Anchored Variable Region of Llama Heavy Chain-Only Antibody JM4 Efficiently Blocks both Cell-Free and T Cell-T Cell Transmission of Human Immunodeficiency Virus Type 1.

    Science.gov (United States)

    Liu, Lihong; Wang, Weiming; Matz, Julie; Ye, Chaobaihui; Bracq, Lucie; Delon, Jerome; Kimata, Jason T; Chen, Zhiwei; Benichou, Serge; Zhou, Paul

    2016-12-01

    The variable regions (VHHs) of two heavy chain-only antibodies, JM2 and JM4, from llamas that have been immunized with a trimeric gp140 bound to a CD4 mimic have been recently isolated (here referred to as VHH JM2 and VHH JM4, respectively). JM2 binds the CD4-binding site of gp120 and neutralizes HIV-1 strains from subtypes B, C, and G. JM4 binds gp120 and neutralizes HIV-1 strains from subtypes A, B, C, A/E, and G in a CD4-dependent manner. In the present study, we constructed glycosylphosphatidylinositol (GPI)-anchored VHH JM2 and JM4 along with an E4 control and transduced them into human CD4 + cell lines and primary CD4 T cells. We report that by genetically linking the VHHs with a GPI attachment signal, VHHs are targeted to the lipid rafts of the plasma membranes. Expression of GPI-VHH JM4, but not GPI-VHH E4 and JM2, on the surface of transduced TZM.bl cells potently neutralizes multiple subtypes of HIV-1 isolates, including tier 2 or 3 strains, transmitted founders, quasispecies, and soluble single domain antibody (sdAb) JM4-resistant viruses. Moreover, transduction of CEMss-CCR5 cells with GPI-VHH JM4, but not with GPI-VHH E4, confers resistance to both cell-free and T cell-T cell transmission of HIV-1 and HIV-1 envelope-mediated fusion. Finally, GPI-VHH JM4-transduced human primary CD4 T cells efficiently resist both cell-free and T cell-T cell transmission of HIV-1. Thus, we conclude that VHH JM4, when targeted to the lipid rafts of the plasma membrane, efficiently neutralizes HIV-1 infection via both cell-free and T cell-T cell transmission. Our findings should have important implications for GPI-anchored antibody-based therapy against HIV-1. Lipid rafts are specialized dynamic microdomains of the plasma membrane and have been shown to be gateways for HIV-1 budding as well as entry into T cells and macrophages. In nature, many glycosylphosphatidylinositol (GPI)-anchored proteins localize in the lipid rafts. In the present study, we developed GPI-anchored

  1. Protein synthesis in body wall and midgut during the larval moulting cycle of the silkworm, Bombyx mori

    International Nuclear Information System (INIS)

    Nagata, Masao

    1976-01-01

    14 C-leucine was injected into larvae at various points of time during the fourth moulting cycle, and four hours after the injection, dissection was carried out. The intake of radioactivity into the proteins of body wall and midgut was investigated. The specific activity of the proteins changed with the moulting of larvae, but the specific activity of the amino acid pool in tissues also changed. Accordingly, the change of specific activity in proteins was not able to be regarded as the variation of synthetic capability. The ratio of the specific activity of proteins to that of amino acid pool was determined, and it was revealed that the capability of protein synthesis of body wall and midgut was high in feeding period and lowered in moulting period, and it was different according to various stages of moulting period. As the result of investigation on the time course of the specific activity of proteins of body wall and midgut after the injection of 14 C-leucine, it was shown that the specific activity reached the maximum earlier in feeding period than in moulting period. The intake of 14 C-leucine into body wall and midgut was examined with 20 min. labeling, consequently the specific activity of proteins was high in feeding period and low in moulting period. The capability of protein synthesis was active in feeding period and dwindled in moulting period in case of the whole body of larvae. (Kako, I.)

  2. Cadmium-binding proteins in midgut gland of freshwater crayfish Procambarus clarkii

    Energy Technology Data Exchange (ETDEWEB)

    Del Ramo, J.; Pastor, A.; Torreblanca, A.; Medina, J.; Diza-Mayans, J.

    1989-02-01

    Metallothioneins, metal binding proteins, were originally isolated and characterized by Margoshes and Vallee. These proteins have a high affinity for various heavy metals, particularly cadmium and mercury and have extensively been studied in mammals. Metal binding proteins have been observed in a variety of marine invertebrates; however, there is very little information available on metal binding proteins in freshwater invertebrates, and particularly in freshwater crustaceans. Cadmium is an ubiquitous non essential element which possesses high toxicity to aquatic organisms. Cadmium binding proteins observed in invertebrates have similar characteristics to mammalian metallothioneins. In 1978, the American red crayfish appeared in Albufera Lake and the surrounding rice fields (Valencia, Spain). Albufera Lake and the surrounding rice fields waters are subjected to very heavy loads of sewage and toxic industrial residues (including heavy metals) from the many urban and wastewaters in this area. In previous reports the authors studied the toxicity and accumulation of cadmium on Procambarus clarkii of Albufera Lake. This crayfish shows a high resistance to cadmium and a great accumulation rate of this metal in several tissues, including midgut gland. Since Procambarus clarkii shows a high resistance to cadmium, the presence of cadmium binding proteins (Cd-BP) in midgut gland of these crayfish would be expected. This report describes results on the characterization of Cd-BPs obtained from cadmium exposed crayfish Procambarus clarkii, demonstrating their presence in this freshwater crayfish.

  3. Protein-anchoring therapy to target extracellular matrix proteins to their physiological destinations.

    Science.gov (United States)

    Ito, Mikako; Ohno, Kinji

    2018-02-20

    Endplate acetylcholinesterase (AChE) deficiency is a form of congenital myasthenic syndrome (CMS) caused by mutations in COLQ, which encodes collagen Q (ColQ). ColQ is an extracellular matrix (ECM) protein that anchors AChE to the synaptic basal lamina. Biglycan, encoded by BGN, is another ECM protein that binds to the dystrophin-associated protein complex (DAPC) on skeletal muscle, which links the actin cytoskeleton and ECM proteins to stabilize the sarcolemma during repeated muscle contractions. Upregulation of biglycan stabilizes the DPAC. Gene therapy can potentially ameliorate any disease that can be recapitulated in cultured cells. However, the difficulty of tissue-specific and developmental stage-specific regulated expression of transgenes, as well as the difficulty of introducing a transgene into all cells in a specific tissue, prevents us from successfully applying gene therapy to many human diseases. In contrast to intracellular proteins, an ECM protein is anchored to the target tissue via its specific binding affinity for protein(s) expressed on the cell surface within the target tissue. Exploiting this unique feature of ECM proteins, we developed protein-anchoring therapy in which a transgene product expressed even in remote tissues can be delivered and anchored to a target tissue using specific binding signals. We demonstrate the application of protein-anchoring therapy to two disease models. First, intravenous administration of adeno-associated virus (AAV) serotype 8-COLQ to Colq-deficient mice, resulting in specific anchoring of ectopically expressed ColQ-AChE at the NMJ, markedly improved motor functions, synaptic transmission, and the ultrastructure of the neuromuscular junction (NMJ). In the second example, Mdx mice, a model for Duchenne muscular dystrophy, were intravenously injected with AAV8-BGN. The treatment ameliorated motor deficits, mitigated muscle histopathologies, decreased plasma creatine kinase activities, and upregulated expression

  4. PrP Knockout Cells Expressing Transmembrane PrP Resist Prion Infection.

    Science.gov (United States)

    Marshall, Karen E; Hughson, Andrew; Vascellari, Sarah; Priola, Suzette A; Sakudo, Akikazu; Onodera, Takashi; Baron, Gerald S

    2017-01-15

    Glycosylphosphatidylinositol (GPI) anchoring of the prion protein (PrP C ) influences PrP C misfolding into the disease-associated isoform, PrP res , as well as prion propagation and infectivity. GPI proteins are found in cholesterol- and sphingolipid-rich membrane regions called rafts. Exchanging the GPI anchor for a nonraft transmembrane sequence redirects PrP C away from rafts. Previous studies showed that nonraft transmembrane PrP C variants resist conversion to PrP res when transfected into scrapie-infected N2a neuroblastoma cells, likely due to segregation of transmembrane PrP C and GPI-anchored PrP res in distinct membrane environments. Thus, it remained unclear whether transmembrane PrP C might convert to PrP res if seeded by an exogenous source of PrP res not associated with host cell rafts and without the potential influence of endogenous expression of GPI-anchored PrP C To further explore these questions, constructs containing either a C-terminal wild-type GPI anchor signal sequence or a nonraft transmembrane sequence containing a flexible linker were expressed in a cell line derived from PrP knockout hippocampal neurons, NpL2. NpL2 cells have physiological similarities to primary neurons, representing a novel and advantageous model for studying transmissible spongiform encephalopathy (TSE) infection. Cells were infected with inocula from multiple prion strains and in different biochemical states (i.e., membrane bound as in brain microsomes from wild-type mice or purified GPI-anchorless amyloid fibrils). Only GPI-anchored PrP C supported persistent PrP res propagation. Our data provide strong evidence that in cell culture GPI anchor-directed membrane association of PrP C is required for persistent PrP res propagation, implicating raft microdomains as a location for conversion. Mechanisms of prion propagation, and what makes them transmissible, are poorly understood. Glycosylphosphatidylinositol (GPI) membrane anchoring of the prion protein (PrP C

  5. Generation of a monoclonal antibody against the glycosylphosphatidylinositol-linked protein Rae-1 using genetically engineered tumor cells.

    Science.gov (United States)

    Hu, Jiemiao; Vien, Long T; Xia, Xueqing; Bover, Laura; Li, Shulin

    2014-02-04

    Although genetically engineered cells have been used to generate monoclonal antibodies (mAbs) against numerous proteins, no study has used them to generate mAbs against glycosylphosphatidylinositol (GPI)-anchored proteins. The GPI-linked protein Rae-1, an NKG2D ligand member, is responsible for interacting with immune surveillance cells. However, very few high-quality mAbs against Rae-1 are available for use in multiple analyses, including Western blotting, immunohistochemistry, and flow cytometry. The lack of high-quality mAbs limits the in-depth analysis of Rae-1 fate, such as shedding and internalization, in murine models. Moreover, currently available screening approaches for identifying high-quality mAbs are excessively time-consuming and costly. We used Rae-1-overexpressing CT26 tumor cells to generate 60 hybridomas that secreted mAbs against Rae-1. We also developed a streamlined screening strategy for selecting the best anti-Rae-1 mAb for use in flow cytometry assay, enzyme-linked immunosorbent assay, Western blotting, and immunostaining. Our cell line-based immunization approach can yield mAbs against GPI-anchored proteins, and our streamlined screening strategy can be used to select the ideal hybridoma for producing such mAbs.

  6. Phosphatidylinositol-glycan-phospholipase D is involved in neurodegeneration in prion disease.

    Directory of Open Access Journals (Sweden)

    Jae-Kwang Jin

    Full Text Available PrPSc is formed from a normal glycosylphosphatidylinositol (GPI-anchored prion protein (PrPC by a posttranslational modification. Most GPI-anchored proteins have been shown to be cleaved by GPI phospholipases. Recently, GPI-phospholipase D (GPI-PLD was shown to be a strictly specific enzyme for GPI anchors. To investigate the involvement of GPI-PLD in the processes of neurodegeneration in prion diseases, we examined the mRNA and protein expression levels of GPI-PLD in the brains of a prion animal model (scrapie, and in both the brains and cerebrospinal fluids (CSF of sporadic and familial Creutzfeldt-Jakob disease (CJD patients. We found that compared with controls, the expression of GPI-PLD was dramatically down-regulated in the brains of scrapie-infected mice, especially in the caveolin-enriched membrane fractions. Interestingly, the observed decrease in GPI-PLD expression levels began at the same time that PrPSc began to accumulate in the infected brains and this decrease was also observed in both the brain and CSF of CJD patients; however, no differences in expression were observed in either the brains or CSF specimens from Alzheimer's disease patients. Taken together, these results suggest that the down-regulation of GPI-PLD protein may be involved in prion propagation in the brains of prion diseases.

  7. Redox Status of β2GPI in Different Stages of Diabetic Angiopathy

    Directory of Open Access Journals (Sweden)

    Jun Ma

    2016-01-01

    Full Text Available We explored the redox status of beta 2 glycoprotein I (β2GPI in different stages of diabetic angiopathy. Type 2 diabetes mellitus (T2DM had a significantly lower proportion of reduced β2GPI as compared to healthy controls (p0.05. The mild-A-stenosis group and mild-diabetic retinopathy (DR groups had higher proportion of reduced β2GPI than their severely affected counterparts. The mild-slow nerve conduction velocity (NCVS group had higher proportion of reduced β2GPI than normal nerve conduction velocity (NCVN group and severe-NCVS groups. The proportion of reduced β2GPI was in positive correlation with 24 h urine microalbumin and total urine protein, and the proportion of reduced β2GPI was in negative correlation with serum and skin advanced glycation end products (AGEs. Taken together, our data implicate that the proportion of reduced β2GPI increased in the early stage of angiopathy and decreased with the aggravation of angiopathy.

  8. A novel dimeric inhibitor targeting Beta2GPI in Beta2GPI/antibody complexes implicated in antiphospholipid syndrome.

    Directory of Open Access Journals (Sweden)

    Alexey Kolyada

    2010-12-01

    Full Text Available β2GPI is a major antigen for autoantibodies associated with antiphospholipid syndrome (APS, an autoimmune disease characterized by thrombosis and recurrent pregnancy loss. Only the dimeric form of β2GPI generated by anti-β2GPI antibodies is pathologically important, in contrast to monomeric β2GPI which is abundant in plasma.We created a dimeric inhibitor, A1-A1, to selectively target β2GPI in β2GPI/antibody complexes. To make this inhibitor, we isolated the first ligand-binding module from ApoER2 (A1 and connected two A1 modules with a flexible linker. A1-A1 interferes with two pathologically important interactions in APS, the binding of β2GPI/antibody complexes with anionic phospholipids and ApoER2. We compared the efficiency of A1-A1 to monomeric A1 for inhibition of the binding of β2GPI/antibody complexes to anionic phospholipids. We tested the inhibition of β2GPI present in human serum, β2GPI purified from human plasma and the individual domain V of β2GPI. We demonstrated that when β2GPI/antibody complexes are formed, A1-A1 is much more effective than A1 in inhibition of the binding of β2GPI to cardiolipin, regardless of the source of β2GPI. Similarly, A1-A1 strongly inhibits the binding of dimerized domain V of β2GPI to cardiolipin compared to the monomeric A1 inhibitor. In the absence of anti-β2GPI antibodies, both A1-A1 and A1 only weakly inhibit the binding of pathologically inactive monomeric β2GPI to cardiolipin.Our results suggest that the approach of using a dimeric inhibitor to block β2GPI in the pathological multivalent β2GPI/antibody complexes holds significant promise. The novel inhibitor A1-A1 may be a starting point in the development of an effective therapeutic for antiphospholipid syndrome.

  9. A Novel Dimeric Inhibitor Targeting Beta2GPI in Beta2GPI/Antibody Complexes Implicated in Antiphospholipid Syndrome

    Energy Technology Data Exchange (ETDEWEB)

    A Kolyada; C Lee; A De Biasio; N Beglova

    2011-12-31

    {beta}2GPI is a major antigen for autoantibodies associated with antiphospholipid syndrome (APS), an autoimmune disease characterized by thrombosis and recurrent pregnancy loss. Only the dimeric form of {beta}2GPI generated by anti-{beta}2GPI antibodies is pathologically important, in contrast to monomeric {beta}2GPI which is abundant in plasma. We created a dimeric inhibitor, A1-A1, to selectively target {beta}2GPI in {beta}2GPI/antibody complexes. To make this inhibitor, we isolated the first ligand-binding module from ApoER2 (A1) and connected two A1 modules with a flexible linker. A1-A1 interferes with two pathologically important interactions in APS, the binding of {beta}2GPI/antibody complexes with anionic phospholipids and ApoER2. We compared the efficiency of A1-A1 to monomeric A1 for inhibition of the binding of {beta}2GPI/antibody complexes to anionic phospholipids. We tested the inhibition of {beta}2GPI present in human serum, {beta}2GPI purified from human plasma and the individual domain V of {beta}2GPI. We demonstrated that when {beta}2GPI/antibody complexes are formed, A1-A1 is much more effective than A1 in inhibition of the binding of {beta}2GPI to cardiolipin, regardless of the source of {beta}2GPI. Similarly, A1-A1 strongly inhibits the binding of dimerized domain V of {beta}2GPI to cardiolipin compared to the monomeric A1 inhibitor. In the absence of anti-{beta}2GPI antibodies, both A1-A1 and A1 only weakly inhibit the binding of pathologically inactive monomeric {beta}2GPI to cardiolipin. Our results suggest that the approach of using a dimeric inhibitor to block {beta}2GPI in the pathological multivalent {beta}2GPI/antibody complexes holds significant promise. The novel inhibitor A1-A1 may be a starting point in the development of an effective therapeutic for antiphospholipid syndrome.

  10. Sialic Acid on the Glycosylphosphatidylinositol Anchor Regulates PrP-mediated Cell Signaling and Prion Formation.

    Science.gov (United States)

    Bate, Clive; Nolan, William; Williams, Alun

    2016-01-01

    The prion diseases occur following the conversion of the cellular prion protein (PrP(C)) into disease-related isoforms (PrP(Sc)). In this study, the role of the glycosylphosphatidylinositol (GPI) anchor attached to PrP(C) in prion formation was examined using a cell painting technique. PrP(Sc) formation in two prion-infected neuronal cell lines (ScGT1 and ScN2a cells) and in scrapie-infected primary cortical neurons was increased following the introduction of PrP(C). In contrast, PrP(C) containing a GPI anchor from which the sialic acid had been removed (desialylated PrP(C)) was not converted to PrP(Sc). Furthermore, the presence of desialylated PrP(C) inhibited the production of PrP(Sc) within prion-infected cortical neurons and ScGT1 and ScN2a cells. The membrane rafts surrounding desialylated PrP(C) contained greater amounts of sialylated gangliosides and cholesterol than membrane rafts surrounding PrP(C). Desialylated PrP(C) was less sensitive to cholesterol depletion than PrP(C) and was not released from cells by treatment with glimepiride. The presence of desialylated PrP(C) in neurons caused the dissociation of cytoplasmic phospholipase A2 from PrP-containing membrane rafts and reduced the activation of cytoplasmic phospholipase A2. These findings show that the sialic acid moiety of the GPI attached to PrP(C) modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP(Sc) formation. These results suggest that pharmacological modification of GPI glycosylation might constitute a novel therapeutic approach to prion diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

    International Nuclear Information System (INIS)

    Grose, C.; Jackson, W.; Traugh, J.A.

    1989-01-01

    Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing [γ- 32 P]ATP. The same glycoprotein was phosphorylated when [ 32 P]GTP was substituted for [ 32 P]ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein

  12. CPB1 of Aedes aegypti Interacts with DENV2 E Protein and Regulates Intracellular Viral Accumulation and Release from Midgut Cells

    Directory of Open Access Journals (Sweden)

    Hong-Wai Tham

    2014-12-01

    Full Text Available Aedes aegypti is a principal vector responsible for the transmission of dengue viruses (DENV. To date, vector control remains the key option for dengue disease management. To develop new vector control strategies, a more comprehensive understanding of the biological interactions between DENV and Ae. aegypti is required. In this study, a cDNA library derived from the midgut of female adult Ae. aegypti was used in yeast two-hybrid (Y2H screenings against DENV2 envelope (E protein. Among the many interacting proteins identified, carboxypeptidase B1 (CPB1 was selected, and its biological interaction with E protein in Ae. aegypti primary midgut cells was further validated. Our double immunofluorescent assay showed that CPB1-E interaction occurred in the endoplasmic reticulum (ER of the Ae. aegypti primary midgut cells. Overexpression of CPB1 in mosquito cells resulted in intracellular DENV2 genomic RNA or virus particle accumulation, with a lower amount of virus release. Therefore, we postulated that in Ae. aegypti midgut cells, CPB1 binds to the E protein deposited on the ER intraluminal membranes and inhibits DENV2 RNA encapsulation, thus inhibiting budding from the ER, and may interfere with immature virus transportation to the trans-Golgi network.

  13. AnchorDock for Blind Flexible Docking of Peptides to Proteins.

    Science.gov (United States)

    Slutzki, Michal; Ben-Shimon, Avraham; Niv, Masha Y

    2017-01-01

    Due to increasing interest in peptides as signaling modulators and drug candidates, several methods for peptide docking to their target proteins are under active development. The "blind" docking problem, where the peptide-binding site on the protein surface is unknown, presents one of the current challenges in the field. AnchorDock protocol was developed by Ben-Shimon and Niv to address this challenge.This protocol narrows the docking search to the most relevant parts of the conformational space. This is achieved by pre-folding the free peptide and by computationally detecting anchoring spots on the surface of the unbound protein. Multiple flexible simulated annealing molecular dynamics (SAMD) simulations are subsequently carried out, starting from pre-folded peptide conformations, constrained to the various precomputed anchoring spots.Here, AnchorDock is demonstrated using two known protein-peptide complexes. A PDZ-peptide complex provides a relatively easy case due to the relatively small size of the protein, and a typical peptide conformation and binding region; a more challenging example is a complex between USP7 N-term and a p53-derived peptide, where the protein is larger, and the peptide conformation and a binding site are generally assumed to be unknown. AnchorDock returned native-like solutions ranked first and third for the PDZ and USP7 complexes, respectively. We describe the procedure step by step and discuss possible modifications where applicable.

  14. Glypican-1 mediates both prion protein lipid raft association and disease isoform formation.

    Directory of Open Access Journals (Sweden)

    David R Taylor

    2009-11-01

    Full Text Available In prion diseases, the cellular form of the prion protein, PrP(C, undergoes a conformational conversion to the infectious isoform, PrP(Sc. PrP(C associates with lipid rafts through its glycosyl-phosphatidylinositol (GPI anchor and a region in its N-terminal domain which also binds to heparan sulfate proteoglycans (HSPGs. We show that heparin displaces PrP(C from rafts and promotes its endocytosis, suggesting that heparin competes with an endogenous raft-resident HSPG for binding to PrP(C. We then utilised a transmembrane-anchored form of PrP (PrP-TM, which is targeted to rafts solely by its N-terminal domain, to show that both heparin and phosphatidylinositol-specific phospholipase C can inhibit its association with detergent-resistant rafts, implying that a GPI-anchored HSPG targets PrP(C to rafts. Depletion of the major neuronal GPI-anchored HSPG, glypican-1, significantly reduced the raft association of PrP-TM and displaced PrP(C from rafts, promoting its endocytosis. Glypican-1 and PrP(C colocalised on the cell surface and both PrP(C and PrP(Sc co-immunoprecipitated with glypican-1. Critically, treatment of scrapie-infected N2a cells with glypican-1 siRNA significantly reduced PrP(Sc formation. In contrast, depletion of glypican-1 did not alter the inhibitory effect of PrP(C on the beta-secretase cleavage of the Alzheimer's amyloid precursor protein. These data indicate that glypican-1 is a novel cellular cofactor for prion conversion and we propose that it acts as a scaffold facilitating the interaction of PrP(C and PrP(Sc in lipid rafts.

  15. β-chain of ATP synthase as a lipophorin binding protein and its role in lipid transfer in the midgut of Panstrongylus megistus (Hemiptera: Reduviidae).

    Science.gov (United States)

    Fruttero, Leonardo L; Demartini, Diogo R; Rubiolo, Edilberto R; Carlini, Célia R; Canavoso, Lilián E

    2014-09-01

    Lipophorin, the main lipoprotein in the circulation of the insects, cycles among peripheral tissues to exchange its lipid cargo at the plasma membrane of target cells, without synthesis or degradation of its apolipoprotein matrix. Currently, there are few characterized candidates supporting the functioning of the docking mechanism of lipophorin-mediated lipid transfer. In this work we combined ligand blotting assays and tandem mass spectrometry to characterize proteins with the property to bind lipophorin at the midgut membrane of Panstrongylus megistus, a vector of Chagas' disease. We further evaluated the role of lipophorin binding proteins in the transfer of lipids between the midgut and lipophorin. The β subunit of the ATP synthase complex (β-ATPase) was identified as a lipophorin binding protein. β-ATPase was detected in enriched midgut membrane preparations free of mitochondria. It was shown that β-ATPase partially co-localizes with lipophorin at the plasma membrane of isolated enterocytes and in the sub-epithelial region of the midgut tissue. The interaction of endogenous lipophorin and β-ATPase was also demonstrated by co-immunoprecipitation assays. Blocking of β-ATPase significantly diminished the binding of lipophorin to the isolated enterocytes and to the midgut tissue. In vivo assays injecting the β-ATPase antibody significantly reduced the transfer of [(3)H]-diacylglycerol from the midgut to the hemolymph in insects fed with [9,10-(3)H]-oleic acid, supporting the involvement of lipophorin-β-ATPase association in the transfer of lipids. In addition, the β-ATPase antibody partially impaired the transfer of fatty acids from lipophorin to the midgut, a less important route of lipid delivery to this tissue. Taken together, the findings strongly suggest that β-ATPase plays a role as a docking lipophorin receptor at the midgut of P. megistus. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Histopathological Effects of Bt and TcdA Insecticidal Proteins on the Midgut Epithelium of Western Corn Rootworm Larvae (Diabrotica virgifera virgifera

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    Andrew J. Bowling

    2017-05-01

    Full Text Available Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte is a major corn pest in the United States, causing annual losses of over $1 billion. One approach to protect against crop loss by this insect is the use of transgenic corn hybrids expressing one or more crystal (Cry proteins derived from Bacillus thuringiensis. Cry34Ab1 and Cry35Ab1 together comprise a binary insecticidal toxin with specific activity against WCR. These proteins have been developed as insect resistance traits in commercialized corn hybrids resistant to WCR feeding damage. Cry34/35Ab1 is a pore forming toxin, but the specific effects of Cry34/35Ab1 on WCR cells and tissues have not been well characterized microscopically, and the overall histopathology is poorly understood. Using high-resolution resin-based histopathology methods, the effects of Cry34/35Ab1 as well as Cry3Aa1, Cry6Aa1, and the Photorhabdus toxin complex protein TcdA have been directly visualized and documented. Clear symptoms of intoxication were observed for all insecticidal proteins tested, including swelling and sloughing of enterocytes, constriction of midgut circular muscles, stem cell activation, and obstruction of the midgut lumen. These data demonstrate the effects of these insecticidal proteins on WCR midgut cells, and the collective response of the midgut to intoxication. Taken together, these results advance our understanding of the insect cell biology and pathology of these insecticidal proteins, which should further the field of insect resistance traits and corn rootworm management.

  17. Comprehensive evaluation of Streptococcus sanguinis cell wall-anchored proteins in early infective endocarditis.

    Science.gov (United States)

    Turner, Lauren Senty; Kanamoto, Taisei; Unoki, Takeshi; Munro, Cindy L; Wu, Hui; Kitten, Todd

    2009-11-01

    Streptococcus sanguinis is a member of the viridans group of streptococci and a leading cause of the life-threatening endovascular disease infective endocarditis. Initial contact with the cardiac infection site is likely mediated by S. sanguinis surface proteins. In an attempt to identify the proteins required for this crucial step in pathogenesis, we searched for surface-exposed, cell wall-anchored proteins encoded by S. sanguinis and then used a targeted signature-tagged mutagenesis (STM) approach to evaluate their contributions to virulence. Thirty-three predicted cell wall-anchored proteins were identified-a number much larger than those found in related species. The requirement of each cell wall-anchored protein for infective endocarditis was assessed in the rabbit model. It was found that no single cell wall-anchored protein was essential for the development of early infective endocarditis. STM screening was also employed for the evaluation of three predicted sortase transpeptidase enzymes, which mediate the cell surface presentation of cell wall-anchored proteins. The sortase A mutant exhibited a modest (approximately 2-fold) reduction in competitiveness, while the other two sortase mutants were indistinguishable from the parental strain. The combined results suggest that while cell wall-anchored proteins may play a role in S. sanguinis infective endocarditis, strategies designed to interfere with individual cell wall-anchored proteins or sortases would not be effective for disease prevention.

  18. Novel PIGT Variant in Two Brothers: Expansion of the Multiple Congenital Anomalies-Hypotonia Seizures Syndrome 3 Phenotype

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    Nadia Skauli

    2016-11-01

    Full Text Available Biallelic PIGT variants were previously reported in seven patients from three families with Multiple Congenital Anomalies-Hypotonia Seizures Syndrome 3 (MCAHS3, characterized by epileptic encephalopathy, hypotonia, global developmental delay/intellectual disability, cerebral and cerebellar atrophy, craniofacial dysmorphisms, and skeletal, ophthalmological, cardiac, and genitourinary abnormalities. We report a novel homozygous PIGT missense variant c.1079G>T (p.Gly360Val in two brothers with several of the typical features of MCAHS3, but in addition, pyramidal tract neurological signs. Notably, they are the first patients with MCAHS3 without skeletal, cardiac, or genitourinary anomalies. PIGT encodes a crucial subunit of the glycosylphosphatidylinositol (GPI transamidase complex, which catalyzes the attachment of proteins to GPI-anchors, attaching the proteins to the cell membrane. In vitro studies in cells from the two brothers showed reduced levels of GPI-anchors and GPI-anchored proteins on the cell surface, supporting the pathogenicity of the novel PIGT variant.

  19. Plant Defense Inhibitors Affect the Structures of Midgut Cells in and

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    Hongmei Li-Byarlay

    2016-01-01

    Full Text Available Plants produce proteins such as protease inhibitors and lectins as defenses against herbivorous insects and pathogens. However, no systematic studies have explored the structural responses in the midguts of insects when challenged with plant defensive proteins and lectins across different species. In this study, we fed two kinds of protease inhibitors and lectins to the fruit fly Drosophila melanogaster and alpha-amylase inhibitors and lectins to the cowpea bruchid Callosobruchus maculatus . We assessed the changes in midgut cell structures by comparing them with such structures in insects receiving normal diets or subjected to food deprivation. Using light and transmission electron microscopy in both species, we observed structural changes in the midgut peritrophic matrix as well as shortened microvilli on the surfaces of midgut epithelial cells in D. melanogaster . Dietary inhibitors and lectins caused similar lesions in the epithelial cells but not much change in the peritrophic matrix in both species. We also noted structural damages in the Drosophila midgut after six hours of starvation and changes were still present after 12 hours. Our study provided the first evidence of key structural changes of midguts using a comparative approach between a dipteran and a coleopteran. Our particular observation and discussion on plant–insect interaction and dietary stress are relevant for future mode of action studies of plant defensive protein in insect physiology.

  20. Plasmodium falciparum merozoite surface protein 1 - Glycosylation and localization to low-density, detergent-resistant membranes in the parasitized erythrocyte

    DEFF Research Database (Denmark)

    Hoessli, D.C.; Poincelet, M.; Gupta, Ramneek

    2003-01-01

    In addition to the major carbohydrate moieties of the glycosylphosphatidylinositol (GPI) anchor, we report that Plasmodium falciparum merozoite surface protein 1 (MSP-1) bears O-GlcNAc modifications predominantly in beta-anomeric configuration, in both the C- and N-terminal portions of the protei...

  1. Comprehensive Evaluation of Streptococcus sanguinis Cell Wall-Anchored Proteins in Early Infective Endocarditis▿ †

    Science.gov (United States)

    Turner, Lauren Senty; Kanamoto, Taisei; Unoki, Takeshi; Munro, Cindy L.; Wu, Hui; Kitten, Todd

    2009-01-01

    Streptococcus sanguinis is a member of the viridans group of streptococci and a leading cause of the life-threatening endovascular disease infective endocarditis. Initial contact with the cardiac infection site is likely mediated by S. sanguinis surface proteins. In an attempt to identify the proteins required for this crucial step in pathogenesis, we searched for surface-exposed, cell wall-anchored proteins encoded by S. sanguinis and then used a targeted signature-tagged mutagenesis (STM) approach to evaluate their contributions to virulence. Thirty-three predicted cell wall-anchored proteins were identified—a number much larger than those found in related species. The requirement of each cell wall-anchored protein for infective endocarditis was assessed in the rabbit model. It was found that no single cell wall-anchored protein was essential for the development of early infective endocarditis. STM screening was also employed for the evaluation of three predicted sortase transpeptidase enzymes, which mediate the cell surface presentation of cell wall-anchored proteins. The sortase A mutant exhibited a modest (∼2-fold) reduction in competitiveness, while the other two sortase mutants were indistinguishable from the parental strain. The combined results suggest that while cell wall-anchored proteins may play a role in S. sanguinis infective endocarditis, strategies designed to interfere with individual cell wall-anchored proteins or sortases would not be effective for disease prevention. PMID:19703977

  2. Transcriptional profiling of midgut immunity response and degeneration in the wandering silkworm, Bombyx mori.

    Science.gov (United States)

    Xu, Qiuyun; Lu, Anrui; Xiao, Guohua; Yang, Bing; Zhang, Jie; Li, Xuquan; Guan, Jingmin; Shao, Qimiao; Beerntsen, Brenda T; Zhang, Peng; Wang, Chengshu; Ling, Erjun

    2012-01-01

    Lepidoptera insects have a novel development process comprising several metamorphic stages during their life cycle compared with vertebrate animals. Unlike most Lepidoptera insects that live on nectar during the adult stage, the Bombyx mori silkworm adults do not eat anything and die after egg-laying. In addition, the midguts of Lepidoptera insects produce antimicrobial proteins during the wandering stage when the larval tissues undergo numerous changes. The exact mechanisms responsible for these phenomena remain unclear. We used the silkworm as a model and performed genome-wide transcriptional profiling of the midgut between the feeding stage and the wandering stage. Many genes concerned with metabolism, digestion, and ion and small molecule transportation were down-regulated during the wandering stage, indicating that the wandering stage midgut loses its normal functions. Microarray profiling, qRT-PCR and western blot proved the production of antimicrobial proteins (peptides) in the midgut during the wandering stage. Different genes of the immune deficiency (Imd) pathway were up-regulated during the wandering stage. However, some key genes belonging to the Toll pathway showed no change in their transcription levels. Unlike butterfly (Pachliopta aristolochiae), the midgut of silkworm moth has a layer of cells, indicating that the development of midgut since the wandering stage is not usual. Cell division in the midgut was observed only for a short time during the wandering stage. However, there was extensive cell apoptosis before pupation. The imbalance of cell division and apoptosis probably drives the continuous degeneration of the midgut in the silkworm since the wandering stage. This study provided an insight into the mechanism of the degeneration of the silkworm midgut and the production of innate immunity-related proteins during the wandering stage. The imbalance of cell division and apoptosis induces irreversible degeneration of the midgut. The Imd pathway

  3. Expression of membrane anchored cytokines and B7-1 alters tumor microenvironment and induces protective antitumor immunity in a murine breast cancer model.

    Science.gov (United States)

    Bozeman, Erica N; Cimino-Mathews, Ashley; Machiah, Deepa K; Patel, Jaina M; Krishnamoorthy, Arun; Tien, Linda; Shashidharamurthy, Rangaiah; Selvaraj, Periasamy

    2013-05-07

    Many studies have shown that the systemic administration of cytokines or vaccination with cytokine-secreting tumors augments an antitumor immune response that can result in eradication of tumors. However, these approaches are hampered by the risk of systemic toxicity induced by soluble cytokines. In this study, we have evaluated the efficacy of 4TO7, a highly tumorigenic murine mammary tumor cell line, expressing glycosyl phosphatidylinositol (GPI)-anchored form of cytokine molecules alone or in combination with the costimulatory molecule B7-1 as a model for potential cell or membrane-based breast cancer vaccines. We observed that the GPI-anchored cytokines expressed on the surface of tumor cells greatly reduced the overall tumorigenicity of the 4TO7 tumor cells following direct live cell challenge as evidenced by transient tumor growth and complete regression within 30 days post challenge. Tumors co-expressing B7-1 and GPI-IL-12 grew the least and for the shortest duration, suggesting that this combination of immunostimulatory molecules is most potent. Protective immune responses were also observed following secondary tumor challenge. Further, the 4TO7-B7-1/GPI-IL-2 and 4TO7-B7-1/GPI-IL-12 transfectants were capable of inducing regression of a wild-type tumor growing at a distant site in a concomitant tumor challenge model, suggesting the tumor immunity elicited by the transfectants can act systemically and inhibit the tumor growth at a distant site. Additionally, when used as irradiated whole cell vaccines, 4TO7-B7-1/GPI-IL-12 led to a significant inhibition in tumor growth of day 7 established tumors. Lastly, we observed a significant decrease in the prevalence of myeloid-derived suppressor cells and regulatory T-cells in the tumor microenvironment on day 7 post challenge with 4TO7-B7-1/GPI-IL-12 cells, which provides mechanistic insight into antitumor efficacy of the tumor-cell membrane expressed IL-12. These studies have implications in designing membrane

  4. Transcriptional profiling of midgut immunity response and degeneration in the wandering silkworm, Bombyx mori.

    Directory of Open Access Journals (Sweden)

    Qiuyun Xu

    Full Text Available BACKGROUND: Lepidoptera insects have a novel development process comprising several metamorphic stages during their life cycle compared with vertebrate animals. Unlike most Lepidoptera insects that live on nectar during the adult stage, the Bombyx mori silkworm adults do not eat anything and die after egg-laying. In addition, the midguts of Lepidoptera insects produce antimicrobial proteins during the wandering stage when the larval tissues undergo numerous changes. The exact mechanisms responsible for these phenomena remain unclear. PRINCIPAL FINDINGS: We used the silkworm as a model and performed genome-wide transcriptional profiling of the midgut between the feeding stage and the wandering stage. Many genes concerned with metabolism, digestion, and ion and small molecule transportation were down-regulated during the wandering stage, indicating that the wandering stage midgut loses its normal functions. Microarray profiling, qRT-PCR and western blot proved the production of antimicrobial proteins (peptides in the midgut during the wandering stage. Different genes of the immune deficiency (Imd pathway were up-regulated during the wandering stage. However, some key genes belonging to the Toll pathway showed no change in their transcription levels. Unlike butterfly (Pachliopta aristolochiae, the midgut of silkworm moth has a layer of cells, indicating that the development of midgut since the wandering stage is not usual. Cell division in the midgut was observed only for a short time during the wandering stage. However, there was extensive cell apoptosis before pupation. The imbalance of cell division and apoptosis probably drives the continuous degeneration of the midgut in the silkworm since the wandering stage. CONCLUSIONS: This study provided an insight into the mechanism of the degeneration of the silkworm midgut and the production of innate immunity-related proteins during the wandering stage. The imbalance of cell division and apoptosis

  5. LRP1 controls biosynthetic and endocytic trafficking of neuronal prion protein

    DEFF Research Database (Denmark)

    Parkyn, Celia J; Vermeulen, Esmeralda G M; Mootoosamy, Roy C

    2008-01-01

    The trafficking of normal cellular prion protein (PrP(C)) is believed to control its conversion to the altered conformation (designated PrP(Sc)) associated with prion disease. Although anchored to the membrane by means of glycosylphosphatidylinositol (GPI), PrP(C) on neurons is rapidly and consti......The trafficking of normal cellular prion protein (PrP(C)) is believed to control its conversion to the altered conformation (designated PrP(Sc)) associated with prion disease. Although anchored to the membrane by means of glycosylphosphatidylinositol (GPI), PrP(C) on neurons is rapidly...... required for this process. Moreover, sustained inhibition of LRP1 levels by siRNA leads to the accumulation of PrP(C) in biosynthetic compartments, with a concomitant lowering of surface PrP(C), suggesting that LRP1 expedites the trafficking of PrP(C) to the neuronal surface. PrP(C) and LRP1 can be co......-immunoprecipitated from the endoplasmic reticulum in normal neurons. The N-terminal domain of PrP(C) binds to purified human LRP1 with nanomolar affinity, even in the presence of 1 microM of the LRP-specific chaperone, receptor-associated protein (RAP). Taken together, these data argue that LRP1 controls both the surface...

  6. Covalent glycoinositolphospholipid (GPI binding to hemoglobin is associated with insulin-activation of erythrocyte membrane protease

    Directory of Open Access Journals (Sweden)

    VESNA NIKETIC

    2004-05-01

    Full Text Available Recently, it was demonstrated that prolonged hyperinsulinism associated with hypoglycemia, both in vivo and in vitro, caused covalent glycoinositolphospholipid (GPI binding to the C termini of both hemoglobin b-chains, which resulted in the formation of a novel, hitherto unrecognized, minor hemoglobin fraction (GPI-Hb (Niketic et al., Biochem. Biophys. Res. Commun. 239 (1997 435. In this study it was demonstrated that exposure of erythrocyte membranes to insulin causes the activation of membrane protease as well as that the formation of GPI-Hb parallels its activity. It is suggested that the insulin-activated protease is able to catalyze, albeit slowly, the transpeptidation, i.e., the replacement of the carboxy-terminal amino acid(s residues of the Hb b-chains with GPI as an exogenous nucleophile. To our knowledge the present results show for the first time that insulin stimulates protease activity in erythrocyte membranes, as well as that insulin-activated protease may be involved in post-translational GPI binding to proteins.

  7. An enzyme-linked immunosorbent assay for measuring GPIHBP1 levels in human plasma or serum

    DEFF Research Database (Denmark)

    Miyashita, Kazuya; Fukamachi, Isamu; Nagao, Manabu

    2018-01-01

    Background: Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), a glycosylphosphatidylinositol (GPI)-anchored protein of capillary endothelial cells, transports lipoprotein lipase to the capillary lumen and is essential for the lipolytic processing of trigl...

  8. Brittle Culm1, a COBRA-Like Protein, Functions in Cellulose Assembly through Binding Cellulose Microfibrils

    Science.gov (United States)

    Zhang, Baocai; Liu, Xiangling; Yan, Meixian; Zhang, Lanjun; Shi, Yanyun; Zhang, Mu; Qian, Qian; Li, Jiayang; Zhou, Yihua

    2013-01-01

    Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity. PMID:23990797

  9. Brittle Culm1, a COBRA-like protein, functions in cellulose assembly through binding cellulose microfibrils.

    Directory of Open Access Journals (Sweden)

    Lifeng Liu

    Full Text Available Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1, a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity.

  10. Brittle Culm1, a COBRA-like protein, functions in cellulose assembly through binding cellulose microfibrils.

    Science.gov (United States)

    Liu, Lifeng; Shang-Guan, Keke; Zhang, Baocai; Liu, Xiangling; Yan, Meixian; Zhang, Lanjun; Shi, Yanyun; Zhang, Mu; Qian, Qian; Li, Jiayang; Zhou, Yihua

    2013-01-01

    Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity.

  11. Resistance to Bacillus thuringiensis by the Indian meal moth, Plodia interpunctella: comparison of midgut proteinases from susceptible and resistant larvae.

    Science.gov (United States)

    Johnson, D E; Brookhart, G L; Kramer, K J; Barnett, B D; McGaughey, W H

    1990-03-01

    Midgut homogenates from susceptible and resistant strains of the Indian meal moth, Plodia interpunctella, were compared for their ability to activate the entomocidal parasporal crystal protein from Bacillus thuringiensis. The properties of midgut proteinases from both types of larvae were also examined. Electrophoretic patterns of crystal protein from B. thuringiensis subspecies kurstaki (HD-1) and aizawai (HD-133 and HD-144) were virtually unchanged following digestion by either type of midgut homogenate. Changes in pH (9.5 to 11.5) or midgut homogenate concentration during digestion failed to substantially alter protein electrophoretic patterns of B. thuringiensis HD-1 crystal toxin. In vitro toxicity of crystal protein activated by either type of midgut preparation was equal toward cultured insect cells from either Manduca sexta or Choristoneura fumiferana. Electrophoresis of midgut extracts in polyacrylamide gels containing gelatin as substrate also yielded matching mobility patterns of proteinases from both types of midguts. Quantitation of midgut proteolytic activity using tritiated casein as a substrate revealed variation between midgut preparations, but no statistically significant differences between proteolytic activities from susceptible and resistant Indian meal moth larvae. Inhibition studies indicated that a trypsin-like proteinase with maximal activity at pH 10 is a major constituent of Indian meal moth midguts. The results demonstrated that midguts from susceptible and resistant strains of P. interpunctella are similar both in their ability to activate B. thuringiensis protoxin and in their proteolytic activity.

  12. Induction of IL-12 Production in Human Peripheral Monocytes by Trypanosoma cruzi Is Mediated by Glycosylphosphatidylinositol-Anchored Mucin-Like Glycoproteins and Potentiated by IFN-γ and CD40-CD40L Interactions

    Directory of Open Access Journals (Sweden)

    Lúcia Cristina Jamli Abel

    2014-01-01

    Full Text Available Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi, is characterized by immunopathology driven by IFN-γ secreting Th1-like T cells. T. cruzi has a thick coat of mucin-like glycoproteins covering its surface, which plays an important role in parasite invasion and host immunomodulation. It has been extensively described that T. cruzi or its products—like GPI anchors isolated from GPI-anchored mucins from the trypomastigote life cycle stage (tGPI-mucins—are potent inducers of proinflammatory responses (i.e., cytokines and NO production by IFN-γ primed murine macrophages. However, little is known about whether T. cruzi or GPI-mucins exert a similar action in human cells. We therefore decided to further investigate the in vitro cytokine production profile from human mononuclear cells from uninfected donors exposed to T. cruzi as well as tGPI-mucins. We observed that both living T. cruzi trypomastigotes and tGPI-mucins are potent inducers of IL-12 by human peripheral blood monocytes and this effect depends on CD40-CD40L interaction and IFN-γ. Our findings suggest that the polarized T1-type cytokine profile seen in T. cruzi infected patients might be a long-term effect of IL-12 production induced by lifelong exposure to T. cruzi tGPI-mucins.

  13. New insights into the organization of plasma membrane and its role in signal transduction.

    Science.gov (United States)

    Suzuki, Kenichi G N

    2015-01-01

    Plasma membranes have heterogeneous structures for efficient signal transduction, required to perform cell functions. Recent evidence indicates that the heterogeneous structures are produced by (1) compartmentalization by actin-based membrane skeleton, (2) raft domains, (3) receptor-receptor interactions, and (4) the binding of receptors to cytoskeletal proteins. This chapter provides an overview of recent studies on diffusion, clustering, raft association, actin binding, and signal transduction of membrane receptors, especially glycosylphosphatidylinositol (GPI)-anchored receptors. Studies on diffusion of GPI-anchored receptors suggest that rafts may be small and/or short-lived in plasma membranes. In steady state conditions, GPI-anchored receptors form transient homodimers, which may represent the "standby state" for the stable homodimers and oligomers upon ligation. Furthermore, It is proposed that upon ligation, the binding of GPI-anchored receptor clusters to cytoskeletal actin filaments produces a platform for downstream signaling, and that the pulse-like signaling easily maintains the stability of the overall signaling activity. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Different domains of Bacillus thuringiensis delta-endotoxins can bind to insect midgut membrane proteins on ligand blots

    NARCIS (Netherlands)

    Maagd, de R.A.; Klei, van der H.; Bakker, P.L.; Stiekema, W.J.; Bosch, D.

    1996-01-01

    We investigated the role of the constituent domains of the CryIA(b) and CryIA(c) δ-endotoxins in binding to midgut epithelial cell membrane proteins of Spodoptera exigua and Manduca sexta on ligand blots. A collection of wild- type and CryIC-CryIA hybrid toxins was used for this purpose. As

  15. A systematic evaluation of protein kinase a-a-kinase anchoring protein interaction motifs

    NARCIS (Netherlands)

    Burgers, Pepijn P|info:eu-repo/dai/nl/341566551; van der Heyden, Marcel A G; Kok, Bart; Heck, Albert J R|info:eu-repo/dai/nl/105189332; Scholten, Arjen|info:eu-repo/dai/nl/313939780

    2015-01-01

    Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIβ, RIIα, and RIIβ) each form a homodimer, and their dimerization domain interacts with a small helical region present

  16. A systematic evaluation of protein kinase A-A-kinase anchoring protein interaction motifs

    NARCIS (Netherlands)

    Burgers, Pepijn P; van der Heyden, MAG; Kok, Bart; Heck, Albert J R; Scholten, Arjen

    2015-01-01

    Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIβ, RIIα, and RIIβ) each form a homodimer, and their dimerization domain interacts with a small helical region present

  17. Midgut of the non-hematophagous mosquito Toxorhynchites theobaldi (Diptera, Culicidae).

    Science.gov (United States)

    Godoy, Raquel S M; Fernandes, Kenner M; Martins, Gustavo F

    2015-10-30

    In most mosquito species, the females require a blood-feeding for complete egg development. However, in Toxorhynchites mosquitoes, the eggs develop without blood-feeding, and both females and males exclusively feed on sugary diets. The midgut is a well-understood organ in blood-feeding mosquitoes, but little is known about it in non-blood-feeding ones. In the present study, the detailed morphology of the midgut of Toxorhynchites theobaldi were investigated using histochemical and ultrastructural methods. The midgut of female and male T. theobaldi adults consists of a long, slender anterior midgut (AMG), and a short, dilated posterior midgut (PMG). The AMG is subdivided into AMG1 (short, with folds) and AMG2 (long, without folds). Nerve branches and enteroendocrine cells are present in AMG and PMG, respectively. Compared with the PMG of blood-feeding female mosquitoes, the PMG of T. theobaldi is smaller; however, in both mosquitoes, PMG seems be the main region of food digestion and absorption, and protein secretion. The epithelial folds present in the AMG of T. theobaldi have not been reported in other mosquitoes; however, the midgut muscle organization and endocrine control of the digestion process are conserved in both T. theobaldi and blood-feeding mosquitoes.

  18. Prostasin-dependent activation of epithelial Na+ channels by low plasmin concentrations

    DEFF Research Database (Denmark)

    Svenningsen, Per; Uhrenholt, Torben R; Palarasah, Yaseelan

    2009-01-01

    by which plasmin stimulates ENaC activity. Cy3-labeled plasmin was found to bind to the surface of the mouse cortical collecting duct cell line, M-1. Binding depended on a glycosylphosphatidylinositol (GPI)-anchored protein. Biotin-label transfer showed that plasmin interacted with the GPI-anchored protein...... plasmin-stimulated ENaC current in monolayers of M-1 cells at low plasmin concentration (1-4 microg/ml). At a high plasmin concentration of 30 microg/ml, there was no difference between cell layers treated with or without PI-PLC. Knockdown of prostasin attenuated binding of plasmin to M1 cells and blocked...... labeling of M-1 cells. Pretreatment with plasmin abolished labeling of M-1 cells in a prostasin-dependent way. We conclude that, at low concentrations, plasmin interacts with GPI-anchored prostasin, which leads to cleavage of the gamma-subunit and activation of ENaC, while at higher concentrations, plasmin...

  19. An Accessory Protein Required for Anchoring and Assembly of Amyloid Fibers in B. subtilis Biofilms

    Science.gov (United States)

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

    2011-01-01

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

  20. Malrotation and midgut volvulus: a historical review and current controversies in diagnosis and management

    International Nuclear Information System (INIS)

    Lampl, Brooke; Berdon, Walter E.; Levin, Terry L.; Cowles, Robert A.

    2009-01-01

    The complex and sometimes controversial subject of malrotation and midgut volvulus is reviewed commencing with the 19th-century Bohemian anatomist, Vaclav Treitz, who described the suspensory muscle anchoring of the duodenal-jejunal junction in the left upper quadrant, and continuing with William Ladd, the 20th-century American ''father of pediatric surgery'' who pioneered the surgical treatment of midgut volvulus. In this review, we present the interesting history of malrotation and discuss the current radiologic and surgical controversies surrounding its diagnosis and treatment. In the symptomatic patient with malrotation and possible midgut volvulus, prompt diagnosis is critical. The clinical examination and plain film are often confusing, and delayed diagnosis can lead to significant morbidity and death. Despite recent intense interest in the position of the mesenteric vessels on US and CT scans, the upper gastrointestinal series remains the fastest and most accurate method of demonstrating duodenal obstruction, the position of the ligament of Treitz, and, if the contrast agent is followed distally, cecal malposition. Controversy exists over the management of asymptomatic patients with malrotation in whom the diagnosis is made incidentally during evaluation for nonspecific complaints, prior to reflux surgery, and in those with heterotaxy syndromes. (orig.)

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

    Science.gov (United States)

    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.

  2. Defining the Conformational Features of Anchorless, Poorly Neuroinvasive Prions

    NARCIS (Netherlands)

    Bett, C.; Kurt, T.D.; Lucero, M.; Trejo, M.; Rozemuller, A.J.M.; Kong, Q.Z.; Nilsson, K.P.R.; Masliah, E.; Oldstone, M.B.; Sigurdson, C.J.

    2013-01-01

    Infectious prions cause diverse clinical signs and form an extraordinary range of structures, from amorphous aggregates to fibrils. How the conformation of a prion dictates the disease phenotype remains unclear. Mice expressing GPI-anchorless or GPI-anchored prion protein exposed to the same

  3. Identification and characterization of secreted proteins in Eimeria tenella

    Science.gov (United States)

    Ramlee, Intan Azlinda; Firdaus-Raih, Mohd; Wan, Kiew-Lian

    2015-09-01

    Eimeria tenella is a protozoan parasite that causes coccidiosis, an economically important disease in the poultry industry. The characterization of proteins that are secreted by parasites have been shown to play important roles in parasite invasion and are considered to be potential control agents. In this study, 775 proteins potentially secreted by E. tenella were identified. These proteins were further filtered to remove mitochondrial proteins. Out of 763 putative secreted proteins, 259 proteins possess transmembrane domains while another 150 proteins have GPI (Glycosylphosphatidylinositol) anchors. Homology search revealed that 315 and 448 proteins have matches with known and hypothetical proteins in the database, respectively. Within this data set, previously characterized secretory proteins such as micronemes, rhoptry kinases and dense granules were detected.

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

    Science.gov (United States)

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

    2011-06-01

    Cells within Bacillus subtilis biofilms are held in place by an extracellular matrix that contains cell-anchored amyloid fibres, composed of the amyloidogenic protein TasA. As biofilms age they disassemble because the cells release the amyloid fibres. This release appears to be the consequence of incorporation of D-tyrosine, D-leucine, D-tryptophan and D-methionine into the cell wall. Here, we characterize the in vivo roles of an accessory protein TapA (TasA anchoring/assembly protein; previously YqxM) that serves both to anchor the fibres to the cell wall and to assemble TasA into fibres. TapA is found in discrete foci in the cell envelope and these foci disappear when cells are treated with a mixture of D-amino acids. Purified cell wall sacculi retain a functional form of this anchoring protein such that purified fibres can be anchored to the sacculi in vitro. In addition, we show that TapA is essential for the proper assembly of the fibres. Its absence results in a dramatic reduction in TasA levels and what little TasA is left produces only thin fibres that are not anchored to the cell. © 2011 Blackwell Publishing Ltd.

  5. Lack of Connection Between Midgut Cell Autophagy Gene Expression and BmCPV Infection in the Midgut of Bombyx mori.

    Science.gov (United States)

    Yang, Xiaobing; Wu, Suli; Wu, Yongpeng; Liu, Yang; Qian, Yonghua; Jiao, Feng

    2015-01-01

    Autophagy is associated with multiple biological processes and has protective and defensive functions with respect to immunity, inflammation, and resistance to microbial infection. In this experiment, we wished to investigate whether autophagy is a factor in the midgut cell response of Bombyx mori to infection by the B. mori cytoplasmic polyhedrosis virus (BmCPV). Our results indicated that the expression of three autophagy-related genes (BmAtg8, BmAtg5, and BmAtg7) in the midgut did not change greatly after BmCPV infection in B. mori. Basal ATG8/ATG8PE protein expression was detected in different B. mori tissues by using western blot analysis. Immunohistochemistry showed that the ATG8/ATG8PE proteins were located mainly in the cytoplasm. ATG8/ATG8PE protein levels decreased at 12 and 16 h after BmCPV infection. Our results indicate that autophagy responded slightly to BmCPV infection, but could not prevent the invasion and replication of the virus. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

  6. Protein kinase C-dependent signaling controls the midgut epithelial barrier to malaria parasite infection in anopheline mosquitoes.

    Directory of Open Access Journals (Sweden)

    Nazzy Pakpour

    Full Text Available Anopheline mosquitoes are the primary vectors of parasites in the genus Plasmodium, the causative agents of malaria. Malaria parasites undergo a series of complex transformations upon ingestion by the mosquito host. During this process, the physical barrier of the midgut epithelium, along with innate immune defenses, functionally restrict parasite development. Although these defenses have been studied for some time, the regulatory factors that control them are poorly understood. The protein kinase C (PKC gene family consists of serine/threonine kinases that serve as central signaling molecules and regulators of a broad spectrum of cellular processes including epithelial barrier function and immunity. Indeed, PKCs are highly conserved, ranging from 7 isoforms in Drosophila to 16 isoforms in mammals, yet none have been identified in mosquitoes. Despite conservation of the PKC gene family and their potential as targets for transmission-blocking strategies for malaria, no direct connections between PKCs, the mosquito immune response or epithelial barrier integrity are known. Here, we identify and characterize six PKC gene family members--PKCδ, PKCε, PKCζ, PKD, PKN, and an indeterminate conventional PKC--in Anopheles gambiae and Anopheles stephensi. Sequence and phylogenetic analyses of the anopheline PKCs support most subfamily assignments. All six PKCs are expressed in the midgut epithelia of A. gambiae and A. stephensi post-blood feeding, indicating availability for signaling in a tissue that is critical for malaria parasite development. Although inhibition of PKC enzymatic activity decreased NF-κB-regulated anti-microbial peptide expression in mosquito cells in vitro, PKC inhibition had no effect on expression of a panel of immune genes in the midgut epithelium in vivo. PKC inhibition did, however, significantly increase midgut barrier integrity and decrease development of P. falciparum oocysts in A. stephensi, suggesting that PKC

  7. Malrotation and midgut volvulus: a historical review and current controversies in diagnosis and management

    Energy Technology Data Exchange (ETDEWEB)

    Lampl, Brooke; Berdon, Walter E. [Morgan Stanley Children' s Hospital of New York-Presbyterian, Department of Radiology, Columbia University College of Physicians and Surgeons, New York, NY (United States); Levin, Terry L. [Montefiore Medical Center, Department of Radiology, Mamaroneck, NY (United States); Cowles, Robert A. [Morgan Stanley Children' s Hospital of New York-Presbyterian, Division of Pediatric Surgery, Columbia University College of Physicians and Surgeons, New York, NY (United States)

    2009-04-15

    The complex and sometimes controversial subject of malrotation and midgut volvulus is reviewed commencing with the 19th-century Bohemian anatomist, Vaclav Treitz, who described the suspensory muscle anchoring of the duodenal-jejunal junction in the left upper quadrant, and continuing with William Ladd, the 20th-century American ''father of pediatric surgery'' who pioneered the surgical treatment of midgut volvulus. In this review, we present the interesting history of malrotation and discuss the current radiologic and surgical controversies surrounding its diagnosis and treatment. In the symptomatic patient with malrotation and possible midgut volvulus, prompt diagnosis is critical. The clinical examination and plain film are often confusing, and delayed diagnosis can lead to significant morbidity and death. Despite recent intense interest in the position of the mesenteric vessels on US and CT scans, the upper gastrointestinal series remains the fastest and most accurate method of demonstrating duodenal obstruction, the position of the ligament of Treitz, and, if the contrast agent is followed distally, cecal malposition. Controversy exists over the management of asymptomatic patients with malrotation in whom the diagnosis is made incidentally during evaluation for nonspecific complaints, prior to reflux surgery, and in those with heterotaxy syndromes. (orig.)

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

    Science.gov (United States)

    Ben-Shimon, Avraham; Niv, Masha Y

    2015-05-05

    The huge conformational space stemming from the inherent flexibility of peptides is among the main obstacles to successful and efficient computational modeling of protein-peptide interactions. Current peptide docking methods typically overcome this challenge using prior knowledge from the structure of the complex. Here we introduce AnchorDock, a peptide docking approach, which automatically targets the docking search to the most relevant parts of the conformational space. This is done by precomputing the free peptide's structure and by computationally identifying anchoring spots on the protein surface. Next, a free peptide conformation undergoes anchor-driven simulated annealing molecular dynamics simulations around the predicted anchoring spots. In the challenging task of a completely blind docking test, AnchorDock produced exceptionally good results (backbone root-mean-square deviation ≤ 2.2Å, rank ≤15) for 10 of 13 unbound cases tested. The impressive performance of AnchorDock supports a molecular recognition pathway that is driven via pre-existing local structural elements. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. The role of Gpi-anchored axonal glycoproteins in neural development and neurological disorders.

    Science.gov (United States)

    Gennarini, Gianfranco; Bizzoca, Antonella; Picocci, Sabrina; Puzzo, Daniela; Corsi, Patrizia; Furley, Andrew J W

    2017-06-01

    This review article focuses on the Contactin (CNTN) subset of the Immunoglobulin supergene family (IgC2/FNIII molecules), whose components share structural properties (the association of Immunoglobulin type C2 with Fibronectin type III domains), as well as a general role in cell contact formation and axonal growth control. IgC2/FNIII molecules include 6 highly related components (CNTN 1-6), associated with the cell membrane via a Glycosyl Phosphatidyl Inositol (GPI)-containing lipid tail. Contactin 1 and Contactin 2 share ~50 (49.38)% identity at the aminoacid level. They are components of the cell surface, from which they may be released in soluble forms. They bind heterophilically to multiple partners in cis and in trans, including members of the related L1CAM family and of the Neurexin family Contactin-associated proteins (CNTNAPs or Casprs). Such interactions are important for organising the neuronal membrane, as well as for modulating the growth and pathfinding of axon tracts. In addition, they also mediate the functional maturation of axons by promoting their interactions with myelinating cells at the nodal, paranodal and juxtaparanodal regions. Such interactions also mediate differential ionic channels (both Na + and K + ) distribution, which is of critical relevance in the generation of the peak-shaped action potential. Indeed, thanks to their interactions with Ankyrin G, Na + channels map within the nodal regions, where they drive axonal depolarization. However, no ionic channels are found in the flanking Contactin1-containing paranodal regions, where CNTN1 interactions with Caspr1 and with the Ig superfamily component Neurofascin 155 in cis and in trans, respectively, build a molecular barrier between the node and the juxtaparanode. In this region K + channels are clustered, depending upon molecular interactions with Contactin 2 and with Caspr2. In addition to these functions, the Contactins appear to have also a role in degenerative and inflammatory

  10. Climate Prediction Center(CPC)Daily GOES Precipitation Index (GPI)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — GOES Precipitation Index (GPI) is a precipitation estimation algorithm. The GPI technique estimates tropical rainfall using cloud-top temperature as the sole...

  11. Separation of Binding Protein of Celangulin V from the Midgut of Mythimna separata Walker by Affinity Chromatography

    Directory of Open Access Journals (Sweden)

    Lina Lu

    2015-05-01

    Full Text Available Celangulin V, an insecticidal compound isolated from the root bark of Chinese bittersweet, can affect the digestive system of insects. However, the mechanism of how Celangulin V induces a series of symptoms is still unknown. In this study, affinity chromatography was conducted through coupling of Celangulin V-6-aminoacetic acid ester to the CNBr-activated Sepharose 4B. SDS-PAGE was used to analyze the collected fraction eluted by Celangulin V. Eight binding proteins (Zinc finger protein, Thioredoxin peroxidase (TPx, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, SUMO E3 ligase RanBP2, Transmembrane protein 1, Actin, APN and V-ATPase were obtained and identified by LC/Q-TOF-MS from the midgut of Mythimna separata larvae. The potential of these proteins to serve as target proteins involved in the insecticidal activity of Celangulin V is discussed.

  12. A midgut lysate of the Riptortus pedestris has antibacterial activity against LPS O-antigen-deficient Burkholderia mutants.

    Science.gov (United States)

    Jang, Ho Am; Seo, Eun Sil; Seong, Min Young; Lee, Bok Luel

    2017-02-01

    Riptortus pedestris, a common pest in soybean fields, harbors a symbiont Burkholderia in a specialized posterior midgut region of insects. Every generation of second nymphs acquires new Burkholderia cells from the environment. We compared in vitro cultured Burkholderia with newly in vivo colonized Burkholderia in the host midgut using biochemical approaches. The bacterial cell envelope of in vitro cultured and in vivo Burkholderia differed in structure, as in vivo bacteria lacked lipopolysaccharide (LPS) O-antigen. The LPS O-antigen deficient bacteria had a reduced colonization rate in the host midgut compared with that of the wild-type Burkholderia. To determine why LPS O-antigen-deficient bacteria are less able to colonize the host midgut, we examined in vitro survival rates of three LPS O-antigen-deficient Burkholderia mutants and lysates of five different midgut regions. The LPS O-antigen-deficient mutants were highly susceptible when cultured with the lysate of a specific first midgut region (M1), indicating that the M1 lysate contains unidentified substance(s) capable of killing LPS O-antigen-deficient mutants. We identified a 17 kDa protein from the M1 lysate, which was enriched in the active fractions. The N-terminal sequence of the protein was determined to be a soybean Kunitz-type trypsin inhibitor. These data suggest that the 17 kDa protein, which was originated from a main soybean source of the R. pedestris host, has antibacterial activity against the LPS O-antigen deficient (rough-type) Burkholderia. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Azadirachtin Affects the Growth of Spodoptera litura Fabricius by Inducing Apoptosis in Larval Midgut.

    Science.gov (United States)

    Shu, Benshui; Zhang, Jingjing; Cui, Gaofeng; Sun, Ranran; Yi, Xin; Zhong, Guohua

    2018-01-01

    Azadirachtin, the environmentally friendly botanical pesticide, has been used as an antifeedant and pest growth regulator in integrated pest management for decades. It has shown strong biological activity against Spodoptera litura , but the mechanism of toxicity remains unclear. The present study showed that azadirachtin inhibited the growth of S. litura larvae, which was resulted by structure destroy and size inhibition of the midgut. Digital gene expression (DGE) analysis of midgut suggested that azadirachtin regulated the transcriptional level of multiple unigenes involved in mitogen-activated protein kinase (MAPK) and calcium apoptotic signaling pathways. Simultaneously, the expression patterns of some differentially expressed unigenes were verified by quantitative real time-PCR (qRT-PCR). In addition, the enhanced terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining, the increased expression of caspase family members and apoptosis-binding motif 1 (IBM1) on both gene and protein level and the release of cytochrome c from mitochondria to cytoplasm were induced in midgut after azadirachtin treatment. These results demonstrated that azadirachtin induced structural alteration in S. litura larval midgut by apoptosis activation. These alterations may affect the digestion and absorption of nutrients and eventually lead to the growth inhibition of larvae.

  14. Characterization of a midgut mucin-like glycoconjugate of Lutzomyia longipalpis with a potential role in Leishmania attachment.

    Science.gov (United States)

    Myšková, Jitka; Dostálová, Anna; Pěničková, Lucie; Halada, Petr; Bates, Paul A; Volf, Petr

    2016-07-25

    Leishmania parasites are transmitted by phlebotomine sand flies and a crucial step in their life-cycle is the binding to the sand fly midgut. Laboratory studies on sand fly competence to Leishmania parasites suggest that the sand flies fall into two groups: several species are termed "specific/restricted" vectors that support the development of one Leishmania species only, while the others belong to so-called "permissive" vectors susceptible to a wide range of Leishmania species. In a previous study we revealed a correlation between specificity vs permissivity of the vector and glycosylation of its midgut proteins. Lutzomyia longipalpis and other four permissive species tested possessed O-linked glycoproteins whereas none were detected in three specific vectors examined. We used a combination of biochemical, molecular and parasitological approaches to characterize biochemical and biological properties of O-linked glycoprotein of Lu. longipalpis. Lectin blotting and mass spectrometry revealed that this molecule with an apparent molecular weight about 45-50 kDa corresponds to a putative 19 kDa protein with unknown function detected in a midgut cDNA library of Lu. longipalpis. We produced a recombinant glycoprotein rLuloG with molecular weight around 45 kDa. Anti-rLuloG antibodies localize the native glycoprotein on epithelial midgut surface of Lu. longipalpis. Although we could not prove involvement of LuloG in Leishmania attachment by blocking the native protein with anti-rLuloG during sand fly infections, we demonstrated strong binding of rLuloG to whole surface of Leishmania promastigotes. We characterized a novel O-glycoprotein from sand fly Lutzomyia longipalpis. It has mucin-like properties and is localized on the luminal side of the midgut epithelium. Recombinant form of the protein binds to Leishmania parasites in vitro. We propose a role of this molecule in Leishmania attachment to sand fly midgut.

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

    Science.gov (United States)

    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.

  16. The automated data processing architecture for the GPI Exoplanet Survey

    Science.gov (United States)

    Wang, Jason J.; Perrin, Marshall D.; Savransky, Dmitry; Arriaga, Pauline; Chilcote, Jeffrey K.; De Rosa, Robert J.; Millar-Blanchaer, Maxwell A.; Marois, Christian; Rameau, Julien; Wolff, Schuyler G.; Shapiro, Jacob; Ruffio, Jean-Baptiste; Graham, James R.; Macintosh, Bruce

    2017-09-01

    The Gemini Planet Imager Exoplanet Survey (GPIES) is a multi-year direct imaging survey of 600 stars to discover and characterize young Jovian exoplanets and their environments. We have developed an automated data architecture to process and index all data related to the survey uniformly. An automated and flexible data processing framework, which we term the GPIES Data Cruncher, combines multiple data reduction pipelines together to intelligently process all spectroscopic, polarimetric, and calibration data taken with GPIES. With no human intervention, fully reduced and calibrated data products are available less than an hour after the data are taken to expedite follow-up on potential objects of interest. The Data Cruncher can run on a supercomputer to reprocess all GPIES data in a single day as improvements are made to our data reduction pipelines. A backend MySQL database indexes all files, which are synced to the cloud, and a front-end web server allows for easy browsing of all files associated with GPIES. To help observers, quicklook displays show reduced data as they are processed in real-time, and chatbots on Slack post observing information as well as reduced data products. Together, the GPIES automated data processing architecture reduces our workload, provides real-time data reduction, optimizes our observing strategy, and maintains a homogeneously reduced dataset to study planet occurrence and instrument performance.

  17. Leishmania attachment in permissive vectors and the role of sand fly midgut proteins in parasite-vector interaction

    OpenAIRE

    Dostálová, Anna

    2012-01-01

    of PhD. thesis named "Leishmania attachment in permissive vectors and the role of sand fly midgut proteins in parasite-vector interaction", Anna Dostálová, 2011 This thesis focuses on the development of protozoan parasites of the genus Leishmania in their insect vectors, sand flies. It sums up results of three projects I was involved in during my PhD studies. Main emphasis was put on permissive sand fly species that support development of various species of Leishmania. Using a novel method of...

  18. Methodological developments in US state-level Genuine Progress Indicators: toward GPI 2.0

    Science.gov (United States)

    Bagstad, Kenneth J.; Berik, Günseli; Gaddis, Erica J. Brown

    2014-01-01

    The Genuine Progress Indicator (GPI) has emerged as an important monetary measure of economic well-being. Unlike mainstream economic indicators, primarily Gross Domestic Product (GDP), the GPI accounts for both the benefits and costs of economic production across diverse economic, social, and environmental domains in a more comprehensive manner. Recently, the GPI has gained traction in subnational policy in the United States, with GPI studies being conducted in a number of states and with their formal adoption by several state governments. As the GPI is applied in different locations, new methods are developed, different data sources are available, and new issues of policy relevance are addressed using its component indicators. This has led to a divergence in methods, reducing comparability between studies and yielding results that are of varying methodological sophistication. In this study, we review the “state of the art” in recent US state-level GPI studies, focusing on those from Hawaii, Maryland, Ohio, Utah, and Vermont. Through adoption of a consistent approach, these and future GPI studies could utilize a framework that supports more uniform, comparable, and accurate measurements of progress. We also identify longer-term issues, particularly related to treatment of nonrenewable resource depletion, government spending, income inequality, and ecosystem services. As these issues are successfully addressed and disseminated, a “GPI 2.0” will emerge that better measures economic well-being and has greater accuracy and policy relevance than past GPI measurements. As the GPI expands further into mainstream policy analysis, a more formal process by which methods could be updated, standardized, and applied is needed.

  19. Receptor protein of Lysinibacillus sphaericus mosquito-larvicidal toxin displays amylomaltase activity.

    Science.gov (United States)

    Sharma, Mahima; Gupta, Gagan D; Kumar, Vinay

    2018-02-01

    The activated binary toxin (BinAB) from Lysinibacillus sphaericus binds to surface receptor protein (Cqm1) on the midgut cell membrane and kills Culex quinquefasciatus larvae on internalization. Cqm1 is attached to cells via a glycosyl-phosphatidylinositol (GPI) anchor. It has been classified as a member of glycoside hydrolase family 13 of the CAZy database. Here, we report characterization of the ordered domain (residues 23-560) of Cqm1. Gene expressing Cqm1 of BinAB susceptible mosquito was chemically synthesized and the protein was purified using E. coli expression system. Values for the Michaelis-Menten kinetics parameters towards 4-nitrophenyl α-D-glucopyranoside (α-pNPG) substrate were estimated to be 0.44 mM (Km) and 1.9 s -1 (kcat). Thin layer chromatography experiments established Cqm1 as α-glucosidase competent to cleave α-1,4-glycosidic bonds of maltose and maltotriose with high glycosyltransferase activity to form glucose-oligomers. The observed hydrolysis and synthesis of glucose-oligomers is consistent with open and accessible active-site in the structural model. The protein also hydrolyses glycogen and sucrose. These activities suggest that Cqm1 may be involved in carbohydrate metabolism in mosquitoes. Further, toxic BinA component does not inhibit α-glucosidase activity of Cqm1, while BinB reduced the activity by nearly 50%. The surface plasmon resonance study reveals strong binding of BinB with Cqm1 (Kd, 9.8 nM). BinA interaction with Cqm1 however, is 1000-fold weaker. Notably the estimated Kd values match well with dissociation constants reported earlier with larvae brush border membrane fractions. The Cqm1 protein forms a stable dimer that is consistent with its apical localization in lipid rafts. Its melting temperature (T m ) as observed by thermofluor-shift assay is 51.5 °C and Ca 2+ provides structural stability to the protein. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. piRNA Profiling of Dengue Virus Type 2-Infected Asian Tiger Mosquito and Midgut Tissues

    Directory of Open Access Journals (Sweden)

    Yanhai Wang

    2018-04-01

    Full Text Available The Asian tiger mosquito, Aedes albopictus, is a competent vector for the majority of arboviruses. The mosquito innate immune response is a primary determinant for arthropod-borne virus transmission, and the midgut is the first barrier to pathogen transmission. Mosquito antiviral immunity is primarily mediated by the small interfering RNA pathway. However, the roles that the P-element induced wimpy testis (PIWI-interacting RNA (piRNA pathway play in antiviral immunity in Ae. albopictus and its midgut still need further exploration. This study aimed to explore the profiles of both viral-derived and host-originated piRNAs in the whole body and midgut infected with Dengue virus 2 (DENV-2 in Ae. albopictus, and to elucidate gene expression profile differences of the PIWI protein family between adult females and their midguts. A deep sequencing-based method was used to identify and analyze small non-coding RNAs, especially the piRNA profiles in DENV-2-infected Ae. albopictus and its midgut. The top-ranked, differentially-expressed piRNAs were further validated using Stem-loop qRT-PCR. Bioinformatics analyses and reverse-transcription PCR (RT-PCR methods were used to detect PIWI protein family members, and their expression profiles. DENV-2 derived piRNAs (vpiRNA, 24–30 nts were observed in both infected Ae. albopictus and its midgut; however, only vpiRNA in the whole-body library had a weak preference for adenine at position 10 (10A in the sense molecules as a feature of secondary piRNA. These vpiRNAs were not equally distributed, instead they were derived from a few specific regions of the genome, especially several hot spots, and displayed an obvious positive strand bias. We refer to the differentially expressed host piRNAs after DENV infection as virus-induced host endogenous piRNAs (vepiRNAs. However, we found that vepiRNAs were abundant in mosquito whole-body tissue, but deficient in the midgut. A total of eleven PIWI family genes were

  1. Azadirachtin Affects the Growth of Spodoptera litura Fabricius by Inducing Apoptosis in Larval Midgut

    Directory of Open Access Journals (Sweden)

    Benshui Shu

    2018-02-01

    Full Text Available Azadirachtin, the environmentally friendly botanical pesticide, has been used as an antifeedant and pest growth regulator in integrated pest management for decades. It has shown strong biological activity against Spodoptera litura, but the mechanism of toxicity remains unclear. The present study showed that azadirachtin inhibited the growth of S. litura larvae, which was resulted by structure destroy and size inhibition of the midgut. Digital gene expression (DGE analysis of midgut suggested that azadirachtin regulated the transcriptional level of multiple unigenes involved in mitogen-activated protein kinase (MAPK and calcium apoptotic signaling pathways. Simultaneously, the expression patterns of some differentially expressed unigenes were verified by quantitative real time-PCR (qRT-PCR. In addition, the enhanced terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL staining, the increased expression of caspase family members and apoptosis-binding motif 1 (IBM1 on both gene and protein level and the release of cytochrome c from mitochondria to cytoplasm were induced in midgut after azadirachtin treatment. These results demonstrated that azadirachtin induced structural alteration in S. litura larval midgut by apoptosis activation. These alterations may affect the digestion and absorption of nutrients and eventually lead to the growth inhibition of larvae.

  2. iTRAQ-based quantitative proteomic analysis of midgut in silkworm infected with Bombyx mori cytoplasmic polyhedrosis virus.

    Science.gov (United States)

    Gao, Kun; Deng, Xiang-Yuan; Shang, Meng-Ke; Qin, Guang-Xing; Hou, Cheng-Xiang; Guo, Xi-Jie

    2017-01-30

    Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects the epithelial cells in the midgut of silkworm and causes them to death, which negatively affects the sericulture industry. In order to determine the midgut response at the protein levels to the virus infection, differential proteomes of the silkworm midgut responsive to BmCPV infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). 193, 408, 189 differentially expressed proteins (DEPs) were reliably quantified by iTRAQ analysis in the midgut of BmCPV-infected and control larvae at 24, 48, 72h post infection (hpi) respectively. KEGG enrichment analysis showed that Oxidative phosphorylation, amyotrophic lateral sclerosis, Toll-like receptor signaling pathway, steroid hormone biosynthesis were the significant pathways (Q value≤0.05) both at 24 and 48hpi. qRT-PCR was used to further verify gene transcription of 30 DEPs from iTRAQ, showing that the regulations of 24 genes at the transcript level were consistent with those at the proteomic level. Moreover, the cluster analysis of the three time groups showed that there were seven co-regulated DEPs including BGIBMGA002620-PA, which was a putative p62/sequestosome-1 protein in silkworm. It was upregulated at both the mRNA level and the proteomic level and may play an important role in regulating the autophagy and apoptosis (especially apoptosis) induced by BmCPV infection. This was the first report using an iTRAQ approach to analyze proteomes of the silkworm midgut against BmCPV infection, which contributes to understanding the defense mechanisms of silkworm midgut to virus infection. The domesticated silkworm, Bombyx mori, is renowned for silk production as well as being a traditional lepidopteron model insect served as a subject for morphological, genetic, physiological, and developmental studies. Bombyx mori cytoplasmic polyhedrosis

  3. The four serotypes of dengue recognize the same putative receptors in Aedes aegypti midgut and Ae. albopictus cells

    Directory of Open Access Journals (Sweden)

    Camacho-Nuez Minerva

    2006-10-01

    Full Text Available Abstract Background Dengue viruses (DENV attach to the host cell surface and subsequently enter the cell by receptor-mediated endocytosis. Several primary and low affinity co-receptors for this flavivirus have been identified. However, the presence of these binding molecules on the cell surface does not necessarily render the cell susceptible to infection. Determination of which of them serve as bona fide receptors for this virus in the vector may be relevant to treating DENV infection and in designing control strategies. Results (1 Overlay protein binding assay showed two proteins with molecular masses of 80 and 67 kDa (R80 and R67. (2 Specific antibodies against these two proteins inhibited cell binding and infection. (3 Both proteins were bound by all four serotypes of dengue virus. (4 R80 and R67 were purified by affinity chromatography from Ae. aegypti mosquito midguts and from Ae albopictus C6/36 cells. (5 In addition, a protein with molecular mass of 57 kDa was purified by affinity chromatography from the midgut extracts. (6 R80 and R67 from radiolabeled surface membrane proteins of C6/36 cells were immunoprecipitated by antibodies against Ae. aegypti midgut. Conclusion Our results strongly suggest that R67 and R80 are receptors for the four serotypes of dengue virus in the midgut cells of Ae. aegypti and in C6/36 Ae. albopictus cells.

  4. A novel mutation in PGAP2 gene causes developmental delay, intellectual disability, epilepsy and microcephaly in consanguineous Saudi family.

    Science.gov (United States)

    Naseer, Muhammad Imran; Rasool, Mahmood; Jan, Mohammed M; Chaudhary, Adeel G; Pushparaj, Peter Natesan; Abuzenadah, Adel M; Al-Qahtani, Mohammad H

    2016-12-15

    PGAP2 (Post-GPI Attachment to Proteins 2) gene is involved in lipid remodeling steps of Glycosylphosphatidylinositol (GPI)-anchor maturation. At the surface of the cell this gene is required for proper expression of GPI-anchored proteins. Hyperphosphatasia with mental retardation syndrome-3 is an autosomal recessive disorder usually characterized by severe mental retardation. Mutations in the PGAP2 gene cause hyperphosphatasia mental retardation syndrome-3. We have identified a large consanguineous family from Saudi origin segregating developmental delay, intellectual disability, epilepsy and microcephaly. Whole exome sequencing with 100× coverage was performed on two affected siblings of the family. Data analysis in the patient revealed a novel missense mutation c.191C>T in PGAP2 gene resulting in Alanine to Valine substitution (Ala64Val). The mutation was reconfirmed and validated by subsequent Sanger sequencing method. The mutation was ruled out in 100 unrelated healthy controls. We suggest that this pathogenic mutation disrupts the proper function of the gene proteins resulting in the disease state. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. In vivo binding of the Cry11Bb toxin of Bacillus thuringiensis subsp. medellin to the midgut of mosquito larvae (Diptera: Culicidae

    Directory of Open Access Journals (Sweden)

    Ruiz Lina María

    2004-01-01

    Full Text Available Bacillus thuringiensis subsp. medellin produces numerous proteins among which 94 kDa known as Cry11Bb, has mosquitocidal activity. The mode of action of the Cry11 proteins has been described as similar to those of the Cry1 toxins, nevertheless, the mechanism of action is still not clear. In this study we investigated the in vivo binding of the Cry11Bb toxin to the midgut of the insect species Anopheles albimanus, Aedes aegypti, and Culex quinquefasciatus by immunohistochemical analysis. Spodoptera frugiperda was included as negative control. The Cry11Bb protein was detected on the apical microvilli of the midgut epithelial cells, mostly on the posterior midgut and gastric caeca of the three mosquito species. Additionally, the toxin was detected in the Malpighian tubules of An. albimanus, Ae. aegypti, Cx. quinquefasciatus, and in the basal membrane of the epithelial cells of Ae. aegypti midgut. No toxin accumulation was observed in the peritrophic membrane of any of the mosquito species studied. These results confirm that the primary site of action of the Cry11 toxins is the apical membrane of the midgut epithelial cells of mosquito larvae.

  6. pH control in the midgut of Aedesaegypti under different nutritional conditions.

    Science.gov (United States)

    Nepomuceno, Denise Barguil; Santos, Vânia Cristina; Araújo, Ricardo Nascimento; Pereira, Marcos Horácio; Sant'Anna, Maurício Roberto; Moreira, Luciano Andrade; Gontijo, Nelder Figueiredo

    2017-09-15

    Aedes aegypti is one of the most important disease vectors in the world. Because their gut is the first site of interaction with pathogens, it is important to understand A. aegypti gut physiology. In this study, we investigated the mechanisms of pH control in the midgut of A. aegypti females under different nutritional conditions. We found that unfed females have an acidic midgut (pH ∼6). The midgut of unfed insects is actively maintained at pH 6 regardless of the ingestion of either alkaline or acidic buffered solutions. V-ATPases are responsible for acidification after ingestion of alkaline solutions. In blood-fed females, the abdominal midgut becomes alkaline (pH 7.54), and the luminal pH decreases slightly throughout blood digestion. Only ingested proteins were able to trigger this abrupt increase in abdominal pH. The ingestion of amino acids, even at high concentrations, did not induce alkalinisation. During blood digestion, the thoracic midgut remains acidic, becoming a suitable compartment for carbohydrate digestion, which is in accordance with the higher alpha-glucolytic activity detected in this compartment. Ingestion of blood releases alkalising hormones in the haemolymph, which induce alkalinisation in ex vivo preparations. This study shows that adult A. aegypti females have a very similar gut physiology to that previously described for Lutzomyia longipalpis It is likely that all haematophagous Nematocera exhibit the same type of physiological behaviour. © 2017. Published by The Company of Biologists Ltd.

  7. Intestinal Rotation Abnormalities and Midgut Volvulus.

    Science.gov (United States)

    Langer, Jacob C

    2017-02-01

    Rotation abnormalities may be asymptomatic or may be associated with obstruction caused by bands, midgut volvulus, or associated atresia or web. The most important goal of clinicians is to determine whether the patient has midgut volvulus with intestinal ischemia, in which case an emergency laparotomy should be done. If the patient is not acutely ill, the next goal is to determine whether the patient has a narrow-based small bowel mesentery. In general, the outcomes for children with a rotation abnormality are excellent, unless there has been midgut volvulus with significant intestinal ischemia. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Akinori Matsushika

    Full Text Available 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

  9. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    2011-07-08

    specific phospholipase C (PI-PLC), and the subsequent recognition by antibodies specific for the cross-reacting determinant (CRD), revealed that HBP is glycosylphosphatidylinositol (GPI)-anchored protein and, further, that the ...

  10. Differential expression profiles in the midgut of Triatoma infestans infected with Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    Diego S Buarque

    Full Text Available Chagas disease, or American trypanosomiasis, is a parasitic disease caused by the protozoan Trypanosoma cruzi and is transmitted by insects from the Triatominae subfamily. To identify components involved in the protozoan-vector relationship, we constructed and analyzed cDNA libraries from RNA isolated from the midguts of uninfected and T. cruzi-infected Triatoma infestans, which are major vectors of Chagas disease. We generated approximately 440 high-quality Expressed Sequence Tags (ESTs from each T. infestans midgut cDNA library. The sequences were grouped in 380 clusters, representing an average length of 664.78 base pairs (bp. Many clusters were not classified functionally, representing unknown transcripts. Several transcripts involved in different processes (e.g., detoxification showed differential expression in response to T. cruzi infection. Lysozyme, cathepsin D, a nitrophorin-like protein and a putative 14 kDa protein were significantly upregulated upon infection, whereas thioredoxin reductase was downregulated. In addition, we identified several transcripts related to metabolic processes or immunity with unchanged expressions, including infestin, lipocalins and defensins. We also detected ESTs encoding juvenile hormone binding protein (JHBP, which seems to be involved in insect development and could be a target in control strategies for the vector. This work demonstrates differential gene expression upon T. cruzi infection in the midgut of T. infestans. These data expand the current knowledge regarding vector-parasite interactions for Chagas disease.

  11. Lufenuron impact upon Anthonomus grandis Boheman (Coleoptera: Curculionidae) midgut and its reflection in gametogenesis.

    Science.gov (United States)

    Costa, Hilton Nobre; da Cunha, Franklin Magliano; Cruz, Glaucilane Santos; D'assunção, Carolline Guimarães; Rolim, Guilherme Gomes; Barros, Maria Edna Gomes; Breda, Mariana Oliveira; Teixeira, Alvaro Aguiar Coelho; Teixeira, Valéria Wanderley

    2017-04-01

    The insecticide Match® (lufenuron), one of the main insect growth regulators used in pest control, has been presented as a viable alternative against the boll weevil, Anthonomus grandis (Coleoptera: Curculionidae), by inhibiting chitin synthesis. Thus, this study aimed to examine whether Match® interferes in the synthesis of the peritrophic matrix, leading to changes in the midgut epithelium, resulting in nutritional deficiency and reflecting, thereby, in the gametogenesis process of A. grandis. Floral cotton buds were immersed in the insecticide solution (800μL of Match®+200mL of distilled water) and offered to the adult insects. The midguts of the insects were evaluated after 24 and 120h after feeding. The gonads were evaluated after 120h. The results showed that Match®, in both evaluation periods, induced histopathological alterations such as disorganization, vacuolization and desquamation of the midgut epithelium; histochemical modifications in the distribution patterns of carbohydrates, although without quantitative changes; and a strong decrease in protein levels. No apoptosis were observed, however, there was an increase in the number of regenerative cell nests. In the testicles, a reduction in the amount of spermatozoids and reduced carbohydrate levels were observed, but no difference in protein levels. The ovarioles presented structural disorganization of follicular cells, yolk reduction and decrease in protein levels, however, no change in carbohydrates levels was noted. Therefore, it is concluded that Match® performs histopathologic and histochemical alterations in the midgut epithelium and the gonads of A. grandis adults, reflecting in the gametogenesis process, presenting itself as a promising tool in the management of this pest on cotton crops. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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.

  13. Phospholipase D specific for the phosphatidylinositol anchor of cell-surface proteins is abundant in plasma

    International Nuclear Information System (INIS)

    Low, M.G.; Prasad, A.R.S.

    1988-01-01

    An enzyme activity capable of degrading the glycosyl-phosphatidylinositol membrane anchor of cell-surface proteins has previously been reported in a number of mammalian tissues. The experiments reported here demonstrate that this anchor-degrading activity is also abundant in mammalian plasma. The activity was inhibited by EGTA or 1,10-phenanthroline. It was capable of removing the anchor from alkaline phosphatase, 5'-nucleotidase, and variant surface glycoprotein but had little or no activity toward phosphatidylinositol or phosphatidylcholine. Phosphatidic acid was the only 3 H-labeled product when this enzyme hydrolyzed [ 3 H]myristate-labeled variant surface glycoprotein. It could be distinguished from the Ca 2 =-dependent inositol phospholipid-specific phospholipase C activity in several rat tissues on the basis of its molecular size and its sensitivity to 1,10-phenanthroline. The data therefore suggest that this activity is due to a phospholipase D with specificity for glycosylphosphatidylinositol structures. Although the precise physiological function of this anchor-specific phospholipase D remains to be determined, these findings indicate that it could play an important role in regulating the expression and release of cell-surface proteins in vivo

  14. Insight into the Mode of Action of Celangulin V on the Transmembrane Potential of Midgut Cells in Lepidopteran Larvae

    Directory of Open Access Journals (Sweden)

    Yingying Wang

    2017-12-01

    Full Text Available Celangulin V (CV is the main insecticidal constituent of Celastrus angulatus. The V-ATPase H subunit of the midgut cells of lepidopteran larvae is the putative target protein of CV. Here, we compared the effects of CV on the midgut membrane potentials of Mythimna separata and Agrotis ipsilon larvae with those of the Cry1Ab toxin from Bacillus thuringiensis and with those of inactive CV-MIA, a synthetic derivative of CV. We investigated the changes in the apical membrane potentials (Vam and basolateral membrane potentials (Vbm of the midguts of sixth-instar larvae force-fed with the test toxins. We also measured the Vam and Vbm of larval midguts that were directly incubated with the test toxins. Similar to the effect of Cry1Ab, the Vam of CV-treated midguts rapidly decayed over time in a dose-dependent manner. By contrast, CV-MIA did not influence Vam. Meanwhile, the Vam of A. ipsilon larval midguts directly incubated with CV decayed less than that of M. separata larval midguts, whereas that of larvae force-fed with CV did not significantly change. Similar to Cry1Ab, CV did not affect the Vbm of isolated midguts. CV significantly inhibited V-ATPase activity in a dose-dependent manner. Therefore, CV initially inhibits V-ATPase in the apical membrane and affects intracellular pH, homeostasis, and nutrient transport mechanisms in lepidopteran midgut cells.

  15. Altered dynamics of a lipid raft associated protein in a kidney model of Fabry disease.

    Science.gov (United States)

    Labilloy, Anatália; Youker, Robert T; Bruns, Jennifer R; Kukic, Ira; Kiselyov, Kirill; Halfter, Willi; Finegold, David; do Monte, Semiramis Jamil Hadad; Weisz, Ora A

    2014-02-01

    Accumulation of globotriaosylceramide (Gb3) and other neutral glycosphingolipids with galactosyl residues is the hallmark of Fabry disease, a lysosomal storage disorder caused by deficiency of the enzyme alpha-galactosidase A (α-gal A). These lipids are incorporated into the plasma membrane and intracellular membranes, with a preference for lipid rafts. Disruption of raft mediated cell processes is implicated in the pathogenesis of several human diseases, but little is known about the effects of the accumulation of glycosphingolipids on raft dynamics in the context of Fabry disease. Using siRNA technology, we have generated a polarized renal epithelial cell model of Fabry disease in Madin-Darby canine kidney cells. These cells present increased levels of Gb3 and enlarged lysosomes, and progressively accumulate zebra bodies. The polarized delivery of both raft-associated and raft-independent proteins was unaffected by α-gal A knockdown, suggesting that accumulation of Gb3 does not disrupt biosynthetic trafficking pathways. To assess the effect of α-gal A silencing on lipid raft dynamics, we employed number and brightness (N&B) analysis to measure the oligomeric status and mobility of the model glycosylphosphatidylinositol (GPI)-anchored protein GFP-GPI. We observed a significant increase in the oligomeric size of antibody-induced clusters of GFP-GPI at the plasma membrane of α-gal A silenced cells compared with control cells. Our results suggest that the interaction of GFP-GPI with lipid rafts may be altered in the presence of accumulated Gb3. The implications of our results with respect to the pathogenesis of Fabry disease are discussed. © 2013 Elsevier Inc. All rights reserved.

  16. Neurodegeneration and unfolded-protein response in mice expressing a membrane-tethered flexible tail of PrP.

    Directory of Open Access Journals (Sweden)

    Paolo Dametto

    Full Text Available The cellular prion protein (PrPC consists of a flexible N-terminal tail (FT, aa 23-128 hinged to a membrane-anchored globular domain (GD, aa 129-231. Ligation of the GD with antibodies induces rapid neurodegeneration, which is prevented by deletion or functional inactivation of the FT. Therefore, the FT is an allosteric effector of neurotoxicity. To explore its mechanism of action, we generated transgenic mice expressing the FT fused to a GPI anchor, but lacking the GD (PrPΔ141-225, or "FTgpi". Here we report that FTgpi mice develop a progressive, inexorably lethal neurodegeneration morphologically and biochemically similar to that triggered by anti-GD antibodies. FTgpi was mostly retained in the endoplasmic reticulum, where it triggered a conspicuous unfolded protein response specifically activating the PERK pathway leading to phosphorylation of eIF2α and upregulation of CHOP ultimately leading to neurodegeration similar to what was observed in prion infection.

  17. GPI-repetitive control for linear systems with parameter uncertainty / variation

    Directory of Open Access Journals (Sweden)

    John A. Cortés-Romero

    2015-01-01

    Full Text Available Robust repetitive control problems for uncertain linear systems have been considered by different approaches. This article proposes the use of Repetitive Control and Generalized Proportional Integral (GPI Control in a complementary fashion. The conditioning and coupling of these techniques has been done in a time discrete context. Repetitive control is a control technique, based on the internal model principle, which yields perfect asymptotic tracking and rejection of periodic signals. On the other hand, GPI control is established as a robust linear control system design technique that is able to reject structured time polynomial additive perturbation, in particular, parameter uncertainty that can be locally approximated by time polynomial signal. GPI control provides a suitable stability and robustness conditions for the proper Repetitive Control operation. A stability analysis is presented under the frequency response framework using plant samples for different parameter uncertainty conditions. We carry out some comparative stability analysis with other complementary control approaches that has been effective for this kind of task, enhancing a better robustness and an improved performance for the GPI case. Illustrative simulation examples are presented which validate the proposed approach.

  18. Spatial and sex-specific dissection of the Anopheles gambiae midgut transcriptome

    Directory of Open Access Journals (Sweden)

    Mahairaki Vassiliki

    2007-01-01

    Full Text Available Abstract Background The midgut of hematophagous insects, such as disease transmitting mosquitoes, carries out a variety of essential functions that mostly relate to blood feeding. The midgut of the female malaria vector mosquito Anopheles gambiae is a major site of interactions between the parasite and the vector. Distinct compartments and cell types of the midgut tissue carry out specific functions and vector borne pathogens interact and infect different parts of the midgut. Results A microarray based global gene expression approach was used to compare transcript abundance in the four major female midgut compartments (cardia, anterior, anterior part of posterior and posterior part of posterior midgut and between the male and female Anopheles gambiae midgut. Major differences between the female and male midgut gene expression relate to digestive processes and immunity. Each compartment has a distinct gene function profile with the posterior midgut expressing digestive enzyme genes and the cardia and anterior midgut expressing high levels of antimicrobial peptide and other immune gene transcripts. Interestingly, the cardia expressed several known anti-Plasmodium factors. A parallel peptidomic analysis of the cardia identified known mosquito antimicrobial peptides as well as several putative short secreted peptides that are likely to represent novel antimicrobial factors. Conclusion The A. gambiae sex specific midgut and female midgut compartment specific transcriptomes correlates with their known functions. The significantly greater functional diversity of the female midgut relate to hematophagy that is associated with digestion and nutrition uptake as well as exposes it to a variety of pathogens, and promotes growth of its endogenous microbial flora. The strikingly high proportion of immunity related factors in the cardia tissue most likely serves the function to increase sterility of ingested sugar and blood. A detailed characterization of the

  19. Conditional Depletion of Nuclear Proteins by the Anchor Away System (ms# CP-10-0125)

    Science.gov (United States)

    Fan, Xiaochun; Geisberg, Joseph V.; Wong, Koon Ho; Jin, Yi

    2011-01-01

    Nuclear proteins play key roles in the regulation of many important cellular processes. In Saccharomyces cerevisiae, many genes encoding nuclear proteins are essential. Here we describe a method termed Anchor Away that can be used to conditionally and rapidly deplete nuclear proteins of interest. It involves conditional export of the protein of interest out of the nucleus and its subsequent sequestration in the cytoplasm. This method can be used to simultaneously deplete multiple proteins from nucleus. PMID:21225637

  20. Clathrin-independent pathways do not contribute significantly to endocytic flux.

    Science.gov (United States)

    Bitsikas, Vassilis; Corrêa, Ivan R; Nichols, Benjamin J

    2014-09-17

    Several different endocytic pathways have been proposed to function in mammalian cells. Clathrin-coated pits are well defined, but the identity, mechanism and function of alternative pathways have been controversial. Here we apply universal chemical labelling of plasma membrane proteins to define all primary endocytic vesicles, and labelling of specific proteins with a reducible SNAP-tag substrate. These approaches provide high temporal resolution and stringent discrimination between surface-connected and intracellular membranes. We find that at least 95% of the earliest detectable endocytic vesicles arise from clathrin-coated pits. GPI-anchored proteins, candidate cargoes for alternate pathways, are also found to enter the cell predominantly via coated pits. Experiments employing a mutated clathrin adaptor reveal distinct mechanisms for sorting into coated pits, and thereby explain differential effects on the uptake of transferrin and GPI-anchored proteins. These data call for a revision of models for the activity and diversity of endocytic pathways in mammalian cells.

  1. First trimester fetal physiologic midgut herniation: Transvaginal sonographic findings

    International Nuclear Information System (INIS)

    Kwon, Tae Hee; Park, Yong Hyun

    1999-01-01

    To evaluate the sonographic features and appearance time of the physiologic midgut herniation early in pregnancy. Sonograms of 87 features ranging from 7 to 13 weeks were obtained over a 2-month period. The presence or absence, the size and echogenecity of the physiologic midgut herniation were evaluate on each examination. Disappearance of the midgut herniation was confirmed on follow-up sonogram at 13-20 weeks, 3-12 weeks after the first sonogram. The results were analyzed in terms of appearance or disappearance time of midgut herniation. In all cases of physiologic midgut herination, an echogenic mass measuring 0.4-0.7 cm wa demonstrated within the base of the umbilical cord at its insertion into the fetal abdomen. This herniation was detected in 3/6 cases (50%) at 8 weeks, in 15/16 cases (94%) at 9 weeks, in 22/24 cases (92%) at 10 weeks and in 12/27 cases (44%) at 11 weeks gestation. None of the features studied at 7 weeks and 12 weeks had a midgut herniation. Sonographic findings of a 0.4-0.7 cm sized echogenic mass within the base of the umbilical cord which appears from 8 weeks to 12 weeks pregnancy represent physiologic midgut herniation in early pregnancy and should not be confused with pathologic ventral wall defected such as omphalocele or gastroschisis.

  2. First trimester fetal physiologic midgut herniation: Transvaginal sonographic findings

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Tae Hee; Park, Yong Hyun [CHA General Hospital. Pochon College of Medicine, Seoul (Korea, Republic of)

    1999-06-15

    To evaluate the sonographic features and appearance time of the physiologic midgut herniation early in pregnancy. Sonograms of 87 features ranging from 7 to 13 weeks were obtained over a 2-month period. The presence or absence, the size and echogenecity of the physiologic midgut herniation were evaluate on each examination. Disappearance of the midgut herniation was confirmed on follow-up sonogram at 13-20 weeks, 3-12 weeks after the first sonogram. The results were analyzed in terms of appearance or disappearance time of midgut herniation. In all cases of physiologic midgut herination, an echogenic mass measuring 0.4-0.7 cm wa demonstrated within the base of the umbilical cord at its insertion into the fetal abdomen. This herniation was detected in 3/6 cases (50%) at 8 weeks, in 15/16 cases (94%) at 9 weeks, in 22/24 cases (92%) at 10 weeks and in 12/27 cases (44%) at 11 weeks gestation. None of the features studied at 7 weeks and 12 weeks had a midgut herniation. Sonographic findings of a 0.4-0.7 cm sized echogenic mass within the base of the umbilical cord which appears from 8 weeks to 12 weeks pregnancy represent physiologic midgut herniation in early pregnancy and should not be confused with pathologic ventral wall defected such as omphalocele or gastroschisis.

  3. Midgut proteinases of Sitotroga cerealella (Oliver) (Lepidoptera:Gelechiidae): Characterization and relationship to resistance in cereals

    International Nuclear Information System (INIS)

    Wu, Lan.

    1989-01-01

    Midgut proteinases are vital to the insects which digest ingested food in the midgut. Insect midgut proteinases, therefore, have been considered as possible targets for the control of insect pests. Proteinaceous proteinase inhibitors are very attractive for their potential use in developing insect resistant plant varieties via genetic engineering. Sitotroga cerealella is one of the major storage pests of cereals, and no antibiotic resistance in wheat against this insect has been identified to date. A series of diagnostic inhibitors, thiol-reducing agents and a metal-ion chelator were used in the identification of proteinases in crude extracts from S. cerealella larval midguts with both protein and ester substrates. The partial inhibition of proteolytic activity in crude midgut extract toward [ 3 H]-methemoglobin by pepstatin A suggested the presence of another proteinase which was sensitive to pepstatin A. The optimum pH range for the proteolytic activity, however, indicated that the major midgut proteinases were not carboxyl proteinases. Two proteinases were successfully purified by a combination of fractionation with ammonium sulfate, gel permeation and anion exchange chromatography. Characterization of the enzymes with the purified enzyme preparations confirmed that the two major proteinases were serine endoproteinases with trypsin-like and chymotrypsin-like specificities respectively. Bioassays were conducted using the artificial seeds to test naturally occurring proteinaceous proteinase inhibitors of potential value. Soybean trypsin inhibitor and the Bowman-Birk proteinase inhibitor had adverse effects on the development of the insect. A predictive model was constructed to evaluate effects of seed resistance in conjunction with other control methods on S. cerealella population dynamics

  4. Recurrent midgut volvulus following a Ladd procedure

    International Nuclear Information System (INIS)

    Panghaal, Vikash; Levin, Terry L.; Han, Bokyung

    2008-01-01

    We present a case of recurrent midgut volvulus in a 3-year-old girl with a history of midgut volvulus repair as an infant. Awareness of the possibility of recurrence even several years following an initial Ladd procedure is crucial to ensure prompt treatment in these children. (orig.)

  5. Recurrent midgut volvulus following a Ladd procedure

    Energy Technology Data Exchange (ETDEWEB)

    Panghaal, Vikash; Levin, Terry L.; Han, Bokyung [Montefiore Medical Center, Department of Radiology, Bronx, NY (United States)

    2008-04-15

    We present a case of recurrent midgut volvulus in a 3-year-old girl with a history of midgut volvulus repair as an infant. Awareness of the possibility of recurrence even several years following an initial Ladd procedure is crucial to ensure prompt treatment in these children. (orig.)

  6. A physiologically-oriented transcriptomic analysis of the midgut of Tenebrio molitor.

    Science.gov (United States)

    Moreira, Nathalia R; Cardoso, Christiane; Dias, Renata O; Ferreira, Clelia; Terra, Walter R

    2017-05-01

    Physiological data showed that T. molitor midgut is buffered at pH 5.6 at the two anterior thirds and at 7.9 at the posterior third. Furthermore, water is absorbed and secreted at the anterior and posterior midgut, respectively, driving a midgut counter flux of fluid. To look for the molecular mechanisms underlying these phenomena and nutrient absorption as well, a transcriptomic approach was used. For this, 11 types of transporters were chosen from the midgut transcriptome obtained by pyrosequencing (Roche 454). After annotation with the aid of databanks and manual curation, the sequences were validated by RT-PCR. The expression level of each gene at anterior, middle and posterior midgut and carcass (larva less midgut) was evaluated by RNA-seq taking into account reference sequences based on 454 contigs and reads obtained by Illumina sequencing. The data showed that sugar and amino acid uniporters and symporters are expressed along the whole midgut. In the anterior midgut are found transporters for NH 3 and NH 4 + that with a chloride channel may be responsible for acidifying the lumen. At the posterior midgut, bicarbonate-Cl - antiporter with bicarbonate supplied by carbonic anhydrase may alkalinize the lumen. Water absorption caused mainly by an anterior Na + -K + -2Cl - symporter and water secretion caused by a posterior K + -Cl - may drive the midgut counter flux. Transporters that complement the action of those described were also found. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Transcriptomic survey of the midgut of Anthonomus grandis (Coleoptera: Curculionidae).

    Science.gov (United States)

    Salvador, Ricardo; Príncipi, Darío; Berretta, Marcelo; Fernández, Paula; Paniego, Norma; Sciocco-Cap, Alicia; Hopp, Esteban

    2014-01-01

    Anthonomus grandis Boheman is a key pest in cotton crops in the New World. Its larval stage develops within the flower bud using it as food and as protection against its predators. This behavior limits the effectiveness of its control using conventional insecticide applications and biocontrol techniques. In spite of its importance, little is known about its genome sequence and, more important, its specific expression in key organs like the midgut. Total mRNA isolated from larval midguts was used for pyrosequencing. Sequence reads were assembled and annotated to generate a unigene data set. In total, 400,000 reads from A. grandis midgut with an average length of 237 bp were assembled and combined into 20,915 contigs. The assembled reads fell into 6,621 genes models. BlastX search using the NCBI-NR database showed that 3,006 unigenes had significant matches to known sequences. Gene Ontology (GO) mapping analysis evidenced that A. grandis is able to transcripts coding for proteins involved in catalytic processing of macromolecules that allows its adaptation to very different feeding source scenarios. Furthermore, transcripts encoding for proteins involved in detoxification mechanisms such as p450 genes, glutathione-S-transferase, and carboxylesterases are also expressed. This is the first report of a transcriptomic study in A. grandis and the largest set of sequence data reported for this species. These data are valuable resources to expand the knowledge of this insect group and could be used in the design of new control strategies based in molecular information. © The Author 2014. Published by Oxford University Press on behalf of the Entomological Society of America.

  8. Mutations in PIGY: expanding the phenotype of inherited glycosylphosphatidylinositol deficiencies.

    Science.gov (United States)

    Ilkovski, Biljana; Pagnamenta, Alistair T; O'Grady, Gina L; Kinoshita, Taroh; Howard, Malcolm F; Lek, Monkol; Thomas, Brett; Turner, Anne; Christodoulou, John; Sillence, David; Knight, Samantha J L; Popitsch, Niko; Keays, David A; Anzilotti, Consuelo; Goriely, Anne; Waddell, Leigh B; Brilot, Fabienne; North, Kathryn N; Kanzawa, Noriyuki; Macarthur, Daniel G; Taylor, Jenny C; Kini, Usha; Murakami, Yoshiko; Clarke, Nigel F

    2015-11-01

    Glycosylphosphatidylinositol (GPI)-anchored proteins are ubiquitously expressed in the human body and are important for various functions at the cell surface. Mutations in many GPI biosynthesis genes have been described to date in patients with multi-system disease and together these constitute a subtype of congenital disorders of glycosylation. We used whole exome sequencing in two families to investigate the genetic basis of disease and used RNA and cellular studies to investigate the functional consequences of sequence variants in the PIGY gene. Two families with different phenotypes had homozygous recessive sequence variants in the GPI biosynthesis gene PIGY. Two sisters with c.137T>C (p.Leu46Pro) PIGY variants had multi-system disease including dysmorphism, seizures, severe developmental delay, cataracts and early death. There were significantly reduced levels of GPI-anchored proteins (CD55 and CD59) on the surface of patient-derived skin fibroblasts (∼20-50% compared with controls). In a second, consanguineous family, two siblings had moderate development delay and microcephaly. A homozygous PIGY promoter variant (c.-540G>A) was detected within a 7.7 Mb region of autozygosity. This variant was predicted to disrupt a SP1 consensus binding site and was shown to be associated with reduced gene expression. Mutations in PIGY can occur in coding and non-coding regions of the gene and cause variable phenotypes. This article contributes to understanding of the range of disease phenotypes and disease genes associated with deficiencies of the GPI-anchor biosynthesis pathway and also serves to highlight the potential importance of analysing variants detected in 5'-UTR regions despite their typically low coverage in exome data. © The Author 2015. Published by Oxford University Press.

  9. Strong alkalinization in the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti): involvement of luminal Na+/K+-ATPase.

    Science.gov (United States)

    Onken, Horst; Patel, Malay; Javoroncov, Margarita; Izeirovski, Sejmir; Moffett, Stacia B; Moffett, David F

    2009-03-01

    Recently, Na(+)/K(+)-ATPase has been detected in the luminal membrane of the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti) with immunohistochemical techniques. In this study, the possible involvement of this ATPase in strong alkalinization was investigated on the level of whole larvae, isolated and perfused midgut preparations and on the molecular level of the Na(+)/K(+)-ATPase protein. Ouabain (5 mM) did not inhibit the capability of intact larval mosquitoes to alkalinize their anterior midgut. Also in isolated and perfused midgut preparations the perfusion of the lumen with ouabain (5 mM) did not result in a significant change of the transepithelial voltage or the capacity of luminal alkalinization. Na(+)/K(+)-ATPase activity was completely abolished when KCl was substituted with choline chloride, suggesting that the enzyme cannot act as an ATP-driven Na(+)/H(+)-exchanger. Altogether the results of the present investigation indicate that apical Na(+)/K(+)-ATPase is not of direct importance for strong luminal alkalinization in the anterior midgut of larval yellow fever mosquitoes.

  10. Molecular and biochemical responses in the midgut of the silkworm, Bombyx mori, infected with Nosema bombycis.

    Science.gov (United States)

    Li, Zhi; Wang, Yu; Wang, Linling; Zhou, Zeyang

    2018-03-06

    Microsporidia are a group of eukaryotic intracellular parasites that infect almost all vertebrates and invertebrates. However, there is little information available of how microsporidia obtain nutrients and energy from host cells. The purpose of this study was to investigate the energy and material requirements of Nosema bombycis for the invasion procedure through analyzing the global variation of the gene expression, protein abundance, fatty acids level and ATP flux induced by the microsporidia N. bombycis infection in the midgut of the silkworm Bombyx mori. A suppression subtractive hybridization (SSH) and quantitative real-time PCR (qPCR) analysis were performed to identify the genes upregulated in the midgut of B. mori 48 h following N. bombycis infection. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to annotate and summarize the differentially expressed genes, according to the categories 'molecular function', 'cellular component' and 'biological process'. To evaluate the nutrition material and energy costs in B.mori infected by N. bombycis, biochemical analysis was performed to determine the variation of protein abundance, fatty acid levels and ATP flux with or without the microsporidia N. bombycis infection in the midgut of the silkworm B. mori. A total of 744 clones were obtained, 288 clones were randomly selected for sequencing, and 110 unigenes were generated. Amongst these, 49.21%, 30.16% and 14.29% genes were involved in 19 molecular functions, 19 biological processes and nine cellular components, respectively. A total of 11 oxidative phosphorylation- and eight proton-coupled ATP synthesis-related genes were upregulated. Seven protein degradation-, three fat degradation-related genes were upregulated, and no genes related to the de novo synthesis of amino acids and fatty acids were significantly upregulated. The data from the biochemical analysis showed the contents of total protein and ATP of B. mori

  11. Andrographolide powder treatment as antifeedant decreased digestive enzyme activity from Plutella xylostella (L.) larvae midgut

    Science.gov (United States)

    Madihah, Malini, Desak Made; Roviani, Hana; Rani, Nessa Vidya; Hermawan, Wawan

    2018-02-01

    Andrographolide, an active compound of Andrographis paniculata, has shown antifeedant activity against Plutella xylostella larvae by disrupting the midgut histological structures. This study aims to determine the activity of andrographolide in crystallized powder form against several digestive enzymes from the midgut of 4th instar P. xylostella larvae. The concentrations used were 0 (control), 1000, 1600, 2500, 4000 and 6500 ppm with four replications each. No-choice antifeedant test with leaf disc method is used in a bioassay for 24 hours. The midgut was dissected from 2nd until 6th segment of 4th instar larvae and was homogenized in iced-buffer solution. Furthermore, larvae's midgut samples were centrifuged at 10,000 rpm, 4°C for 20 min and the supernatant is used as enzyme source. The results showed that andrographolide significantly reduces the amylase, invertase, protease and trypsin activity, as well as total protein concentration compared with control (p<0.05) in a dose-dependent manner. This study provides information about the mode of action of andrographolide in inhibiting feed activity by the reduced digestive enzyme activity of 4th instar P. xylostella larvae.

  12. Midgut absorption of proteins by tsetse flies, Glossina morsitans morsitans Westwood (Diptera: Glossinidae)

    International Nuclear Information System (INIS)

    Nogge, G.; Giannetti, M.

    1980-01-01

    Experiments have been conducted to determine how far tsetse flies are able to absorb undigested proteins from the midgut and to transport into the haemolymph. Comparison of the protein pattern of blood serum and haemolymph as revealed by electrophoresis shows no congruency except for the albumin fraction, which is about 10% of the total protein contents. The peritrophic membrane of tsetse flies allows the passage of FITC-conjugated dextrans up to but not greater than a molecular weight of approximately 45,000. It can be concluded therefore that complete serum globulins cannot pass through the peritrophic membrane, but fragments of globulins and albumin might do so. The presence of serum proteins in the haemolymph of tsetse flies after oral administration was investigated by immunological techniques. With the help of immunoelectrophoresis as well as Ouchter-Lony tests, albumin, Fab-, and Fc-fragments were found to be present in the haemolymph. Tsetse flies usually fed on bovine blood replace bovine albumin present in their haemolymph by human albumin when fed on human blood. Quantitative determinations with the aid of rocket immunoelectrophoresis after Laurell revealed the occurrence of human albumin in relation to the size and number of blood meals. When fed continuously on human blood, the amount of human albumin rises following eight to ten blood meals after which the achieved level is maintained. Human albumin disappears from the haemolymph and is replaced by bovine albumin when the flies are fed again with bovine blood. On the basis of these results the amount of albumin absorbed from a single blood meal lies in the range of 0.01 to 0.035% of its albumin contents. (author)

  13. Epididymosomes: transfer of fertility-modulating proteins to the sperm surface.

    Science.gov (United States)

    Martin-DeLeon, Patricia A

    2015-01-01

    A variety of glycosylphosphatidylinositol (GPI)-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP) which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane (TM) proteins, including plasma membrane Ca 2 + -ATPase 4 (PMCA4). Synthesized in the testis, PMCA4 is an essential protein and the major Ca 2 + efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion-competent sites on membranes. On the basis of knowledge of PMCA4's interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca 2 + -dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach.

  14. Epididymosomes: transfer of fertility-modulating proteins to the sperm surface

    Directory of Open Access Journals (Sweden)

    Patricia A Martin-DeLeon

    2015-01-01

    Full Text Available A variety of glycosylphosphatidylinositol (GPI-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane (TM proteins, including plasma membrane Ca 2 + -ATPase 4 (PMCA4. Synthesized in the testis, PMCA4 is an essential protein and the major Ca 2 + efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion-competent sites on membranes. On the basis of knowledge of PMCA4′s interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca 2 + -dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach.

  15. Comprehensive Evaluation of Streptococcus sanguinis Cell Wall-Anchored Proteins in Early Infective Endocarditis▿ †

    OpenAIRE

    Turner, Lauren Senty; Kanamoto, Taisei; Unoki, Takeshi; Munro, Cindy L.; Wu, Hui; Kitten, Todd

    2009-01-01

    Streptococcus sanguinis is a member of the viridans group of streptococci and a leading cause of the life-threatening endovascular disease infective endocarditis. Initial contact with the cardiac infection site is likely mediated by S. sanguinis surface proteins. In an attempt to identify the proteins required for this crucial step in pathogenesis, we searched for surface-exposed, cell wall-anchored proteins encoded by S. sanguinis and then used a targeted signature-tagged mutagenesis (STM) a...

  16. MIDGUT MALROTATION IN OLDER CHILDREN

    Directory of Open Access Journals (Sweden)

    H. Nahvi Z. Khorgami

    2008-05-01

    Full Text Available Midgut malrotation is typically presented during the first few months of life but sometimes may encounter later in life, causing difficulties and mistakes in diagnosis. We reviewed records of eleven rare patients with midgut malrotation older than one year of age and extracted their clinical and paraclinical data. The most common presenting symptoms were bilious vomiting, recurrent abdominal pain and constipation. Five of eleven patients had presented from neonatal period. The average interval between first symptoms and surgical correction of malrotation was about 22 months. Some of the patients had been undergone false treatments. Most cases were diagnosed by contrast studies (upper gastrointestinal series and barium enema. Diagnosing midgut malrotation in older children is often delayed. This anomaly should be suspected in all children with signs and symptoms of small bowel obstruction, chronic abdominal pain and vague abdominal discomfort and in all patients of any age with abdominal discomfort who do not respond to other therapies. Contrast studies may be necessary to rule out malrotation in suspected patients.

  17. Immunogenic Eimeria tenella glycosylphosphatidylinositol-anchored surface antigens (SAGs induce inflammatory responses in avian macrophages.

    Directory of Open Access Journals (Sweden)

    Yock-Ping Chow

    Full Text Available 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.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.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.

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

    Science.gov (United States)

    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.

  19. The midgut transcriptome of Phlebotomus (Larroussius) perniciosus, a vector of Leishmania infantum: comparison of sugar fed and blood fed sand flies.

    Science.gov (United States)

    Dostálová, Anna; Votýpka, Jan; Favreau, Amanda J; Barbian, Kent D; Volf, Petr; Valenzuela, Jesus G; Jochim, Ryan C

    2011-05-10

    Parasite-vector interactions are fundamental in the transmission of vector-borne diseases such as leishmaniasis. Leishmania development in the vector sand fly is confined to the digestive tract, where sand fly midgut molecules interact with the parasites. In this work we sequenced and analyzed two midgut-specific cDNA libraries from sugar fed and blood fed female Phlebotomus perniciosus and compared the transcript expression profiles. A total of 4111 high quality sequences were obtained from the two libraries and assembled into 370 contigs and 1085 singletons. Molecules with putative roles in blood meal digestion, peritrophic matrix formation, immunity and response to oxidative stress were identified, including proteins that were not previously reported in sand flies. These molecules were evaluated relative to other published sand fly transcripts. Comparative analysis of the two libraries revealed transcripts differentially expressed in response to blood feeding. Molecules up regulated by blood feeding include a putative peritrophin (PperPer1), two chymotrypsin-like proteins (PperChym1 and PperChym2), a putative trypsin (PperTryp3) and four putative microvillar proteins (PperMVP1, 2, 4 and 5). Additionally, several transcripts were more abundant in the sugar fed midgut, such as two putative trypsins (PperTryp1 and PperTryp2), a chymotrypsin (PperChym3) and a microvillar protein (PperMVP3). We performed a detailed temporal expression profile analysis of the putative trypsin transcripts using qPCR and confirmed the expression of blood-induced and blood-repressed trypsins. Trypsin expression was measured in Leishmania infantum-infected and uninfected sand flies, which identified the L. infantum-induced down regulation of PperTryp3 at 24 hours post-blood meal. This midgut tissue-specific transcriptome provides insight into the molecules expressed in the midgut of P. perniciosus, an important vector of visceral leishmaniasis in the Old World. Through the comparative

  20. MacoNPV baculovirus midgut-specific gene expression during infection of the bertha armyworm, Mamestra configurata

    Energy Technology Data Exchange (ETDEWEB)

    Donly, B. Cameron, E-mail: Cam.Donly@agr.gc.ca [London Research and Development Centre, AAFC, London, ON (Canada); Kaplanoglu, Emine [London Research and Development Centre, AAFC, London, ON (Canada); Theilmann, David A. [Summerland Research and Development Centre, AAFC, Summerland, BC (Canada); Baldwin, Doug; Sieminska, Edyta; Hegedus, Dwayne D.; Erlandson, Martin A. [Saskatoon Research and Development Centre, AAFC, Saskatoon, SK (Canada)

    2016-12-15

    Baculoviruses have two forms, occlusion derived virus (ODV) which is responsible for primary infection in host midgut tissue and budded virus (BV), which infects all other host tissues during secondary infection. This study examined the primary infection by ODV of midgut cells of bertha armyworm Mamestra configurata fourth instar larvae and measured the expression of viral genes over a time course of infection. Both digital PCR and RNA sequencing methods showed the profile of transcription to be different from those produced by AcMNPV BV infection of in vitro cell cultures. This included having unique collections of genes expressed early, as well as much greater late gene expression of p6.9 and much reduced expression of polh and p10. These differences likely reflect characteristics unique to the critical step of in vivo midgut cell infection, and provide insights into the processes that regulate viral gene expression in different host tissues. -- Highlights: •The transcriptome of MacoNPV ODV in larval midgut was measured by RNA-seq and digital PCR. •The earliest genes expressed included fusion protein, hoar, and me53. •p6.9 was highly expressed late but polH and p10 were less so. •These patterns are unique from BV of other baculoviruses in tissue culture cells.

  1. MacoNPV baculovirus midgut-specific gene expression during infection of the bertha armyworm, Mamestra configurata

    International Nuclear Information System (INIS)

    Donly, B. Cameron; Kaplanoglu, Emine; Theilmann, David A.; Baldwin, Doug; Sieminska, Edyta; Hegedus, Dwayne D.; Erlandson, Martin A.

    2016-01-01

    Baculoviruses have two forms, occlusion derived virus (ODV) which is responsible for primary infection in host midgut tissue and budded virus (BV), which infects all other host tissues during secondary infection. This study examined the primary infection by ODV of midgut cells of bertha armyworm Mamestra configurata fourth instar larvae and measured the expression of viral genes over a time course of infection. Both digital PCR and RNA sequencing methods showed the profile of transcription to be different from those produced by AcMNPV BV infection of in vitro cell cultures. This included having unique collections of genes expressed early, as well as much greater late gene expression of p6.9 and much reduced expression of polh and p10. These differences likely reflect characteristics unique to the critical step of in vivo midgut cell infection, and provide insights into the processes that regulate viral gene expression in different host tissues. -- Highlights: •The transcriptome of MacoNPV ODV in larval midgut was measured by RNA-seq and digital PCR. •The earliest genes expressed included fusion protein, hoar, and me53. •p6.9 was highly expressed late but polH and p10 were less so. •These patterns are unique from BV of other baculoviruses in tissue culture cells.

  2. PhaR, a Negative Regulator of PhaP, Modulates the Colonization of a Burkholderia Gut Symbiont in the Midgut of the Host Insect, Riptortus pedestris.

    Science.gov (United States)

    Jang, Seong Han; Jang, Ho Am; Lee, Junbeom; Kim, Jong Uk; Lee, Seung Ah; Park, Kyoung-Eun; Kim, Byung Hyun; Jo, Yong Hun; Lee, Bok Luel

    2017-06-01

    Five genes encoding PhaP family proteins and one phaR gene have been identified in the genome of Burkholderia symbiont strain RPE75. PhaP proteins function as the surface proteins of polyhydroxyalkanoate (PHA) granules, and the PhaR protein acts as a negative regulator of PhaP biosynthesis. Recently, we characterized one phaP gene to understand the molecular cross talk between Riptortus insects and Burkholderia gut symbionts. In this study, we constructed four other phaP gene-depleted mutants (Δ phaP1 , Δ phaP2 , Δ phaP3 , and Δ phaP4 mutants), one phaR gene-depleted mutant, and a phaR -complemented mutant (Δ phaR/phaR mutant). To address the biological roles of four phaP family genes and the phaR gene during insect-gut symbiont interaction, these Burkholderia mutants were fed to the second-instar nymphs, and colonization ability and fitness parameters were examined. In vitro , the Δ phaP3 and Δ phaR mutants cannot make a PHA granule normally in a stressful environment. Furthermore, the Δ phaR mutation decreased the colonization ability in the host midgut and negatively affected the host insect's fitness compared with wild-type Burkholderia -infected insects. However, other phaP family gene-depleted mutants colonized well in the midgut of the fifth-instar nymph insects. However, in the case of females, the colonization rate of the Δ phaP3 mutant was decreased and the host's fitness parameters were decreased compared with the wild-type-infected host, suggesting that the environment of the female midgut may be more hostile than that of the male midgut. These results demonstrate that PhaR plays an important role in the biosynthesis of PHA granules and that it is significantly related to the colonization of the Burkholderia gut symbiont in the host insects' midgut. IMPORTANCE Bacterial polyhydroxyalkanoate (PHA) biosynthesis is a complex process requiring several enzymes. The biological roles of PHA granule synthesis enzymes and the surface proteins of PHA

  3. Amino acid metabolic signaling influences Aedes aegypti midgut microbiome variability.

    Directory of Open Access Journals (Sweden)

    Sarah M Short

    2017-07-01

    Full Text Available The mosquito midgut microbiota has been shown to influence vector competence for multiple human pathogens. The microbiota is highly variable in the field, and the sources of this variability are not well understood, which limits our ability to understand or predict its effects on pathogen transmission. In this work, we report significant variation in female adult midgut bacterial load between strains of A. aegypti which vary in their susceptibility to dengue virus. Composition of the midgut microbiome was similar overall between the strains, with 81-92% of reads coming from the same five bacterial families, though we did detect differences in the presence of some bacterial families including Flavobacteriaceae and Entobacteriaceae. We conducted transcriptomic analysis on the two mosquito strains that showed the greatest difference in bacterial load, and found that they differ in transcript abundance of many genes implicated in amino acid metabolism, in particular the branched chain amino acid degradation pathway. We then silenced this pathway by targeting multiple genes using RNA interference, which resulted in strain-specific bacterial proliferation, thereby eliminating the difference in midgut bacterial load between the strains. This suggests that the branched chain amino acid (BCAA degradation pathway controls midgut bacterial load, though the mechanism underlying this remains unclear. Overall, our results indicate that amino acid metabolism can act to influence the midgut microbiota. Moreover, they suggest that genetic or physiological variation in BCAA degradation pathway activity may in part explain midgut microbiota variation in the field.

  4. Chilling of Dormant Buds Hyperinduces FLOWERING LOCUS T and Recruits GA-Inducible 1,3-β-Glucanases to Reopen Signal Conduits and Release Dormancy in Populus[W][OA

    Science.gov (United States)

    Rinne, Päivi L.H.; Welling, Annikki; Vahala, Jorma; Ripel, Linda; Ruonala, Raili; Kangasjärvi, Jaakko; van der Schoot, Christiaan

    2011-01-01

    In trees, production of intercellular signals and accessibility of signal conduits jointly govern dormancy cycling at the shoot apex. We identified 10 putative cell wall 1,3-β-glucanase genes (glucan hydrolase family 17 [GH17]) in Populus that could turn over 1,3-β-glucan (callose) at pores and plasmodesmata (PD) and investigated their regulation in relation to FT and CENL1 expression. The 10 genes encode orthologs of Arabidopsis thaliana BG_ppap, a PD-associated glycosylphosphatidylinositol (GPI) lipid-anchored protein, the Arabidopsis PD callose binding protein PDCB, and a birch (Betula pendula) putative lipid body (LB) protein. We found that these genes were differentially regulated by photoperiod, by chilling (5°C), and by feeding of gibberellins GA3 and GA4. GA3 feeding upregulated all LB-associated GH17s, whereas GA4 upregulated most GH17s with a GPI anchor and/or callose binding motif, but only GA4 induced true bud burst. Chilling upregulated a number of GA biosynthesis and signaling genes as well as FT, but not CENL1, while the reverse was true for both GA3 and GA4. Collectively, the results suggest a model for dormancy release in which chilling induces FT and both GPI lipid-anchored and GA3-inducible GH17s to reopen signaling conduits in the embryonic shoot. When temperatures rise, the reopened conduits enable movement of FT and CENL1 to their targets, where they drive bud burst, shoot elongation, and morphogenesis. PMID:21282527

  5. Chilling of dormant buds hyperinduces FLOWERING LOCUS T and recruits GA-inducible 1,3-beta-glucanases to reopen signal conduits and release dormancy in Populus.

    Science.gov (United States)

    Rinne, Päivi L H; Welling, Annikki; Vahala, Jorma; Ripel, Linda; Ruonala, Raili; Kangasjärvi, Jaakko; van der Schoot, Christiaan

    2011-01-01

    In trees, production of intercellular signals and accessibility of signal conduits jointly govern dormancy cycling at the shoot apex. We identified 10 putative cell wall 1,3-β-glucanase genes (glucan hydrolase family 17 [GH17]) in Populus that could turn over 1,3-β-glucan (callose) at pores and plasmodesmata (PD) and investigated their regulation in relation to FT and CENL1 expression. The 10 genes encode orthologs of Arabidopsis thaliana BG_ppap, a PD-associated glycosylphosphatidylinositol (GPI) lipid-anchored protein, the Arabidopsis PD callose binding protein PDCB, and a birch (Betula pendula) putative lipid body (LB) protein. We found that these genes were differentially regulated by photoperiod, by chilling (5°C), and by feeding of gibberellins GA(3) and GA(4). GA(3) feeding upregulated all LB-associated GH17s, whereas GA(4) upregulated most GH17s with a GPI anchor and/or callose binding motif, but only GA(4) induced true bud burst. Chilling upregulated a number of GA biosynthesis and signaling genes as well as FT, but not CENL1, while the reverse was true for both GA(3) and GA(4). Collectively, the results suggest a model for dormancy release in which chilling induces FT and both GPI lipid-anchored and GA(3)-inducible GH17s to reopen signaling conduits in the embryonic shoot. When temperatures rise, the reopened conduits enable movement of FT and CENL1 to their targets, where they drive bud burst, shoot elongation, and morphogenesis.

  6. Neuroimmunophilin GPI-1046 reduces ethanol consumption in part through activation of GLT1 in alcohol-preferring rats.

    Science.gov (United States)

    Sari, Y; Sreemantula, S N

    2012-12-27

    We have previously shown that ceftriaxone, β-lactam antibiotic known to upregulate glutamate transporter 1 (GLT1), reduced ethanol intake in alcohol-preferring (P) rats. GLT1 is a glial glutamate transporter that regulates the majority of extracellular glutamate uptake. We tested in this study the effects of neuroimmunophilin GPI-1046 (3-(3-pyridyl)-1-propyl (2S)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidinecarboxylate), known also to upregulate GLT1 expression, in ethanol intake in P rats. Male P rats had concurrent access to free choice of 15% and 30% ethanol, water, and food for five weeks. On Week 6, P rats continued in this drinking and food regimen and they were administered either 10 or 20mg/kg GPI-1046 (i.p.), or a vehicle for five consecutive days. Body weight, ethanol intake, and water consumption were measured daily for 8 days starting on Day 1 of GPI-1046 or vehicle i.p. injections. We have also tested the effect of GPI-1046 (20mg/kg) on daily sucrose (10%) intake. The data revealed significant dose-dependent effects in the reduction of ethanol intake starting 48 h after the first treatment with GPI-1046 throughout treatment and post-treatment periods. There were also dose-dependent increases in water intake. However, GPI-1046 treatment did not affect the body weight of all animals nor sucrose intake. Importantly, GPI-1046 (20mg/kg) increased GLT1 level compared to all groups in nucleus accumbens core (NAc-core). Alternatively, GPI-1046 (10mg/kg) upregulated GLT1 level in NAc-core compared to vehicle (ethanol naïve) group. Moreover, both doses of GPI-1046 increased significantly GLT1 level in the prefrontal cortex (PFC) compared to ethanol naïve vehicle group. GPI-1046 (20mg/kg) increased GLT1 level in PFC compared to naïve control group that was exposed to water and food only. These findings demonstrated that neuroimmunophilin GPI-1046 attenuates ethanol intake in part through the upregulation of GLT1 in PFC and NAc-core. Copyright © 2012 IBRO

  7. Anchoring protein crystals to mounting loops with hydrogel using inkjet technology.

    Science.gov (United States)

    Shinoda, Akira; Tanaka, Yoshikazu; Yao, Min; Tanaka, Isao

    2014-11-01

    X-ray crystallography is an important technique for structure-based drug discovery, mainly because it is the only technique that can reveal whether a ligand binds to the target protein as well as where and how it binds. However, ligand screening by X-ray crystallography involves a crystal-soaking experiment, which is usually performed manually. Thus, the throughput is not satisfactory for screening large numbers of candidate ligands. In this study, a technique to anchor protein crystals to mounting loops by using gel and inkjet technology has been developed; the method allows soaking of the mounted crystals in ligand-containing solution. This new technique may assist in the design of a fully automated drug-screening pipeline.

  8. Midgut Protease Activity During Larval Development of Anastrepha obliqua (Diptera: Tephritidae) Fed With Natural and Artificial Diet

    Science.gov (United States)

    Rivera-Ciprian, José Pedro; Aceituno-Medina, Marysol; Guillen, Karina

    2017-01-01

    Abstract In this study, we examined the activity of two serine proteases (chymotrypsin and trypsin) and two metalloproteases (carboxypeptidases A and B) during larval development in Anastrepha obliqua fed natural (mango fruit) and artificial (formulation used in mass-rearing) diets. Proteolytic activity of chymotrypsin, trypsin, carboxypeptidase A, and carboxypeptidase B was detected in the midgut of different instars of A. obliqua and was strongly affected by the pH and diet type. The protein content of the natural and artificial diets was similar. Enzymatic activity was higher in the midgut of the larvae fed the natural diet than in larvae fed the artificial diet. The activity of the endopeptidases (chymotrypsin and trypsin) was lower than those of the exopeptidases (carboxypeptidases A and B). The pH of the midgut varied from acidic to neutral. The results indicate that in the midgut of the larvae reared on both types of diet, the level of carboxypeptidase activity was approximately 100-fold greater than the level of chymotrypsin activity and 10,000-fold greater than the level of trypsin. In conclusion, carboxypeptidase A and B are the main proteases involved in the digestion of proteins in the larvae of A. obliqua. The natural diet showed a high bioaccessibility. A clear tendency to express high activities of chymotrypsin and trypsin was observed by the third instar. Our research contributes to the planning and development of novel bioaccessibility assays to understand the nutrition processing of A. obliqua larvae under mass-rearing conditions for sterile insect technique.

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

    Science.gov (United States)

    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.

  10. The midgut transcriptome of Lutzomyia longipalpis: comparative analysis of cDNA libraries from sugar-fed, blood-fed, post-digested and Leishmania infantum chagasi-infected sand flies

    Directory of Open Access Journals (Sweden)

    Elnaiem Dia-Eldin

    2008-01-01

    Full Text Available Abstract Background In the life cycle of Leishmania within the alimentary canal of sand flies the parasites have to survive the hostile environment of blood meal digestion, escape the blood bolus and attach to the midgut epithelium before differentiating into the infective metacyclic stages. The molecular interactions between the Leishmania parasites and the gut of the sand fly are poorly understood. In the present work we sequenced five cDNA libraries constructed from midgut tissue from the sand fly Lutzomyia longipalpis and analyzed the transcripts present following sugar feeding, blood feeding and after the blood meal has been processed and excreted, both in the presence and absence of Leishmania infantum chagasi. Results Comparative analysis of the transcripts from sugar-fed and blood-fed cDNA libraries resulted in the identification of transcripts differentially expressed during blood feeding. This included upregulated transcripts such as four distinct microvillar-like proteins (LuloMVP1, 2, 4 and 5, two peritrophin like proteins, a trypsin like protein (Lltryp1, two chymotrypsin like proteins (LuloChym1A and 2 and an unknown protein. Downregulated transcripts by blood feeding were a microvillar-like protein (LuloMVP3, a trypsin like protein (Lltryp2 and an astacin-like metalloprotease (LuloAstacin. Furthermore, a comparative analysis between blood-fed and Leishmania infected midgut cDNA libraries resulted in the identification of the transcripts that were differentially expressed due to the presence of Leishmania in the gut of the sand fly. This included down regulated transcripts such as four microvillar-like proteins (LuloMVP1,2, 4 and 5, a Chymotrypsin (LuloChym1A and a carboxypeptidase (LuloCpepA1, among others. Upregulated midgut transcripts in the presence of Leishmania were a peritrophin like protein (LuloPer1, a trypsin-like protein (Lltryp2 and an unknown protein. Conclusion This transcriptome analysis represents the largest set

  11. Awa1p on the cell surface of sake yeast inhibits biofilm formation and the co-aggregation between sake yeasts and Lactobacillus plantarum ML11-11.

    Science.gov (United States)

    Hirayama, Satoru; Shimizu, Masashi; Tsuchiya, Noriko; Furukawa, Soichi; Watanabe, Daisuke; Shimoi, Hitoshi; Takagi, Hiroshi; Ogihara, Hirokazu; Morinaga, Yasushi

    2015-05-01

    We examined mixed-species biofilm formation between Lactobacillus plantarum ML11-11 and both foaming and non-foaming mutant strains of Saccharomyces cerevisiae sake yeasts. Wild-type strains showed significantly lower levels of biofilm formation compared with the non-foaming mutants. Awa1p, a protein involved in foam formation during sake brewing, is a glycosylphosphatidylinositol (GPI)-anchored protein and is associated with the cell wall of sake yeasts. The AWA1 gene of the non-foaming mutant strain Kyokai no. 701 (K701) has lost the C-terminal sequence that includes the GPI anchor signal. Mixed-species biofilm formation and co-aggregation of wild-type strain Kyokai no. 7 (K7) were significantly lower than K701 UT-1 (K701 ura3/ura3 trp1/trp1), while the levels of strain K701 UT-1 carrying the AWA1 on a plasmid were comparable to those of K7. The levels of biofilm formation and co-aggregation of the strain K701 UT-1 harboring AWA1 with a deleted GPI anchor signal were similar to those of K701 UT-1. These results clearly demonstrate that Awa1p present on the surface of sake yeast strain K7 inhibits adhesion between yeast cells and L. plantarum ML11-11, consequently impeding mixed-species biofilm formation. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  12. DRAGON, a GPI-anchored membrane protein, inhibits BMP signaling in C2C12 myoblasts.

    Science.gov (United States)

    Kanomata, Kazuhiro; Kokabu, Shoichiro; Nojima, Junya; Fukuda, Toru; Katagiri, Takenobu

    2009-06-01

    Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation of myoblasts via binding to cell surface receptors. Repulsive guidance molecules (RGMs) have been identified as BMP co-receptors. We report here that DRAGON/RGMb, a member of the RGM family, suppressed BMP signaling in C2C12 myoblasts via a novel mechanism. All RGMs were expressed in C2C12 cells that were differentiated into myocytes and osteoblastic cells, but RGMc was not detected in immature cells. In C2C12 cells, only DRAGON suppressed ALP and Id1 promoter activities induced by BMP-4 or by constitutively activated BMP type I receptors. This inhibition by DRAGON was dependent on the secretory form of the von Willbrand factor type D domain. DRAGON even suppressed BMP signaling induced by constitutively activated Smad1. Over-expression of neogenin did not alter the inhibitory capacity of DRAGON. Taken together, these findings indicate that DRAGON may be an inhibitor of BMP signaling in C2C12 myoblasts. We also suggest that a novel molecule(s) expressed on the cell membrane may mediate the signal transduction of DRAGON in order to suppress BMP signaling in C2C12 myoblasts.

  13. GPIHBP1 and Plasma Triglyceride Metabolism

    DEFF Research Database (Denmark)

    Fong, Loren G; Young, Stephen G; Beigneux, Anne P

    2016-01-01

    GPIHBP1, a GPI-anchored protein in capillary endothelial cells, is crucial for the lipolytic processing of triglyceride-rich lipoproteins (TRLs). GPIHBP1 shuttles lipoprotein lipase (LPL) to its site of action in the capillary lumen and is essential for the margination of TRLs along capillaries -...

  14. A unifying mechanism accounts for sensing of membrane curvature by BAR domains, amphipathic helices and membrane-anchored proteins

    DEFF Research Database (Denmark)

    Bhatia, Vikram Kjøller; Hatzakis, Nikos; Stamou, Dimitrios

    2010-01-01

    itself. We thus anticipate that membrane curvature will promote the redistribution of proteins that are anchored in membranes through any type of hydrophobic moiety, a thesis that broadens tremendously the implications of membrane curvature for protein sorting, trafficking and signaling in cell biology....

  15. Experimental vaccination of chicks with Plasmodium gallinaceum sporozoites. I. Circumsporozoite proteins are expressed by sporozoites recovered from both salivary glands and midguts of mosquitoes

    International Nuclear Information System (INIS)

    Daher, V.R.; Krettli, A.U.

    1987-01-01

    Immunogenicity of Plasmodium gallinaceum sporozoites for chicks and their in vitro reactivity with normal and specific immune sera were studied. Two sporozoite populations recovered from experimentally infected Aedes fluviatilis were used: sporozoites from salivary glands and sporozoites from midgut oocysts. Populations seven to nine days old of sporozoites recovered from salivary glands were infective for all chicks until the chicks were three weeks old; however, sporozoites recovered from midguts containing oocysts infected these chicks only if isolated on days 8-9, but not on day 7 after the mosquitoes' infective blood meal. Infectivity of the sporozoites was lost after exposure to ultraviolet (UV) light (30 min) or X-rays (13 krad). Inactivated sporozoites from both sources proved highly immunogenic to chicks that were immunized by several intravenous or intramuscular injections. These parasites elicited a strong humoral immune response in the chicks, as measured by the circumsporozoite precipitation (CSP) reaction. The levels of the CSP antibodies were similar with sporozoites from both sources, there being no detectable differences in the percentage of reactive sporozoites or the intensity of the CSP reaction with sera containing antibodies to either sporozoites from salivary glands or sporozoites from oocysts. These results provide the first evidence that avian malaria sporozoites express the circumsporozoite protein that has been extensively characterized in mammalian malaria (rodent, simian, human sporozoites). Furthermore, we observed that the yields of sporozoites obtained from mosquito midguts, on days 8 and 9 of the P. gallinaceum infection, were at least twice as great as those obtained by salivary gland dissection, even 20 days after a blood meal

  16. Induction of rat alkaline phosphatase isozymes bearing a glycan-phosphatidylinositol anchor modified by in vivo treatment with a benzimidazole derivative linked to ethylbenzene.

    Science.gov (United States)

    Harada, T; Koyama, I; Sato, K; Komoda, T

    2000-10-01

    Serum alkaline phosphatase (ALP) is detected in soluble-form as a result of translocation from the membrane site by cleavage at the glycosyl-phosphatidylinositol moiety (GPI anchor). It is known that membrane-bound ALP (mALP) can be detected in serum in certain pathological and physiological conditions, and that it can be solubilized in vitro to soluble-ALP (sALP) by phosphatidylinositol-specific phospholipase C (PIPLC), phospholipase D, bile salt, detergent, etc. We observed a marked increase in ALP activity in the serum of rats given a benzimidazole derivative by gavage, and detected it as slow-migrating ALPs (SM-ALPs), which were mALP-like but resistant to PIPLC and n-butanol treatment on disc PAGE. On the other hand, ficin treatment made SM-ALPs shift to the sALP position. The molecular size of the SM-ALPs was smaller than that of sALP on sodium dodecyl sulphide-polyacrylamide slab-gel electrophoresis (SDS-PAGE), and immunoreactivity revealed the intestinal type. SM-ALPs were also detected in the duodenum and jejunum. The main sugar chain structure of SM-ALPs was the biantennary complex-type, which was coincided with intestinal sALP sugar chain. These results suggest that intestinal ALPs induced by the benzimidazole derivative were modified in their C-terminus or GPI anchor region and modification of this region may also participate in translocation into the bloodstream.

  17. The comprehensive native interactome of a fully functional tagged prion protein.

    Directory of Open Access Journals (Sweden)

    Dorothea Rutishauser

    Full Text Available The enumeration of the interaction partners of the cellular prion protein, PrP(C, may help clarifying its elusive molecular function. Here we added a carboxy proximal myc epitope tag to PrP(C. When expressed in transgenic mice, PrP(myc carried a GPI anchor, was targeted to lipid rafts, and was glycosylated similarly to PrP(C. PrP(myc antagonized the toxicity of truncated PrP, restored prion infectibility of PrP(C-deficient mice, and was physically incorporated into PrP(Sc aggregates, indicating that it possessed all functional characteristics of genuine PrP(C. We then immunopurified myc epitope-containing protein complexes from PrP(myc transgenic mouse brains. Gentle differential elution with epitope-mimetic decapeptides, or a scrambled version thereof, yielded 96 specifically released proteins. Quantitative mass spectrometry with isotope-coded tags identified seven proteins which co-eluted equimolarly with PrP(C and may represent component of a multiprotein complex. Selected PrP(C interactors were validated using independent methods. Several of these proteins appear to exert functions in axomyelinic maintenance.

  18. A Case of Midgut Volvulus Associated with a Jejunal Diverticulum

    Directory of Open Access Journals (Sweden)

    Joseph Gutowski

    2017-01-01

    Full Text Available Midgut volvulus in adults is a rare entity that may present with intermittent colicky abdominal pain mixed with completely asymptomatic episodes. This small bowel twist may result in complications of obstruction, ischemia, hemorrhage, or perforation. With a midgut volvulus, complications may be life-threatening, and emergent surgical intervention is the mainstay of treatment. This current case involves an 80-year-old woman with intermittent abdominal pain with increasing severity and decreasing interval of time to presentation. A CAT scan revealed mesenteric swirling with possible internal hernia. A diagnostic laparoscopy followed by laparotomy revealed a midgut volvulus, extensive adhesions involving the root of the mesentery, and a large jejunal diverticulum. The adhesions were lysed enabling untwisting of the bowel, allowing placement of the small bowel in the correct anatomic position and resection of the jejunal diverticulum. This is a rare case of midgut volvulus with intermittent abdominal pain, associated with jejunal diverticulum managed successfully. A midgut volvulus should be considered in the differential diagnosis of a patient who present with a small bowel obstruction secondary to an internal hernia, especially when a swirl sign is present on the CAT scan.

  19. Pyrosequencing the Manduca sexta larval midgut transcriptome: messages for digestion, detoxification and defence.

    Science.gov (United States)

    Pauchet, Y; Wilkinson, P; Vogel, H; Nelson, D R; Reynolds, S E; Heckel, D G; ffrench-Constant, R H

    2010-02-01

    The tobacco hornworm Manduca sexta is an important model for insect physiology but genomic and transcriptomic data are currently lacking. Following a recent pyrosequencing study generating immune related expressed sequence tags (ESTs), here we use this new technology to define the M. sexta larval midgut transcriptome. We generated over 387,000 midgut ESTs, using a combination of Sanger and 454 sequencing, and classified predicted proteins into those involved in digestion, detoxification and immunity. In many cases the depth of 454 pyrosequencing coverage allowed us to define the entire cDNA sequence of a particular gene. Many new M. sexta genes are described including up to 36 new cytochrome P450s, some of which have been implicated in the metabolism of host plant-derived nicotine. New lepidopteran gene families such as the beta-fructofuranosidases, previously thought to be restricted to Bombyx mori, are also described. An unexpectedly high number of ESTs were involved in immunity, for example 39 contigs encoding serpins, and the increasingly appreciated role of the midgut in insect immunity is discussed. Similar studies of other tissues will allow for a tissue by tissue description of the M. sexta transcriptome and will form an essential complimentary step on the road to genome sequencing and annotation.

  20. A-kinase anchoring protein 150 in the mouse brain is concentrated in areas involved in learning and memory

    NARCIS (Netherlands)

    Ostroveanu, Anghelus; Van der Zee, Eddy A.; Dolga, Amalia M.; Luiten, Paul G. M.; Eisel, Ulrich L. M.; Nijholt, Ingrid M.

    2007-01-01

    A-kinase anchoring proteins (AKAPs) form large macromolecular signaling complexes that specifically target cAMP-dependent protein kinase (PKA) to unique subcellular compartments and thus, provide high specificity to PKA signaling. For example, the AKAP79/150 family tethers PKA, PKC and PP2B to

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

    Science.gov (United States)

    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.

  2. Revealing the structure and dust content of debris disks on solar systems scales with GPI

    Science.gov (United States)

    Duchene, Gaspard; Fitzgerald, Michael P.; Kalas, Paul; Graham, James R.; Arriaga, Pauline; Bruzzone, Sebastian; Chen, Christine; Dawson, Rebekah Ilene; Dong, Ruobing; Draper, Zachary; Esposito, Thomas; Follette, Katherine; Hung, Li-Wei; Lawler, Samantha; Metchev, Stanimir; Millar-Blanchaer, Max; Murray-Clay, Ruth; Perrin, Marshall D.; Rameau, Julien; Wang, Jason; Wolff, Schuyler; Macintosh, Bruce; GPIES Team

    2016-01-01

    High contrast scattered light images offer the best prospect to assess the detailed geometry and structure of dusty debris disks. In turn, such images can yield profound insight on the architecture of the underlying planetary system as dust grains respond to the gravitational pull of planetary bodies. A new generation of extreme adaptive optics systems now enables an unprecedented exploration of circumstellar disks on solar system scales. Here we review the new science derived from over a dozen debris disks imaged with the Gemini Planet Imager (GPI) as part of the GPI Exoplanet Survey (GPIES). In addition to its exquisite imaging capability, GPI's polarimetric mode provides invaluable insight on the dust content of each disk, in most cases for the very first time. These early results typically reveal narrow belts of material with evacuated regions roughly 50-100 AU in radius, subtle asymmetries in structure and high degree of linear polarization. We will provide an overview of the disk observations made during the GPIES campaign to date and will discuss in more detail some of the most remarkable systems.This work is supported by grants NSF AST-0909188, -1411868, -1413718; NASA NNX-15AD95G, -14AJ80G, -11AD21G; and the NExSS research network.

  3. Morphometry of the midgut of Melipona quadrifasciata anthidioides (Lepeletier) (Hymenoptera: Apidae) during metamorphosis.

    Science.gov (United States)

    Cruz, L C; Araújo, V A; Dolder, H; Araújo, A P A; Serrão, J E; Neves, C A

    2011-01-01

    In Hymenoptera, midgut changes begin in the last instar. At this stage, the larval epithelial digestive cells degenerate, leaving only the basal membrane and the regenerative cells which will develop into a new epithelium during the pupal stage and in the adult. Epithelium renewal is followed by changes in volume and shape of the midgut. Morphometric analysis of digestive cells and total midgut volume of Melipona quadrifasciata anthidioides (Lepeletier) were conducted to verify whether cell volume increase are sufficient to account for the total midgut volume increase that occurs during metamorphosis. An increase in midgut volume was verified in spite of the scarcity of cell proliferation found during metamorphosis. At the end of metamorphosis, the increase in cell volume was not sufficient to explain the increase in volume of the midgut, indicating that an increase in the number of digestive cells is apparently necessary. Nevertheless, the mechanism by which regenerative cells reconstitute the epithelium during metamorphosis remains unknown.

  4. The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut.

    Science.gov (United States)

    Beton, Daniela; Guzzo, Cristiane R; Ribeiro, Alberto F; Farah, Chuck S; Terra, Walter R

    2012-09-01

    Cathepsin L-like proteinases (CAL) are major digestive proteinases in the beetle Tenebrio molitor. Procathepsin Ls 2 (pCAL2) and 3 (pCAL3) were expressed as recombinant proteins in Escherichia coli, purified and activated under acidic conditions. Immunoblot analyses of different T. molitor larval tissues demonstrated that a polyclonal antibody to pCAL3 recognized pCAL3 and cathepsin L 3 (CAL3) only in the anterior two-thirds of midgut tissue and midgut luminal contents of T. molitor larvae. Furthermore, immunocytolocalization data indicated that pCAL3 occurs in secretory vesicles and microvilli in anterior midgut. Therefore CAL3, like cathepsin L 2 (CAL2), is a digestive enzyme secreted by T. molitor anterior midgut. CAL3 hydrolyses Z-FR-MCA and Z-RR-MCA (typical cathepsin substrates), whereas CAL2 hydrolyses only Z-FR-MCA. Active site mutants (pCAL2C25S and pCAL3C26S) were constructed by replacing the catalytic cysteine with serine to prevent autocatalytic processing. Recombinant pCAL2 and pCAL3 mutants (pCAL2C25S and pCAL3C26S) were prepared, crystallized and their 3D structures determined at 1.85 and 2.1 Å, respectively. While the overall structure of these enzymes is similar to other members of the papain superfamily, structural differences in the S2 subsite explain their substrate specificities. The data also supported models for CAL trafficking to lysosomes and to secretory vesicles to be discharged into midgut contents. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Plasmodium ookinetes coopt mammalian plasminogen to invade the mosquito midgut

    DEFF Research Database (Denmark)

    Ghosh, Anil K; Coppens, Isabelle; Gårdsvoll, Henrik

    2011-01-01

    Ookinete invasion of the mosquito midgut is an essential step for the development of the malaria parasite in the mosquito. Invasion involves recognition between a presumed mosquito midgut receptor and an ookinete ligand. Here, we show that enolase lines the ookinete surface. An antienolase antibody...

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

    Science.gov (United States)

    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...

  7. Larval midgut modifications associated with Bti resistance in the yellow fever mosquito using proteomic and transcriptomic approaches

    Directory of Open Access Journals (Sweden)

    Tetreau Guillaume

    2012-06-01

    Full Text Available Abstract Background Bacillus thuringiensis var. israelensis (Bti is a natural larval mosquito pathogen producing pore-forming toxins targeting the midgut of Diptera larvae. It is used worldwide for mosquito control. Resistance mechanisms of an Aedes aegypti laboratory strain selected for 30 generations with field-collected leaf litter containing Bti toxins were investigated in larval midguts at two levels: 1. gene transcription using DNA microarray and RT-qPCR and 2. differential expression of brush border membrane proteins using DIGE (Differential In Gel Electrophoresis. Results Several Bti Cry toxin receptors including alkaline phosphatases and N-aminopeptidases and toxin-binding V-ATPases exhibited altered expression levels in the resistant strain. The under-expression of putative Bti-receptors is consistent with Bt-resistance mechanisms previously described in Lepidoptera. Four soluble metalloproteinases were found under-transcribed together with a drastic decrease of metalloproteinases activity in the resistant strain, suggesting a role in resistance by decreasing the amount of activated Cry toxins in the larval midgut. Conclusions By combining transcriptomic and proteomic approaches, we detected expression changes at nearly each step of the ingestion-to-infection process, providing a short list of genes and proteins potentially involved in Bti-resistance whose implication needs to be validated. Collectively, these results open the way to further functional analyses to better characterize Bti-resistance mechanisms in mosquitoes.

  8. Intestinal stem cells in the adult Drosophila midgut

    International Nuclear Information System (INIS)

    Jiang, Huaqi; Edgar, Bruce A.

    2011-01-01

    Drosophila has long been an excellent model organism for studying stem cell biology. Notably, studies of Drosophila's germline stem cells have been instrumental in developing the stem cell niche concept. The recent discovery of somatic stem cells in adult Drosophila, particularly the intestinal stem cells (ISCs) of the midgut, has established Drosophila as an exciting model to study stem cell-mediated adult tissue homeostasis and regeneration. Here, we review the major signaling pathways that regulate the self-renewal, proliferation and differentiation of Drosophila ISCs, discussing how this regulation maintains midgut homeostasis and mediates regeneration of the intestinal epithelium after injury. -- Highlights: ► The homeostasis and regeneration of adult fly midguts are mediated by ISCs. ► Damaged enterocytes induce the proliferation of intestinal stem cells (ISC). ► EGFR and Jak/Stat signalings mediate compensatory ISC proliferation. ► Notch signaling regulates ISC self-renewal and differentiation.

  9. Role of sialic acids in the midguts of Trypanosoma congolense ...

    African Journals Online (AJOL)

    Administrator

    total sialic acid concentration. The relevance of these findings to the role of sialic acids in the midgut of. T. congolense infected C.p. pipiense mosquitoes is discussed in this paper. Key words: Trypanosoma congolense, Culex pipiense pipiense, sialic acid, midgut. INTRODUCTION. The Culex pipiense pipiense mosquito is ...

  10. A novel germline PIGA mutation in Ferro-Cerebro-Cutaneous syndrome: a neurodegenerative X-linked epileptic encephalopathy with systemic iron-overload.

    Science.gov (United States)

    Swoboda, Kathryn J; Margraf, Rebecca L; Carey, John C; Zhou, Holly; Newcomb, Tara M; Coonrod, Emily; Durtschi, Jacob; Mallempati, Kalyan; Kumanovics, Attila; Katz, Ben E; Voelkerding, Karl V; Opitz, John M

    2014-01-01

    Three related males presented with a newly recognized x-linked syndrome associated with neurodegeneration, cutaneous abnormalities, and systemic iron overload. Linkage studies demonstrated that they shared a haplotype on Xp21.3-Xp22.2 and exome sequencing was used to identify candidate variants. Of the segregating variants, only a PIGA mutation segregated with disease in the family. The c.328_330delCCT PIGA variant predicts, p.Leu110del (or c.1030_1032delCTT, p.Leu344del depending on the reference sequence). The unaffected great-grandfather shared his X allele with the proband but he did not have the PIGA mutation, indicating that the mutation arose de novo in his daughter. A single family with a germline PIGA mutation has been reported; affected males had a phenotype characterized by multiple congenital anomalies and severe neurologic impairment resulting in infantile lethality. In contrast, affected boys in the family described here were born without anomalies and were neurologically normal prior to onset of seizures after 6 months of age, with two surviving to the second decade. PIGA encodes an enzyme in the GPI anchor biosynthesis pathway. An affected individual in the family studied here was deficient in GPI anchor proteins on granulocytes but not erythrocytes. In conclusion, the PIGA mutation in this family likely causes a reduction in GPI anchor protein cell surface expression in various cell types, resulting in the observed pleiotropic phenotype involving central nervous system, skin, and iron metabolism. © 2013 Wiley Periodicals, Inc.

  11. Interaction of angiotensin-converting enzyme (ACE) with membrane-bound carboxypeptidase M (CPM) - a new function of ACE.

    Science.gov (United States)

    Sun, Xiaoou; Wiesner, Burkhard; Lorenz, Dorothea; Papsdorf, Gisela; Pankow, Kristin; Wang, Po; Dietrich, Nils; Siems, Wolf-Eberhard; Maul, Björn

    2008-12-01

    Angiotensin-converting enzyme (ACE) demonstrates, besides its typical dipeptidyl-carboxypeptidase activity, several unusual functions. Here, we demonstrate with molecular, biochemical, and cellular techniques that the somatic wild-type murine ACE (mACE), stably transfected in Chinese Hamster Ovary (CHO) or Madin-Darby Canine Kidney (MDCK) cells, interacts with endogenous membranal co-localized carboxypeptidase M (CPM). CPM belongs to the group of glycosylphosphatidylinositol (GPI)-anchored proteins. Here we report that ACE, completely independent of its known dipeptidase activities, has GPI-targeted properties. Our results indicate that the spatial proximity between mACE and the endogenous CPM enables an ACE-evoked release of CPM. These results are discussed with respect to the recently proposed GPI-ase activity and function of sperm-bound ACE.

  12. Therapeutic efficacy of PD-L1 blockade in a breast cancer model is enhanced by cellular vaccines expressing B7-1 and glycolipid-anchored IL-12.

    Science.gov (United States)

    Bozeman, Erica N; He, Sara; Shafizadeh, Yalda; Selvaraj, Periasamy

    2016-01-01

    Immunotherapeutic approaches have emerged as promising strategies to treat various cancers, including breast cancer. A single approach, however, is unlikely to effectively combat the complex, immune evasive strategies found within the tumor microenvironment, thus novel, effective combination treatments must be explored. In this study, we investigated the efficacy of a combination therapy consisting of PD-L1 immune checkpoint blockade and whole cell vaccination in a HER-2 positive mouse model of breast cancer. We demonstrate that tumorigenicity is completely abrogated when adjuvanted with immune stimulatory molecules (ISMs) B7-1 and a cell-surface anchored (GPI) form of IL-12 or GM-CSF. Irradiated cellular vaccines expressing the combination of adjuvants B7-1 and GPI-IL-12 completely inhibited tumor formation which was correlative with robust HER-2 specific CTL activity. However, in a therapeutic setting, both cellular vaccination and PD-L1 blockade induced only 10-20% tumor regression when administered alone but resulted in 50% tumor regression as a combination therapy. This protection was significantly hindered following CD4 or CD8 depletion indicating the essential role played by cellular immunity. Collectively, these pre-clinical studies provide a strong rationale for further investigation into the efficacy of combination therapy with tumor cell vaccines adjuvanted with membrane-anchored ISMs along with PD-L1 blockade for the treatment of breast cancer.

  13. A hypothetical model of crossing Bombyx mori nucleopolyhedrovirus through its host midgut physical barrier.

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    Yang Cheng

    Full Text Available Bombyx mori nucleopolyhedrovirus (BmNPV is a primary pathogen of silkworm (B. mori that causes severe economic losses each year. However, the molecular mechanisms of silkworm-BmNPV interactions, especially the silkworm proteins that can interact with the virus, are still largely unknown. In this study, the total and membrane proteins of silkworm midguts were displayed using one- and two-dimensional electrophoresis. A virus overlay assay was used to detect B. mori proteins that specifically bind to BmNPV particles. Twelve proteins were located and identified using mass spectrometry, and the different expression of the corresponding genes in BmNPV susceptible and resistant silkworm strains also indicated their involvement in BmNPV infection. The 12 proteins are grouped based on their potential roles in viral infection, for example, endocytosis, intracellular transportation, and host responses. Based on these results, we hypothesize the following: I vacuolar ATP synthase catalytic subunit A and subunit B may be implicated in the process of the membrane fusion of virus and the release of the nucleocapsid into cytoplasm; II actin, enolase and phosphoglycerate kinase are cytoskeleton associated proteins and may play an important role in BmNPV intracellular transportation; III mitochondrial prohibitin complex protein 2, ganglioside-induced differentiation-associated protein, calreticulin, regucalcin-like isoform X1 and 60 kDa heat shock protein are involved in cell apoptosis regulation during BmNPV infection in larvae midguts; IV ribosomal P0 may be associated with BmNPV infection by regulating gene expression of BmNPV; V arginine kinase has a role in the antiviral activities against BmNPV. Our work should prove informative by providing multiple protein targets and a novel direction to investigate the molecular mechanisms of the interactions between silkworms and BmNPV.

  14. A Hypothetical Model of Crossing Bombyx mori Nucleopolyhedrovirus through Its Host Midgut Physical Barrier

    Science.gov (United States)

    Cheng, Yang; Wang, Xue-Yang; Hu, Hao; Killiny, Nabil; Xu, Jia-Ping

    2014-01-01

    Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen of silkworm (B. mori) that causes severe economic losses each year. However, the molecular mechanisms of silkworm-BmNPV interactions, especially the silkworm proteins that can interact with the virus, are still largely unknown. In this study, the total and membrane proteins of silkworm midguts were displayed using one- and two-dimensional electrophoresis. A virus overlay assay was used to detect B. mori proteins that specifically bind to BmNPV particles. Twelve proteins were located and identified using mass spectrometry, and the different expression of the corresponding genes in BmNPV susceptible and resistant silkworm strains also indicated their involvement in BmNPV infection. The 12 proteins are grouped based on their potential roles in viral infection, for example, endocytosis, intracellular transportation, and host responses. Based on these results, we hypothesize the following: I) vacuolar ATP synthase catalytic subunit A and subunit B may be implicated in the process of the membrane fusion of virus and the release of the nucleocapsid into cytoplasm; II) actin, enolase and phosphoglycerate kinase are cytoskeleton associated proteins and may play an important role in BmNPV intracellular transportation; III) mitochondrial prohibitin complex protein 2, ganglioside-induced differentiation-associated protein, calreticulin, regucalcin-like isoform X1 and 60 kDa heat shock protein are involved in cell apoptosis regulation during BmNPV infection in larvae midguts; IV) ribosomal P0 may be associated with BmNPV infection by regulating gene expression of BmNPV; V) arginine kinase has a role in the antiviral activities against BmNPV. Our work should prove informative by providing multiple protein targets and a novel direction to investigate the molecular mechanisms of the interactions between silkworms and BmNPV. PMID:25502928

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

    Science.gov (United States)

    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.

  16. Malrotation with midgut volvulus associated with perforated ileal duplication

    Directory of Open Access Journals (Sweden)

    Anand Pandey

    2013-01-01

    Full Text Available Duplication of the alimentary tract is an important surgical condition. It may occur anywhere in the gastrointestinal tract. An important complication of this entity is perforation of the normal or abnormal gut. Malrotation with midgut volvulus can be a surgical emergency. We present a patient, who presented as malrotation with midgut volvulus associated with perforated ileal duplication. The patient was successfully managed.

  17. Midgut morphological changes and autophagy during metamorphosis in sand flies.

    Science.gov (United States)

    Malta, Juliana; Heerman, Matthew; Weng, Ju Lin; Fernandes, Kenner M; Martins, Gustavo Ferreira; Ramalho-Ortigão, Marcelo

    2017-06-01

    During metamorphosis, holometabolous insects undergo significant remodeling of their midgut and become able to cope with changes in dietary requirements between larval and adult stages. At this stage, insects must be able to manage and recycle available food resources in order to develop fully into adults, especially when no nutrients are acquired from the environment. Autophagy has been previously suggested to play a crucial role during metamorphosis of the mosquito. Here, we investigate the overall morphological changes of the midgut of the sand fly during metamorphosis and assess the expression profiles of the autophagy-related genes ATG1, ATG6, and ATG8, which are associated with various steps of the autophagic process. Morphological changes in the midgut start during the fourth larval instar, with epithelial degeneration followed by remodeling via the differentiation of regenerative cells in pre-pupal and pupal stages. The changes in the midgut epithelium are paired with the up-regulation of ATG1, ATG6 and ATG8 during the larva-adult transition. Vein, a putative epidermal growth factor involved in regulating epithelial midgut regeneration, is also up-regulated. Autophagy has further been confirmed in sand flies via the presence of autophagosomes residing within the cytoplasmic compartment of the pupal stages. An understanding of the underlying mechanisms of this process should aid the future management of this neglected tropical vector.

  18. Intestinal stem cells in the adult Drosophila midgut

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Huaqi, E-mail: Huaqi.Jiang@UTSouthwestern.edu [Department of Developmental Biology, UT Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, TX, 75235 (United States); Edgar, Bruce A., E-mail: b.edgar@dkfz.de [ZMBH-DKFZ Alliance, Im Neuenheimer Feld 282, D-69120 Heidelberg (Germany); Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109 (United States)

    2011-11-15

    Drosophila has long been an excellent model organism for studying stem cell biology. Notably, studies of Drosophila's germline stem cells have been instrumental in developing the stem cell niche concept. The recent discovery of somatic stem cells in adult Drosophila, particularly the intestinal stem cells (ISCs) of the midgut, has established Drosophila as an exciting model to study stem cell-mediated adult tissue homeostasis and regeneration. Here, we review the major signaling pathways that regulate the self-renewal, proliferation and differentiation of Drosophila ISCs, discussing how this regulation maintains midgut homeostasis and mediates regeneration of the intestinal epithelium after injury. -- Highlights: Black-Right-Pointing-Pointer The homeostasis and regeneration of adult fly midguts are mediated by ISCs. Black-Right-Pointing-Pointer Damaged enterocytes induce the proliferation of intestinal stem cells (ISC). Black-Right-Pointing-Pointer EGFR and Jak/Stat signalings mediate compensatory ISC proliferation. Black-Right-Pointing-Pointer Notch signaling regulates ISC self-renewal and differentiation.

  19. Expression of human FcgammaRIIIa as a GPI-linked molecule on CHO cells to enable measurement of human IgG binding.

    Science.gov (United States)

    Armour, Kathryn L; Smith, Cheryl S; Clark, Michael R

    2010-03-31

    The efficacy of a therapeutic IgG molecule may be as dependent on the optimisation of the constant region to suit its intended indication as on the selection of its variable regions. A crucial effector function to be maximised or minimised is antibody-dependent cell-mediated cytotoxicity by natural killer cells. Traditional assays of ADCC activity suffer from considerable inter-donor and intra-donor variability, which makes the measurement of antibody binding to human FcgammaRIIIa, the key receptor for ADCC, an attractive alternative method of assessment. Here, we describe the development of cell lines and assays for this purpose. The transmembrane receptor, FcgammaRIIIa, requires co-expression with signal transducing subunits to prevent its degradation, unlike the homologous receptor FcgammaRIIIb that is expressed as a GPI-anchored molecule. Therefore, to simplify the production of cell lines as reliable assay components, we expressed FcgammaRIIIa as a GPI-anchored molecule. Separate, stable CHO cell lines that express either the 158F or the higher-affinity 158V allotype of FcgammaRIIIa were isolated using fluorescence-activated cell sorting. The identities of the expressed receptors were confirmed using a panel of monoclonal antibodies that distinguish between subclasses and allotypes of FcgammaRIII and the cell lines were shown to have slightly higher levels of receptor than FcgammaRIII-positive peripheral blood mononuclear cells. Because the affinity of FcgammaRIIIa for IgG is intermediate amongst the receptors that bind IgG, we were able to use these cell lines to develop flow cytometric assays to measure the binding of both complexed and monomeric immunoglobulin. Thus, by choosing the appropriate method, weakly- or strongly-binding IgG can be efficiently compared. We have quantified the difference in the binding of wildtype IgG1 and IgG3 molecules to the two functional allotypes of the receptor and report that the FcgammaRIIIa-158V-antibody interaction is 3

  20. Increased centrosome amplification in aged stem cells of the Drosophila midgut

    International Nuclear Information System (INIS)

    Park, Joung-Sun; Pyo, Jung-Hoon; Na, Hyun-Jin; Jeon, Ho-Jun; Kim, Young-Shin; Arking, Robert; Yoo, Mi-Ae

    2014-01-01

    Highlights: • Increased centrosome amplification in ISCs of aged Drosophila midguts. • Increased centrosome amplification in ISCs of oxidative stressed Drosophila midguts. • Increased centrosome amplification in ISCs by overexpression of PVR, EGFR, and AKT. • Supernumerary centrosomes can be responsible for abnormal ISC polyploid cells. • Supernumerary centrosomes can be a useful marker for aging stem cells. - Abstract: Age-related changes in long-lived tissue-resident stem cells may be tightly linked to aging and age-related diseases such as cancer. Centrosomes play key roles in cell proliferation, differentiation and migration. Supernumerary centrosomes are known to be an early event in tumorigenesis and senescence. However, the age-related changes of centrosome duplication in tissue-resident stem cells in vivo remain unknown. Here, using anti-γ-tubulin and anti-PH3, we analyzed mitotic intestinal stem cells with supernumerary centrosomes in the adult Drosophila midgut, which may be a versatile model system for stem cell biology. The results showed increased centrosome amplification in intestinal stem cells of aged and oxidatively stressed Drosophila midguts. Increased centrosome amplification was detected by overexpression of PVR, EGFR, and AKT in intestinal stem cells/enteroblasts, known to mimic age-related changes including hyperproliferation of intestinal stem cells and hyperplasia in the midgut. Our data show the first direct evidence for the age-related increase of centrosome amplification in intestinal stem cells and suggest that the Drosophila midgut is an excellent model for studying molecular mechanisms underlying centrosome amplification in aging adult stem cells in vivo

  1. Increased centrosome amplification in aged stem cells of the Drosophila midgut

    Energy Technology Data Exchange (ETDEWEB)

    Park, Joung-Sun; Pyo, Jung-Hoon; Na, Hyun-Jin; Jeon, Ho-Jun; Kim, Young-Shin [Department of Molecular Biology, Pusan National University, Busan 609-735 (Korea, Republic of); Arking, Robert, E-mail: aa2210@wayne.edu [Department of Biological Sciences, Wayne State University, Detroit, MI 48202 (United States); Yoo, Mi-Ae, E-mail: mayoo@pusan.ac.kr [Department of Molecular Biology, Pusan National University, Busan 609-735 (Korea, Republic of)

    2014-07-25

    Highlights: • Increased centrosome amplification in ISCs of aged Drosophila midguts. • Increased centrosome amplification in ISCs of oxidative stressed Drosophila midguts. • Increased centrosome amplification in ISCs by overexpression of PVR, EGFR, and AKT. • Supernumerary centrosomes can be responsible for abnormal ISC polyploid cells. • Supernumerary centrosomes can be a useful marker for aging stem cells. - Abstract: Age-related changes in long-lived tissue-resident stem cells may be tightly linked to aging and age-related diseases such as cancer. Centrosomes play key roles in cell proliferation, differentiation and migration. Supernumerary centrosomes are known to be an early event in tumorigenesis and senescence. However, the age-related changes of centrosome duplication in tissue-resident stem cells in vivo remain unknown. Here, using anti-γ-tubulin and anti-PH3, we analyzed mitotic intestinal stem cells with supernumerary centrosomes in the adult Drosophila midgut, which may be a versatile model system for stem cell biology. The results showed increased centrosome amplification in intestinal stem cells of aged and oxidatively stressed Drosophila midguts. Increased centrosome amplification was detected by overexpression of PVR, EGFR, and AKT in intestinal stem cells/enteroblasts, known to mimic age-related changes including hyperproliferation of intestinal stem cells and hyperplasia in the midgut. Our data show the first direct evidence for the age-related increase of centrosome amplification in intestinal stem cells and suggest that the Drosophila midgut is an excellent model for studying molecular mechanisms underlying centrosome amplification in aging adult stem cells in vivo.

  2. Characterization of recombinant Trypanosoma brucei gambiense Translationally Controlled Tumor Protein (rTbgTCTP) and its interaction with Glossina midgut bacteria.

    Science.gov (United States)

    Bossard, Géraldine; Bartoli, Manon; Fardeau, Marie-Laure; Holzmuller, Philippe; Ollivier, Bernard; Geiger, Anne

    2017-09-03

    In humans, sleeping sickness (i.e. Human African Trypanosomiasis) is caused by the protozoan parasites Trypanosoma brucei gambiense (Tbg) in West and Central Africa, and T. b. rhodesiense in East Africa. We previously showed in vitro that Tbg is able to excrete/secrete a large number of proteins, including Translationally Controlled Tumor Protein (TCTP). Moreover, the tctp gene was described previously to be expressed in Tbg-infected flies. Aside from its involvement in diverse cellular processes, we have investigated a possible alternative role within the interactions occurring between the trypanosome parasite, its tsetse fly vector, and the associated midgut bacteria. In this context, the Tbg tctp gene was synthesized and cloned into the baculovirus vector pAcGHLT-A, and the corresponding protein was produced using the baculovirus Spodoptera frugicola (strain 9) / insect cell system. The purified recombinant protein rTbgTCTP was incubated together with bacteria isolated from the gut of tsetse flies, and was shown to bind to 24 out of the 39 tested bacteria strains belonging to several genera. Furthermore, it was shown to affect the growth of the majority of these bacteria, especially when cultivated under microaerobiosis and anaerobiosis. Finally, we discuss the potential for TCTP to modulate the fly microbiome composition toward favoring trypanosome survival.

  3. Membrane skeletal proteins and their integral membrane protein anchors are targets for tyrosine and threonine kinases in Euglena.

    Science.gov (United States)

    Fazio, M J; Da Silva, A C; Rosiere, T K; Bouck, G B

    1995-01-01

    Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [32P]-orthophosphate or gamma-[32P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.

  4. Protein kinase C zeta suppresses low- or high-grade colorectal cancer (CRC) phenotypes by interphase centrosome anchoring.

    Science.gov (United States)

    Deevi, Ravi Kiran; Javadi, Arman; McClements, Jane; Vohhodina, Jekaterina; Savage, Kienan; Loughrey, Maurice Bernard; Evergren, Emma; Campbell, Frederick Charles

    2018-04-01

    Histological grading provides prognostic stratification of colorectal cancer (CRC) by scoring heterogeneous phenotypes. Features of aggressiveness include aberrant mitotic spindle configurations, chromosomal breakage, and bizarre multicellular morphology, but pathobiology is poorly understood. Protein kinase C zeta (PKCz) controls mitotic spindle dynamics, chromosome segregation, and multicellular patterns, but its role in CRC phenotype evolution remains unclear. Here, we show that PKCz couples genome segregation to multicellular morphology through control of interphase centrosome anchoring. PKCz regulates interdependent processes that control centrosome positioning. Among these, interaction between the cytoskeletal linker protein ezrin and its binding partner NHERF1 promotes the formation of a localized cue for anchoring interphase centrosomes to the cell cortex. Perturbation of these phenomena induced different outcomes in cells with single or extra centrosomes. Defective anchoring of a single centrosome promoted bipolar spindle misorientation, multi-lumen formation, and aberrant epithelial stratification. Collectively, these disturbances induce cribriform multicellular morphology that is typical of some categories of low-grade CRC. By contrast, defective anchoring of extra centrosomes promoted multipolar spindle formation, chromosomal instability (CIN), disruption of glandular morphology, and cell outgrowth across the extracellular matrix interface characteristic of aggressive, high-grade CRC. Because PKCz enhances apical NHERF1 intensity in 3D epithelial cultures, we used an immunohistochemical (IHC) assay of apical NHERF1 intensity as an indirect readout of PKCz activity in translational studies. We show that apical NHERF1 IHC intensity is inversely associated with multipolar spindle frequency and high-grade morphology in formalin-fixed human CRC samples. To conclude, defective PKCz control of interphase centrosome anchoring may underlie distinct categories of

  5. Hemoglobinúria paroxística noturna: relato de dois casos Paroxysmal nocturnal hemoglobinuria: two case reports

    Directory of Open Access Journals (Sweden)

    Carlos J. Araújo

    2002-12-01

    Full Text Available A Hemoglobina Paroxística Noturna (HPN é uma doença adquirida da stem cell hematopoética caracterizada por anemia hemolítica crônica, episódios trombóticos, e com freqüência pancitopenia. É uma desordem clonal, causada por mutação somática do gene PIG-A ligado ao cromossomo X, o qual é requisitado para a formação da estrutura da âncora glicosil-fosfatidil-inositol (GPI. A deficiência da GPI ancorada á proteína CD59 explica a hemólise intravascular na PNH, resultando da inabilidade dos eritrócitos inativar a superfície do complemento. Uma imensa relação clínica existe entre HPN e a anemia aplástica (AA. A ausência de GPI ancorada às proteínas é facilmente detectada pelos métodos de citometria de fluxo aplicados aos eritrócitos e leucócitos; os testes de Ham a da sucrose são absolutos. Em algumas vezes o tratamento com corticóides e/ou androgênio é útil. O transplante de medula óssea alogênico é curativo. O objetivo deste artigo é relatar dois casos de HPN com revisão, enfatizando os aspectos fisiopatológicos, clínicos, diagnósticos e tratamento da HPN.Paroxysmal nocturnal hemoglobinuria (PNH is an acquired hematopoietic stem cell disease characterized by chronic hemolytic anemia, thrombotic episodes and often pancytopenia. It is a chronic disorder caused by a somatic mutation of the X-linked gene PIG-A, which is required for formation of the glycosylphosphatidylinositols (GPI - anchor structure. Deficiency of the GPI-anchored protein CD59 explains intravascular hemolysis in PNH, which results from the inability of erythrocytes to inactivate the surface complement. A very strong clinical relationship exists between aplastic anemia (AA and PNH. Absence of GPI-anchored proteins is easily detected by flow cytometric methods applied to both erythrocytes and leukocytes; the Ham and sucrose tests are now obsolete. Treatment with glucocorticoids and / or androgen is sometimes helpful. Allogeneic

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

    Science.gov (United States)

    Balachandran, Manasi; Giannone, Richard J; Bemis, David A; Kania, Stephen A

    2017-01-01

    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 with 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.

  7. Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus.

    Science.gov (United States)

    Lan, Hanhong; Chen, Hongyan; Liu, Yuyan; Jiang, Chaoyang; Mao, Qianzhuo; Jia, Dongsheng; Chen, Qian; Wei, Taiyun

    2016-01-15

    Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. Infection by a rice virus (Southern rice black streaked dwarf virus [SRBSDV]) of its incompetent vector (the small brown planthopper [SBPH]) is restricted to the midgut epithelium. Here, we show that the siRNA pathway is triggered by SRBSDV infection in continuously cultured cells derived from the SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of the siRNA pathway by RNA interference strongly increased the ability of SRBSDV to propagate in continuously cultured SBPH cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/μg of midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. Silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results reveal the new finding that the siRNA pathway can control the initial infection of the insect midgut epithelium by a virus, which finally affects the competence of the virus's vector. Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the midgut epithelium of an

  8. PINK1 is required for timely cell-type specific mitochondrial clearance during Drosophila midgut metamorphosis.

    Science.gov (United States)

    Liu, Yan; Lin, Jingjing; Zhang, Minjie; Chen, Kai; Yang, Shengxi; Wang, Qun; Yang, Hongqin; Xie, Shusen; Zhou, Yongjian; Zhang, Xi; Chen, Fei; Yang, Yufeng

    2016-11-15

    Mitophagy is the selective degradation of mitochondria by autophagy, which is an important mitochondrial quality and quantity control process. During Drosophila metamorphosis, the degradation of midgut involves a large change in length and organization, which is mediated by autophagy. Here we noticed a cell-type specific mitochondrial clearance process that occurs in enterocytes (ECs), while most mitochondria remain in intestinal stem cells (ISCs) during metamorphosis. Although PINK1/PARKIN represent the canonical pathway for the elimination of impaired mitochondria in varied pathological conditions, their roles in developmental processes or normal physiological conditions have been less studied. To examine the potential contribution of PINK1 in developmental processes, we monitored the dynamic expression pattern of PINK1 in the midgut development by taking advantage of a newly CRISPR/Cas9 generated knock-in fly strain expressing PINK1-mCherry fusion protein that presumably recapitulates the endogenous expression pattern of PINK1. We disclosed a spatiotemporal correlation between the expression pattern of PINK1 and the mitochondrial clearance or persistence in ECs or ISCs respectively. By mosaic genetic analysis, we then demonstrated that PINK1 and PARKIN function epistatically to mediate the specific timely removal of mitochondria, and are involved in global autophagy in ECs during Drosophila midgut metamorphosis, with kinase-dead PINK1 exerting dominant negative effects. Taken together, our studies concluded that the PINK1/PARKIN is crucial for timely cell-type specific mitophagy under physiological conditions and demonstrated again that Drosophila midgut metamorphosis might serve as an elegant in vivo model to study autophagy. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport

    Science.gov (United States)

    Hirata, Tetsuya; Fujita, Morihisa; Nakamura, Shota; Gotoh, Kazuyoshi; Motooka, Daisuke; Murakami, Yoshiko; Maeda, Yusuke; Kinoshita, Taroh

    2015-01-01

    The importance of endosome-to–trans-Golgi network (TGN) retrograde transport in the anterograde transport of proteins is unclear. In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport. Knockout (KO) of each of the four GARP subunits, VPS51–VPS54, in HEK293 cells caused severely defective anterograde transport of both glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins from the TGN. Overexpression of VAMP4, v-SNARE, in VPS54-KO cells partially restored not only endosome-to-TGN retrograde transport, but also anterograde transport of both GPI-anchored and transmembrane proteins. Further screening for genes whose overexpression normalized the VPS54-KO phenotype identified TMEM87A, encoding an uncharacterized Golgi-resident membrane protein. Overexpression of TMEM87A or its close homologue TMEM87B in VPS54-KO cells partially restored endosome-to-TGN retrograde transport and anterograde transport. Therefore GARP- and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for efficient post-Golgi anterograde transport of cell-surface integral membrane proteins. In addition, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport. PMID:26157166

  10. Plasmodium falciparum ookinetes require mosquito midgut chondroitin sulfate proteoglycans for cell invasion.

    NARCIS (Netherlands)

    Dinglasan, R.R.; Alaganan, A.; Ghosh, A.K.; Saito, A.; Kuppevelt, A.H.M.S.M. van; Jacobs-Lorena, M.

    2007-01-01

    Malaria transmission entails development of the Plasmodium parasite in its insect vector, the Anopheles mosquito. Parasite invasion of the mosquito midgut is the critical first step and involves adhesion to host epithelial cell ligands. Partial evidence suggests that midgut oligosaccharides are

  11. Polyphenol-Rich Diets Exacerbate AMPK-Mediated Autophagy, Decreasing Proliferation of Mosquito Midgut Microbiota, and Extending Vector Lifespan.

    Directory of Open Access Journals (Sweden)

    Rodrigo Dutra Nunes

    2016-10-01

    Full Text Available Mosquitoes feed on plant-derived fluids such as nectar and sap and are exposed to bioactive molecules found in this dietary source. However, the role of such molecules on mosquito vectorial capacity is unknown. Weather has been recognized as a major determinant of the spread of dengue, and plants under abiotic stress increase their production of polyphenols.Here, we show that including polyphenols in mosquito meals promoted the activation of AMP-dependent protein kinase (AMPK. AMPK positively regulated midgut autophagy leading to a decrease in bacterial proliferation and an increase in vector lifespan. Suppression of AMPK activity resulted in a 6-fold increase in midgut microbiota. Similarly, inhibition of polyphenol-induced autophagy induced an 8-fold increase in bacterial proliferation. Mosquitoes maintained on the polyphenol diet were readily infected by dengue virus.The present findings uncover a new direct route by which exacerbation of autophagy through activation of the AMPK pathway leads to a more efficient control of mosquito midgut microbiota and increases the average mosquito lifespan. Our results suggest for the first time that the polyphenol content and availability of the surrounding vegetation may increase the population of mosquitoes prone to infection with arboviruses.

  12. Induction of Manduca sexta Larvae Caspases Expression in Midgut Cells by Bacillus thuringiensis Cry1Ab Toxin

    Directory of Open Access Journals (Sweden)

    Helena Porta

    2011-01-01

    Full Text Available Bacillus thuringiensis produces crystal toxins known as Cry that are highly selective against important agricultural and human health-related insect pests. Cry proteins are pore-forming toxins that interact with specific receptors in the midgut cell membrane of susceptible larvae making pores that cause osmotic shock, leading finally to insect death. In the case of pore-forming toxins that are specific to mammalian cells, death responses at low doses may induce apoptosis or pyroptosis, depending on the cell type. The death mechanism induced by Cry toxins in insect midgut cells is poorly understood. Here, we analyze the caspases expression by RT-PCR analysis, showing that the initial response of Manduca sexta midgut cells after low dose of Cry1Ab toxin administration involves a fast and transient accumulation of caspase-1 mRNA, suggesting that pyroptosis was activated by Cry1Ab toxin as an initial response but was repressed later. In contrast, caspase-3 mRNA requires a longer period of time of toxin exposure to be activated but presents a sustained activation, suggesting that apoptosis may be a cell death mechanism induced also at low dose of toxin.

  13. Malrotation with midgut volvulus: CT findings of bowel infarction

    International Nuclear Information System (INIS)

    Aidlen, Jeremy; Anupindi, Sudha A.; Jaramillo, Diego; Doody, Daniel P.

    2005-01-01

    Midgut volvulus, the most common serious complication of malrotation, can be diagnosed using conventional contrast fluoroscopy, US or CT. CT is a quick and comprehensive examination in the evaluation of complex acute abdominal pathology in children. Contrast-enhanced CT can readily help the radiologist recognize perfusion abnormalities of the bowel, which is vital for reducing morbidity and mortality in affected children. Our case emphasizes and demonstrates additional CT features of bowel infarction in a child with a proven malrotation with midgut volvulus. (orig.)

  14. Malrotation with midgut volvulus: CT findings of bowel infarction

    Energy Technology Data Exchange (ETDEWEB)

    Aidlen, Jeremy [University of Massachusetts Medical Center, Department of Surgery, Worchester (United States); Anupindi, Sudha A.; Jaramillo, Diego [Massachusetts General Hospital, Pediatric Radiology, Boston, MA (United States); Doody, Daniel P. [Massachusetts General Hospital, Department of Pediatric Surgery, Boston, MA (United States)

    2005-05-01

    Midgut volvulus, the most common serious complication of malrotation, can be diagnosed using conventional contrast fluoroscopy, US or CT. CT is a quick and comprehensive examination in the evaluation of complex acute abdominal pathology in children. Contrast-enhanced CT can readily help the radiologist recognize perfusion abnormalities of the bowel, which is vital for reducing morbidity and mortality in affected children. Our case emphasizes and demonstrates additional CT features of bowel infarction in a child with a proven malrotation with midgut volvulus. (orig.)

  15. Tissue-specific Proteogenomic Analysis of Plutella xylostella Larval Midgut Using a Multialgorithm Pipeline.

    Science.gov (United States)

    Zhu, Xun; Xie, Shangbo; Armengaud, Jean; Xie, Wen; Guo, Zhaojiang; Kang, Shi; Wu, Qingjun; Wang, Shaoli; Xia, Jixing; He, Rongjun; Zhang, Youjun

    2016-06-01

    The diamondback moth, Plutella xylostella (L.), is the major cosmopolitan pest of brassica and other cruciferous crops. Its larval midgut is a dynamic tissue that interfaces with a wide variety of toxicological and physiological processes. The draft sequence of the P. xylostella genome was recently released, but its annotation remains challenging because of the low sequence coverage of this branch of life and the poor description of exon/intron splicing rules for these insects. Peptide sequencing by computational assignment of tandem mass spectra to genome sequence information provides an experimental independent approach for confirming or refuting protein predictions, a concept that has been termed proteogenomics. In this study, we carried out an in-depth proteogenomic analysis to complement genome annotation of P. xylostella larval midgut based on shotgun HPLC-ESI-MS/MS data by means of a multialgorithm pipeline. A total of 876,341 tandem mass spectra were searched against the predicted P. xylostella protein sequences and a whole-genome six-frame translation database. Based on a data set comprising 2694 novel genome search specific peptides, we discovered 439 novel protein-coding genes and corrected 128 existing gene models. To get the most accurate data to seed further insect genome annotation, more than half of the novel protein-coding genes, i.e. 235 over 439, were further validated after RT-PCR amplification and sequencing of the corresponding transcripts. Furthermore, we validated 53 novel alternative splicings. Finally, a total of 6764 proteins were identified, resulting in one of the most comprehensive proteogenomic study of a nonmodel animal. As the first tissue-specific proteogenomics analysis of P. xylostella, this study provides the fundamental basis for high-throughput proteomics and functional genomics approaches aimed at deciphering the molecular mechanisms of resistance and controlling this pest. © 2016 by The American Society for Biochemistry and

  16. Tissue-specific Proteogenomic Analysis of Plutella xylostella Larval Midgut Using a Multialgorithm Pipeline*

    Science.gov (United States)

    Zhu, Xun; Xie, Shangbo; Armengaud, Jean; Xie, Wen; Guo, Zhaojiang; Kang, Shi; Wu, Qingjun; Wang, Shaoli; Xia, Jixing; He, Rongjun; Zhang, Youjun

    2016-01-01

    The diamondback moth, Plutella xylostella (L.), is the major cosmopolitan pest of brassica and other cruciferous crops. Its larval midgut is a dynamic tissue that interfaces with a wide variety of toxicological and physiological processes. The draft sequence of the P. xylostella genome was recently released, but its annotation remains challenging because of the low sequence coverage of this branch of life and the poor description of exon/intron splicing rules for these insects. Peptide sequencing by computational assignment of tandem mass spectra to genome sequence information provides an experimental independent approach for confirming or refuting protein predictions, a concept that has been termed proteogenomics. In this study, we carried out an in-depth proteogenomic analysis to complement genome annotation of P. xylostella larval midgut based on shotgun HPLC-ESI-MS/MS data by means of a multialgorithm pipeline. A total of 876,341 tandem mass spectra were searched against the predicted P. xylostella protein sequences and a whole-genome six-frame translation database. Based on a data set comprising 2694 novel genome search specific peptides, we discovered 439 novel protein-coding genes and corrected 128 existing gene models. To get the most accurate data to seed further insect genome annotation, more than half of the novel protein-coding genes, i.e. 235 over 439, were further validated after RT-PCR amplification and sequencing of the corresponding transcripts. Furthermore, we validated 53 novel alternative splicings. Finally, a total of 6764 proteins were identified, resulting in one of the most comprehensive proteogenomic study of a nonmodel animal. As the first tissue-specific proteogenomics analysis of P. xylostella, this study provides the fundamental basis for high-throughput proteomics and functional genomics approaches aimed at deciphering the molecular mechanisms of resistance and controlling this pest. PMID:26902207

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

    Directory of Open Access Journals (Sweden)

    Manasi Balachandran

    Full Text Available 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 with 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.

  18. Intra-specific diversity of Serratia marcescens in Anopheles mosquito midgut defines Plasmodium transmission capacity

    Science.gov (United States)

    Bando, Hironori; Okado, Kiyoshi; Guelbeogo, Wamdaogo M.; Badolo, Athanase; Aonuma, Hiroka; Nelson, Bryce; Fukumoto, Shinya; Xuan, Xuenan; Sagnon, N'Fale; Kanuka, Hirotaka

    2013-01-01

    A critical stage in malaria transmission occurs in the Anopheles mosquito midgut, when the malaria parasite, Plasmodium, ingested with blood, first makes contact with the gut epithelial surface. To understand the response mechanisms within the midgut environment, including those influenced by resident microbiota against Plasmodium, we focus on a midgut bacteria species' intra-specific variation that confers diversity to the mosquito's competency for malaria transmission. Serratia marcescens isolated from either laboratory-reared mosquitoes or wild populations in Burkina Faso shows great phenotypic variation in its cellular and structural features. Importantly, this variation is directly correlated with its ability to inhibit Plasmodium development within the mosquito midgut. Furthermore, this anti-Plasmodium function conferred by Serratia marcescens requires increased expression of the flagellum biosynthetic pathway that is modulated by the motility master regulatory operon, flhDC. These findings point to new strategies for controlling malaria through genetic manipulation of midgut bacteria within the mosquito. PMID:23571408

  19. Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism

    Science.gov (United States)

    Caccia, Silvia; Di Lelio, Ilaria; La Storia, Antonietta; Marinelli, Adriana; Varricchio, Paola; Franzetti, Eleonora; Banyuls, Núria; Tettamanti, Gianluca; Casartelli, Morena; Giordana, Barbara; Ferré, Juan; Gigliotti, Silvia; Pennacchio, Francesco

    2016-01-01

    Bacillus thuringiensis is a widely used bacterial entomopathogen producing insecticidal toxins, some of which are expressed in insect-resistant transgenic crops. Surprisingly, the killing mechanism of B. thuringiensis remains controversial. In particular, the importance of the septicemia induced by the host midgut microbiota is still debated as a result of the lack of experimental evidence obtained without drastic manipulation of the midgut and its content. Here this key issue is addressed by RNAi-mediated silencing of an immune gene in a lepidopteran host Spodoptera littoralis, leaving the midgut microbiota unaltered. The resulting cellular immunosuppression was characterized by a reduced nodulation response, which was associated with a significant enhancement of host larvae mortality triggered by B. thuringiensis and a Cry toxin. This was determined by an uncontrolled proliferation of midgut bacteria, after entering the body cavity through toxin-induced epithelial lesions. Consequently, the hemolymphatic microbiota dramatically changed upon treatment with Cry1Ca toxin, showing a remarkable predominance of Serratia and Clostridium species, which switched from asymptomatic gut symbionts to hemocoelic pathogens. These experimental results demonstrate the important contribution of host enteric flora in B. thuringiensis-killing activity and provide a sound foundation for developing new insect control strategies aimed at enhancing the impact of biocontrol agents by reducing the immunocompetence of the host. PMID:27506800

  20. Anopheles Midgut Epithelium Evades Human Complement Activity by Capturing Factor H from the Blood Meal

    Science.gov (United States)

    Khattab, Ayman; Barroso, Marta; Miettinen, Tiera; Meri, Seppo

    2015-01-01

    Hematophagous vectors strictly require ingesting blood from their hosts to complete their life cycles. Exposure of the alimentary canal of these vectors to the host immune effectors necessitates efficient counteractive measures by hematophagous vectors. The Anopheles mosquito transmitting the malaria parasite is an example of hematophagous vectors that within seconds can ingest human blood double its weight. The innate immune defense mechanisms, like the complement system, in the human blood should thereby immediately react against foreign cells in the mosquito midgut. A prerequisite for complement activation is that the target cells lack complement regulators on their surfaces. In this work, we analyzed whether human complement is active in the mosquito midgut, and how the mosquito midgut cells protect themselves against complement attack. We found that complement remained active for a considerable time and was able to kill microbes within the mosquito midgut. However, the Anopheles mosquito midgut cells were not injured. These cells were found to protect themselves by capturing factor H, the main soluble inhibitor of the alternative complement pathway. Factor H inhibited complement on the midgut cells by promoting inactivation of C3b to iC3b and preventing the activity of the alternative pathway amplification C3 convertase enzyme. An interference of the FH regulatory activity by monoclonal antibodies, carried to the midgut via blood, resulted in increased mosquito mortality and reduced fecundity. By using a ligand blotting assay, a putative mosquito midgut FH receptor could be detected. Thereby, we have identified a novel mechanism whereby mosquitoes can tolerate human blood. PMID:25679788

  1. Anopheles midgut epithelium evades human complement activity by capturing factor H from the blood meal.

    Directory of Open Access Journals (Sweden)

    Ayman Khattab

    2015-02-01

    Full Text Available Hematophagous vectors strictly require ingesting blood from their hosts to complete their life cycles. Exposure of the alimentary canal of these vectors to the host immune effectors necessitates efficient counteractive measures by hematophagous vectors. The Anopheles mosquito transmitting the malaria parasite is an example of hematophagous vectors that within seconds can ingest human blood double its weight. The innate immune defense mechanisms, like the complement system, in the human blood should thereby immediately react against foreign cells in the mosquito midgut. A prerequisite for complement activation is that the target cells lack complement regulators on their surfaces. In this work, we analyzed whether human complement is active in the mosquito midgut, and how the mosquito midgut cells protect themselves against complement attack. We found that complement remained active for a considerable time and was able to kill microbes within the mosquito midgut. However, the Anopheles mosquito midgut cells were not injured. These cells were found to protect themselves by capturing factor H, the main soluble inhibitor of the alternative complement pathway. Factor H inhibited complement on the midgut cells by promoting inactivation of C3b to iC3b and preventing the activity of the alternative pathway amplification C3 convertase enzyme. An interference of the FH regulatory activity by monoclonal antibodies, carried to the midgut via blood, resulted in increased mosquito mortality and reduced fecundity. By using a ligand blotting assay, a putative mosquito midgut FH receptor could be detected. Thereby, we have identified a novel mechanism whereby mosquitoes can tolerate human blood.

  2. Intrauterine midgut volvulus without malrotation: Diagnosis from the ‘coffee bean sign’

    Science.gov (United States)

    Park, Jun Seok; Cha, Seong Jae; Kim, Beom Gyu; Kim, Yong Seok; Choi, Yoo Shin; Chang, In Taik; Kim, Gwang Jun; Lee, Woo Seok; Kim, Gi Hyeon

    2008-01-01

    Fetal midgut volvulus is quite rare, and most cases are associated with abnormalities of intestinal rotation or fixation. We report a case of midgut volvulus without malrotation, associated with a meconium pellet, during the gestation period. This 2.79 kg, 33-wk infant was born via a spontaneous vaginal delivery caused by preterm labor. Prenatal ultrasound showed dilated bowel loops with the appearance of a ‘coffee bean sign’. This patient had an unusual presentation with a distended abdomen showing skin discoloration. An emergency laparotomy revealed a midgut volvulus and a twisted small bowel, caused by complicated meconium ileus. Such nonspecific prenatal radiological signs and a low index of suspicion of a volvulus during gestation might delay appropriate surgical management and result in ischemic necrosis of the bowel. Preterm labor, specific prenatal sonographic findings (for example, the coffee bean sign) and bluish discoloration of the abdominal wall could suggest intrauterine midgut volvulus requiring prompt surgical intervention. PMID:18322966

  3. Clinical roundtable monograph: Paroxysmal nocturnal hemoglobinuria: a case-based discussion.

    Science.gov (United States)

    Szer, Jeff; Hill, Anita; Weitz, Ilene Ceil

    2012-11-01

    Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired disorder characterized by chronic intravascular hemolysis as the primary clinical manifestation and morbidities that include anemia, thrombosis, renal impairment, pulmonary hypertension, and bone marrow failure. The prevalence of the PNH clone (from <1-100% PNH granulocytes) is approximately 16 per million, and careful monitoring is required. The average age of onset of the clinical disease is the early 30s, although it can present at all ages. PNH is caused by the acquisition of a somatic mutation of the gene phosphatidylinositol glycan anchor (PIG-A) in a multipotent hematopoietic stem cell (HSC), with clonal expansion of the mutated HSC. The mutation causes a deficiency in the synthesis of glycosylphosphatidylinositol (GPI). In cells derived from normal HSCs, the complement regulatory proteins CD55 and CD59 are anchored to the hematopoietic cell membrane surface via GPI, protecting the cells from complement-mediated lysis. However, in patients with PNH, these 2 proteins, along with numerous other GPI-linked proteins, are absent from the cell surface of red cells, granulocytes, monocytes, and platelets, resulting in complement-mediated intravascular hemolysis and other complications. Lysis of red blood cells is the most obvious manifestation, but as other cell lineages are also affected, this complement-mediated attack contributes to additional complications, such as thrombosis. Eculizumab, a humanized monoclonal antibody against the C5 complement protein, is the only effective drug therapy for PNH patients. The antibody prevents cleavage of the C5 protein by C5 convertase, in turn preventing generation of C5b-9 and release of C5a, thereby protecting from hemolysis of cells lacking the CD59 surface protein and other complications associated with complement activation. Drs. Ilene C. Weitz, Anita Hill, and Jeff Szer discuss 3 recent cases of patients with PNH.

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

    Science.gov (United States)

    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

  5. Distinguishing two types of gray mullet, Mugil cephalus L. (Mugiliformes: Mugilidae), by using glucose-6-phosphate isomerase (GPI) allozymes with special reference to enzyme activities.

    Science.gov (United States)

    Huang, C S; Weng, C F; Lee, S C

    2001-06-01

    The resident and migratory types of gray mullet, Mugil cephalus, on the coast of Taiwan can not be separated morphologically. Allozyme analysis was applied to estimate genetic variation between the two types of gray mullet and to test whether they belong to different populations. After starch gel electrophoresis, different allelic frequency spectra of glucose-6-phosphate isomerase-A (GPI-A) between stocks was observed. The resident stock contained Gpi-A(135) and Gpi-A(100), whereas the migratory type contained Gpi-A(100) only. In addition, GPI activities of locus A showed two distinct profiles between the two alleles. The results broadly revealed that Gpi-A allelic frequency was not regulated by temperature changes even after 6 months of thermal acclimation. This suggests that natural selection may play a role in shaping the allelic frequency change during the migratory journey. These findings suggest that the Gpi-A allelic difference can be used for population discrimination.

  6. Midgut of the non-hematophagous mosquito Toxorhynchites theobaldi (Diptera, Culicidae)

    OpenAIRE

    Godoy, Raquel S. M.; Fernandes, Kenner M.; Martins, Gustavo F.

    2015-01-01

    In most mosquito species, the females require a blood-feeding for complete egg development. However, in Toxorhynchites mosquitoes, the eggs develop without blood-feeding, and both females and males exclusively feed on sugary diets. The midgut is a well-understood organ in blood-feeding mosquitoes, but little is known about it in non-blood-feeding ones. In the present study, the detailed morphology of the midgut of Toxorhynchites theobaldi were investigated using histochemical and ultrastructu...

  7. Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism

    OpenAIRE

    Caccia, Silvia; Di Lelio, Ilaria; La Storia, Antonietta; Marinelli, Adriana; Varricchio, Paola; Franzetti, Eleonora; Banyuls, Núria; Tettamanti, Gianluca; Casartelli, Morena; Giordana, Barbara; Ferré, Juan; Gigliotti, Silvia; Ercolini, Danilo; Pennacchio, Francesco

    2016-01-01

    Bacillus thuringiensis and its toxins are widely used for insect control. Notwithstanding the remarkable importance of this insect pathogen, its killing mechanism has yet to be fully elucidated. Here we show that the microbiota resident in the host midgut triggers a lethal septicemia. The infection process is enhanced by reducing the host immune response and its control on replication of midgut bacteria invading the body cavity through toxin-induced epithelial lesions. The experimental approa...

  8. Signal transduction in neurons: effects of cellular prion protein on fyn kinase and ERK1/2 kinase

    Directory of Open Access Journals (Sweden)

    Tomasi Vittorio

    2010-12-01

    Full Text Available Abstract Background It has been reported that cellular prion protein (PrPc co-localizes with caveolin-1 and participates to signal transduction events by recruiting Fyn kinase. As PrPc is a secreted protein anchored to the outer surface membrane through a glycosylphosphatidylinositol (GPI anchor (secPrP and caveolin-1 is located in the inner leaflet of plasma membrane, there is a problem of how the two proteins can physically interact each other and transduce signals. Results By using the GST-fusion proteins system we observed that PrPc strongly interacts with caveolin-1 scaffolding domain and with a caveolin-1 hydrophilic C-terminal region, but not with the caveolin-1 N-terminal region. In vitro binding experiments were also performed to define the site(s of PrPc interacting with cav-1. The results are consistent with a participation of PrPc octapeptide repeats motif in the binding to caveolin-1 scaffolding domain. The caveolar localization of PrPc was ascertained by co-immunoprecipitation, by co-localization after flotation in density gradients and by confocal microscopy analysis of PrPc and caveolin-1 distributions in a neuronal cell line (GN11 expressing caveolin-1 at high levels. Conclusions We observed that, after antibody-mediated cross-linking or copper treatment, PrPc was internalized probably into caveolae. We propose that following translocation from rafts to caveolae or caveolae-like domains, secPrP could interact with caveolin-1 and induce signal transduction events.

  9. Signal transduction in neurons: effects of cellular prion protein on fyn kinase and ERK1/2 kinase.

    Science.gov (United States)

    Tomasi, Vittorio

    2010-12-16

    It has been reported that cellular prion protein (PrPc) co-localizes with caveolin-1 and participates to signal transduction events by recruiting Fyn kinase. As PrPc is a secreted protein anchored to the outer surface membrane through a glycosylphosphatidylinositol (GPI) anchor (secPrP) and caveolin-1 is located in the inner leaflet of plasma membrane, there is a problem of how the two proteins can physically interact each other and transduce signals. By using the GST-fusion proteins system we observed that PrPc strongly interacts with caveolin-1 scaffolding domain and with a caveolin-1 hydrophilic C-terminal region, but not with the caveolin-1 N-terminal region. In vitro binding experiments were also performed to define the site(s) of PrPc interacting with cav-1. The results are consistent with a participation of PrPc octapeptide repeats motif in the binding to caveolin-1 scaffolding domain. The caveolar localization of PrPc was ascertained by co-immunoprecipitation, by co-localization after flotation in density gradients and by confocal microscopy analysis of PrPc and caveolin-1 distributions in a neuronal cell line (GN11) expressing caveolin-1 at high levels. We observed that, after antibody-mediated cross-linking or copper treatment, PrPc was internalized probably into caveolae. We propose that following translocation from rafts to caveolae or caveolae-like domains, secPrP could interact with caveolin-1 and induce signal transduction events.

  10. Endocytosis of GPI-linked membrane folate receptor-alpha.

    Science.gov (United States)

    Rijnboutt, S; Jansen, G; Posthuma, G; Hynes, J B; Schornagel, J H; Strous, G J

    1996-01-01

    GPI-linked membrane folate receptors (MFRs) have been implicated in the receptor-mediated uptake of reduced folate cofactors and folate-based chemotherapeutic drugs. We have studied the biosynthetic transport to and internalization of MFR isoform alpha in KB-cells. MFR-alpha was synthesized as a 32-kD protein and converted in a maturely glycosylated 36-38-kD protein 1 h after synthesis. 32-kD MFR-alpha was completely soluble in Triton X-100 at 0 degree C. In contrast, only 33% of the 36-38-kD species could be solubilized at these conditions whereas complete solubilization was obtained in Triton X-100 at 37 degrees C or in the presence of saponin at 0 degree C. Similar solubilization characteristics were found when MFR-alpha at the plasma membrane was labeled with a crosslinkable 125I-labeled photoaffinity-analog of folic acid as a ligand. Triton X-100-insoluble membrane domains containing MFR-alpha could be separated from soluble MFR-alpha on sucrose flotation gradients. Only Triton X-100 soluble MFR-alpha was internalized from the plasma membrane. The reduced-folate-carrier, an integral membrane protein capable of translocating (anti-)folates across membranes, was completely excluded from the Triton X-100-resistant membrane domains. Internalized MFR-alpha recycled slowly to the cell surface during which it remained soluble in Triton X-100 at 0 degree C. Using immunoelectron microscopy, we found MFR-alpha along the entire endocytic pathway: in clathrin-coated buds and vesicles, and in small and large endosomal vacuoles. In conclusion, our data indicate that a large fraction, if not all, of internalizing MFR-alpha bypasses caveolae.

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

    International Nuclear Information System (INIS)

    Anderie, Ines; Schulz, Irene; Schmid, Andreas

    2007-01-01

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

  12. Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

    Directory of Open Access Journals (Sweden)

    Leah Theresa Sigle

    2013-09-01

    Full Text Available Sandflies (Diptera: Psychodidae are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2. Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.

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

    Science.gov (United States)

    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.

  14. Malrotation complicating midgut volvulus: Ultrasonographic finding

    International Nuclear Information System (INIS)

    Cho, Young Kwon; Jeon, Hae Jeong; Jin, Yong Hyun; Park, Dong Rib; Lee, Chang Hee

    2000-01-01

    The intestinal malrotation is one of disease representing jaundice and intermittent vomiting in neonatal period and its clinical manifestation varies from no symptom to fatal symptom requiring emergency operation. We report one case of malrotation with midgut volvulus representing 'whirl pool sign' on color ultrasound image.

  15. Setting sail for glucose homeostasis with the AKAP150-PP2B-anchor.

    Science.gov (United States)

    Teo, Adrian Kee Keong; Kulkarni, Rohit N

    2012-10-17

    Glucose-stimulated insulin secretion, controlled by multiple protein phosphorylation events, is critical for the regulation of glucose homeostasis. Protein kinase A (PKA) is known to play a role in β cell physiology, but the role of its anchoring protein is not fully understood. Hinke et al (2012) illustrate the significance of A-kinase anchoring protein 150 in tethering protein phosphatase 2B to mediate nutrient-stimulated insulin secretion and thus modulate glucose homeostasis.

  16. Long-Term Effect of GPi-DBS in a Patient With Generalized Dystonia Due to GLUT1 Deficiency Syndrome

    Directory of Open Access Journals (Sweden)

    Idil Hanci

    2018-05-01

    Full Text Available Treatment outcomes from pallidal deep brain stimulation are highly heterogeneous reflecting the phenotypic and etiologic spectrum of dystonia. Treatment stratification to neurostimulation therapy primarily relies on the phenotypic motor presentation; however, etiology including genetic factors are increasingly recognized as modifiers of treatment outcomes. Here, we describe a 53 year-old female patient with a progressive generalized dystonia since age 25. The patient underwent deep brain stimulation of the globus pallidus internus (GPi-DBS at age 44. Since the clinical phenotype included mobile choreo-dystonic features, we expected favorable therapeutic outcome from GPi-DBS. Although mobile dystonia components were slightly improved in the long-term outcome from GPi-DBS the overall therapeutic response 9 years from implantation was limited when comparing “stimulation off” and “stimulation on” despite of proper electrode localization and sufficient stimulation programming. In order to further understand the reason for this limited motor symptom response, we aimed to clarify the etiology of generalized dystonia in this patient. Genetic testing identified a novel heterozygous pathogenic SLC2A1 mutation as cause of glucose transporter type 1 deficiency syndrome (GLUT1-DS. This case report presents the first outcome of GPi-DBS in a patient with GLUT1-DS, and suggests that genotype relations may increasingly complement phenotype-based therapy stratification of GPi-DBS in dystonia.

  17. Discriminating lysosomal membrane protein types using dynamic neural network.

    Science.gov (United States)

    Tripathi, Vijay; Gupta, Dwijendra Kumar

    2014-01-01

    This work presents a dynamic artificial neural network methodology, which classifies the proteins into their classes from their sequences alone: the lysosomal membrane protein classes and the various other membranes protein classes. In this paper, neural networks-based lysosomal-associated membrane protein type prediction system is proposed. Different protein sequence representations are fused to extract the features of a protein sequence, which includes seven feature sets; amino acid (AA) composition, sequence length, hydrophobic group, electronic group, sum of hydrophobicity, R-group, and dipeptide composition. To reduce the dimensionality of the large feature vector, we applied the principal component analysis. The probabilistic neural network, generalized regression neural network, and Elman regression neural network (RNN) are used as classifiers and compared with layer recurrent network (LRN), a dynamic network. The dynamic networks have memory, i.e. its output depends not only on the input but the previous outputs also. Thus, the accuracy of LRN classifier among all other artificial neural networks comes out to be the highest. The overall accuracy of jackknife cross-validation is 93.2% for the data-set. These predicted results suggest that the method can be effectively applied to discriminate lysosomal associated membrane proteins from other membrane proteins (Type-I, Outer membrane proteins, GPI-Anchored) and Globular proteins, and it also indicates that the protein sequence representation can better reflect the core feature of membrane proteins than the classical AA composition.

  18. Chikungunya virus dissemination from the midgut of Aedes aegypti is associated with temporal basal lamina degradation during bloodmeal digestion.

    Directory of Open Access Journals (Sweden)

    Shengzhang Dong

    2017-09-01

    Full Text Available In the mosquito, the midgut epithelium is the initial tissue to become infected with an arthropod-borne virus (arbovirus that has been acquired from a vertebrate host along with a viremic bloodmeal. Following its replication in midgut epithelial cells, the virus needs to exit the midgut and infect secondary tissues including the salivary glands before it can be transmitted to another vertebrate host. The viral exit mechanism from the midgut, the midgut escape barrier (MEB, is poorly understood although it is an important determinant of mosquito vector competence for arboviruses. Using chikungunya virus (CHIKV as a model in Aedes aegypti, we demonstrate that the basal lamina (BL of the extracellular matrix (ECM surrounding the midgut constitutes a potential barrier for the virus. The BL, predominantly consisting of collagen IV and laminin, becomes permissive during bloodmeal digestion in the midgut lumen. Bloodmeal digestion, BL permissiveness, and CHIKV dissemination are coincident with increased collagenase activity, diminished collagen IV abundance, and BL shredding in the midgut between 24-32 h post-bloodmeal. This indicates that there may be a window-of-opportunity during which the MEB in Ae. aegypti becomes permissive for CHIKV. Matrix metalloproteinases (MMPs are the principal extracellular endopeptidases responsible for the degradation/remodeling of the ECM including the BL. We focused on Ae. aegypti (AeMMP1, which is expressed in midgut epithelial cells, is inducible upon bloodfeeding, and shows collagenase (gelatinase activity. However, attempts to inhibit AeMMP activity in general or specifically that of AeMMP1 did not seem to affect its function nor produce an altered midgut escape phenotype. As an alternative, we silenced and overexpressed the Ae. aegypti tissue inhibitor of metalloproteinases (AeTIMP in the mosquito midgut. AeTIMP was highly upregulated in the midgut during bloodmeal digestion and was able to inhibit MMP activity in

  19. Two-point anchoring of a lanthanide-binding peptide to a target protein enhances the paramagnetic anisotropic effect

    International Nuclear Information System (INIS)

    Saio, Tomohide; Ogura, Kenji; Yokochi, Masashi; Kobashigawa, Yoshihiro; Inagaki, Fuyuhiko

    2009-01-01

    Paramagnetic lanthanide ions fixed in a protein frame induce several paramagnetic effects such as pseudo-contact shifts and residual dipolar couplings. These effects provide long-range distance and angular information for proteins and, therefore, are valuable in protein structural analysis. However, until recently this approach had been restricted to metal-binding proteins, but now it has become applicable to non-metalloproteins through the use of a lanthanide-binding tag. Here we report a lanthanide-binding peptide tag anchored via two points to the target proteins. Compared to conventional single-point attached tags, the two-point linked tag provides two to threefold stronger anisotropic effects. Though there is slight residual mobility of the lanthanide-binding tag, the present tag provides a higher anisotropic paramagnetic effect

  20. The cellular prion protein negatively regulates phagocytosis and cytokine expression in murine bone marrow-derived macrophages.

    Directory of Open Access Journals (Sweden)

    Min Wang

    Full Text Available The cellular prion protein (PrP(C is a glycosylphosphatidylinositol (GPI-anchored glycoprotein on the cell surface. Previous studies have demonstrated contradictory roles for PrP(C in connection with the phagocytic ability of macrophages. In the present work, we investigated the function of PrP(C in phagocytosis and cytokine expression in bone marrow-derived macrophages infected with Escherichia coli. E. coli infection induced an increase in the PRNP mRNA level. Knockout of PrP(C promoted bacterial uptake; upregulated Rab5, Rab7, and Eea1 mRNA expression; and increased the recruitment of lysosomal-associated membrane protein-2 to phagosomes, suggesting enhanced microbicidal activity. Remarkably, knockout of PrP(C suppressed the proliferation of internalized bacteria and increased the expression of cytokines such as interleukin-1β. Collectively, our data reveal an important role of PrP(C as a negative regulator for phagocytosis, phagosome maturation, cytokine expression, and macrophage microbicidal activity.

  1. Catalase protects Aedes aegypti from oxidative stress and increases midgut infection prevalence of Dengue but not Zika.

    Science.gov (United States)

    Oliveira, José Henrique M; Talyuli, Octávio A C; Goncalves, Renata L S; Paiva-Silva, Gabriela Oliveira; Sorgine, Marcos Henrique F; Alvarenga, Patricia Hessab; Oliveira, Pedro L

    2017-04-01

    Digestion of blood in the midgut of Aedes aegypti results in the release of pro-oxidant molecules that can be toxic to the mosquito. We hypothesized that after a blood meal, the antioxidant capacity of the midgut is increased to protect cells against oxidative stress. Concomitantly, pathogens present in the blood ingested by mosquitoes, such as the arboviruses Dengue and Zika, also have to overcome the same oxidative challenge, and the antioxidant program induced by the insect is likely to influence infection status of the mosquito and its vectorial competence. We found that blood-induced catalase mRNA and activity in the midgut peaked 24 h after feeding and returned to basal levels after the completion of digestion. RNAi-mediated silencing of catalase (AAEL013407-RB) reduced enzyme activity in the midgut epithelia, increased H2O2 leakage and decreased fecundity and lifespan when mosquitoes were fed H2O2. When infected with Dengue 4 and Zika virus, catalase-silenced mosquitoes showed no alteration in infection intensity (number of plaque forming units/midgut) 7 days after the infectious meal. However, catalase knockdown reduced Dengue 4, but not Zika, infection prevalence (percent of infected midguts). Here, we showed that blood ingestion triggers an antioxidant response in the midgut through the induction of catalase. This protection facilitates the establishment of Dengue virus in the midgut. Importantly, this mechanism appears to be specific for Dengue because catalase silencing did not change Zika virus prevalence. In summary, our data suggest that redox balance in the midgut modulates mosquito vectorial competence to arboviral infections.

  2. Malrotation complicating midgut volvulus: Ultrasonographic finding

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Young Kwon; Jeon, Hae Jeong; Jin, Yong Hyun; Park, Dong Rib; Lee, Chang Hee [Kon Kuk University College of Medicine, Seoul (Korea, Republic of)

    2000-06-15

    The intestinal malrotation is one of disease representing jaundice and intermittent vomiting in neonatal period and its clinical manifestation varies from no symptom to fatal symptom requiring emergency operation. We report one case of malrotation with midgut volvulus representing 'whirl pool sign' on color ultrasound image.

  3. Dynamics and regulation of glycolysis-tricarboxylic acid metabolism in the midgut of Spodoptera litura during metamorphosis.

    Science.gov (United States)

    Hu, D; Luo, W; Fan, L F; Liu, F L; Gu, J; Deng, H M; Zhang, C; Huang, L H; Feng, Q L

    2016-04-01

    Significant changes usually take place in the internal metabolism of insects during metamorphosis. The glycolysis-tricarboxylic acid (glycolysis-TCA) pathway is important for energy metabolism. To elucidate its dynamics, the mRNA levels of genes involved in this pathway were examined in the midgut of Spodoptera litura during metamorphosis, and the pyruvate content was quantified. The expression patterns of these genes in response to starvation were examined, and the interaction between protein phosphatase 1 (PP1) and phosphofructokinase (PFK) was studied. The results revealed that the expression or activities of most glycolytic enzymes was down-regulated in prepupae and then recovered in some degree in pupae, and all TCA-related genes were remarkably suppressed in both the prepupae and pupae. Pyruvate was enriched in the pupal midgut. Taken together, these results suggest that insects decrease both glycolysis and TCA in prepupae to save energy and then up-regulate glycolysis but down-regulate TCA in pupae to increase the supply of intermediates for construction of new organs. The expression of all these genes were down-regulated by starvation, indicating that non-feeding during metamorphosis may be a regulator of glycolysis-TCA pathway in the midgut. Importantly, interaction between PP1 and PFK was identified and is suggested to be involved in the regulation of glycolysis. © 2015 The Royal Entomological Society.

  4. Effect of ionizing radiation on tissue proteins of the desert locust, Schistocerca gregaria Forskal

    International Nuclear Information System (INIS)

    Rao, P.J.; Singh, Y.; Mehrotra, K.N.

    1981-01-01

    Effect of gamma radiation on the protein concentration of thoracic and mandibular muscles and midgut of adult male desert locust, Schistocerca gregaria Forskal at 2.5, 5.0 and 7.5 Krad doses was studied. The protein concentration of thoracic muscles was the highest on a μg/mg wet tissue basis, followed by the mandibular muscles and midgut. Considerable increase in protein concentration of the tissues on 5th and 9th day and depletion on 7th day suggests the existence of a circadian rhythm like phenomenon in tissue proteins. The effect of radiation on protein concentration of thoracic and mandibular muscles was comparatively more pronounced than midgut tissue and was generally dose dependent. (author)

  5. Microbial population analysis of the midgut of Melophagus ovinus via high-throughput sequencing.

    Science.gov (United States)

    Duan, De-Yong; Liu, Guo-Hua; Cheng, Tian-Yin; Wang, Ya-Qin

    2017-08-09

    Melophagus ovinus, one of the most common haematophagous ectoparasites of sheep, can cause anaemia and reductions in weight gain, wool growth and hide value. However, no information is available about the microfloral structure of the midgut of this ectoparasite. In the present study, we investigated the microbial community structure of the midgut contents of fully engorged female and male M. ovinus using Illumina HiSeq. The phylum showing the highest abundance was Proteobacteria (99.9%). The dominant bacterial genera in females and males were Bartonella, Arsenophonus and Wolbachia. Some less abundant bacterial genera were also detected, including Enterobacter, Acinetobacter, Halomonas, Shewanella, Bacillus and Staphylococcus. Bartonella, Arsenophonus and Wolbachia were the dominant bacterial genera in the midgut of female and male M. ovinus. Although detected, Enterobacter, Acinetobacter, Halomonas, Shewanella, Bacillus and Staphylococcus showed low abundances. Importantly, this is the first report of the presence of Arsenophonus, Wolbachia, Enterobacter, Halomonas, Shewanella, Bacillus and Staphylococcus in the midgut of M. ovinus.

  6. Passage of ingested Mansonella ozzardi (Spirurida: Onchocercidae) microfilariae through the midgut of Aedes aegypti (Diptera: Culicidae).

    Science.gov (United States)

    Vaughan, Jefferson A; Bell, Jeffrey A; Turell, Michael J; Chadee, Dave D

    2007-01-01

    When virus and microfilariae are ingested concurrently by a mosquito, microfilariae (mf) may penetrate the mosquito midgut and introduce virus directly into the mosquito hemocoel, allowing mosquitoes to become infectious much sooner than normal and enhancing transmission of viruses by mosquitoes. Mansonella ozzardi (Manson) is a benign filarial nematode parasite of humans in Latin America and is transmitted by black flies (Diptera: Simuliidae) and biting midges (Diptera: Ceratopogonidae). Because M. ozzardi and dengue are sympatric, we wanted to know whether M. ozzardi mf had the ability to penetrate the midgut of Aedes aegypti (L.) (Diptera: Culicidae) and thus play a potential role in the enhancement of dengue transmission. To test this, the F1 progeny from locally collected Ae. aegypti were fed on M. ozzardi-infected human males in an endemic village in northern Trinidad. Mosquitoes were dissected at various times after feeding and examined for mf in the midguts and thoraces. Microfilariae penetrated the midguts of 43% of 63 mosquitoes that ingested mf. Overall, 11% of mf penetrated the midgut by 17 h after being ingested. The intensity of midgut penetration was positively correlated to the numbers of mf ingested. Because midgut penetration is a key requirement for mf enhancement to occur, the potential exists that M. ozzardi could be involved in the enhancement of dengue virus transmission.

  7. Catalase protects Aedes aegypti from oxidative stress and increases midgut infection prevalence of Dengue but not Zika.

    Directory of Open Access Journals (Sweden)

    José Henrique M Oliveira

    2017-04-01

    Full Text Available Digestion of blood in the midgut of Aedes aegypti results in the release of pro-oxidant molecules that can be toxic to the mosquito. We hypothesized that after a blood meal, the antioxidant capacity of the midgut is increased to protect cells against oxidative stress. Concomitantly, pathogens present in the blood ingested by mosquitoes, such as the arboviruses Dengue and Zika, also have to overcome the same oxidative challenge, and the antioxidant program induced by the insect is likely to influence infection status of the mosquito and its vectorial competence.We found that blood-induced catalase mRNA and activity in the midgut peaked 24 h after feeding and returned to basal levels after the completion of digestion. RNAi-mediated silencing of catalase (AAEL013407-RB reduced enzyme activity in the midgut epithelia, increased H2O2 leakage and decreased fecundity and lifespan when mosquitoes were fed H2O2. When infected with Dengue 4 and Zika virus, catalase-silenced mosquitoes showed no alteration in infection intensity (number of plaque forming units/midgut 7 days after the infectious meal. However, catalase knockdown reduced Dengue 4, but not Zika, infection prevalence (percent of infected midguts.Here, we showed that blood ingestion triggers an antioxidant response in the midgut through the induction of catalase. This protection facilitates the establishment of Dengue virus in the midgut. Importantly, this mechanism appears to be specific for Dengue because catalase silencing did not change Zika virus prevalence. In summary, our data suggest that redox balance in the midgut modulates mosquito vectorial competence to arboviral infections.

  8. Bm86 midgut protein sequence variation in South Texas cattle fever ticks

    Directory of Open Access Journals (Sweden)

    Kammlah Diane M

    2010-11-01

    Full Text Available Abstract Background Cattle fever ticks, Rhipicephalus (Boophilus microplus and R. (B. annulatus, vector bovine and equine babesiosis, and have significantly expanded beyond the permanent quarantine zone established in South Texas. Currently, there are no vaccines approved for use within the United States for controlling these vectors. Vaccines developed in Australia and Cuba based on the midgut antigen Bm86 have variable efficacy against cattle fever ticks. A possible explanation for this variation in vaccine efficacy is amino acid sequence divergence between the recombinant Bm86 vaccine component and native Bm86 expressed in ticks from different geographical regions of the world. Results There was 91.8% amino acid sequence identity in Bm86 among R. microplus and R. annulatus sequenced from South Texas infestations. When South Texas isolates were compared to the Australian Yeerongpilly and Cuban Camcord vaccine strains, there was 89.8% and 90.0% identity, respectively. Most of the sequence divergence was focused in one region of the protein, amino acids 206-298. Hydrophilicity profiles revealed that two short regions of Bm86 (amino acids 206-210 and 560-570 appear to be more hydrophilic in South Texas isolates compared to vaccine strains. Only one amino acid difference was found between South Texas and vaccine strains within two previously described B-cell epitopes. A total of 4 amino acid differences were observed within three peptides previously shown to induce protective immune responses in cattle. Conclusions Sequence differences between South Texas isolates and Yeerongpilly and Camcord strains are spread throughout the entire Bm86 sequence, suggesting that geographic variation does exist. Differences within previously described B-cell epitopes between South Texas isolates and vaccine strains are minimal; however, short regions of hydrophilic amino acids found unique to South Texas isolates suggest that additional unique surface exposed

  9. Characterization of proteinases from the midgut of Rhipicephalus (Boophilus microplus involved in the generation of antimicrobial peptides

    Directory of Open Access Journals (Sweden)

    Craik Charles S

    2010-07-01

    Full Text Available Abstract Background Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins. A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins. Results An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1'. Conclusions BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.

  10. Numerical investigation to the dual-fuel spray combustion process in an ethanol direct injection plus gasoline port injection (EDI + GPI) engine

    International Nuclear Information System (INIS)

    Huang, Yuhan; Hong, Guang; Huang, Ronghua

    2015-01-01

    Highlights: • A 5D PDF table was used to model the dual-fuel turbulence–chemistry interactions. • The cooling effect of ethanol direct injection (EDI) was examined. • The higher flame speed of ethanol in EDI + GPI increased the thermal efficiency. • The partially premixed combustion in EDI + GPI reduced the combustion temperature. • Ethanol’s low evaporation rate in low temperature led to incomplete combustion. - Abstract: Ethanol direct injection plus gasoline port injection (EDI + GPI) is a new technology to make the use of ethanol fuel more effective and efficient in spark ignition engines. Multi-dimensional computational fluid dynamics modelling was conducted on an EDI + GPI engine in both single and dual fuelled conditions. The in-cylinder flow field was solved in the realizable k−ε turbulence model with detailed engine geometry. The temporal and spatial distributions of the liquid and vapour fuels were simulated with the spray breakup and evaporation models. The combustion process was modelled with the partially premixed combustion concept in which both mixture fraction and progress variable were solved. The three-dimensional and five-dimensional presumed Probability Density Function (PDF) look-up tables were used to model the single-fraction-mixture and two-fraction-mixture turbulence–chemistry interactions respectively. The model was verified by comparing the numerical and experimental results of spray pattern and cylinder pressure. The simulation results showed that the combustion process of EDI + GPI dual-fuelled condition was partially premixed combustion because of the low evaporation rate of ethanol spray in low temperature environment before combustion. Compared with GPI only, the higher flame speed of ethanol fuel contributed to the greater pressure rise rate and maximum cylinder pressure in EDI + GPI condition, which consequently resulted in higher power output and thermal efficiency. The lower adiabatic flame temperature of

  11. Plasmodium falciparum Plasmodium helical interspersed subtelomeric proteins contribute to cytoadherence and anchor P. falciparum erythrocyte membrane protein 1 to the host cell cytoskeleton

    DEFF Research Database (Denmark)

    Oberli, Alexander; Zurbrügg, Laura; Rusch, Sebastian

    2016-01-01

    is anchored to the cytoskeleton, and the Plasmodium helical interspersed subtelomeric (PHIST) gene family plays a role in many host cell modifications including binding the intracellular domain of PfEMP1. Here, we show that conditional reduction of the PHIST protein PFE1605w strongly reduces adhesion...... interacts with both the intracellular segment of PfEMP1 and with cytoskeletal components. This is the first report of a PHIST protein interacting with key molecules of the cytoadherence complex and the host cytoskeleton, and this functional role seems to play an essential role in the pathology of P...

  12. Discovery of ebselen as an inhibitor of Cryptosporidium parvum glucose-6-phosphate isomerase (CpGPI by high-throughput screening of existing drugs

    Directory of Open Access Journals (Sweden)

    Rana Eltahan

    2018-04-01

    Full Text Available Cryptosporidium parvum is a water-borne and food-borne apicomplexan pathogen. It is one of the top four diarrheal-causing pathogens in children under the age of five in developing countries, and an opportunistic pathogen in immunocompromised individuals. Unlike other apicomplexans, C. parvum lacks Kreb's cycle and cytochrome-based respiration, thus relying mainly on glycolysis to produce ATP. In this study, we characterized the primary biochemical features of the C. parvum glucose-6-phosphate isomerase (CpGPI and determined its Michaelis constant towards fructose-6-phosphate (Km = 0.309 mM, Vmax = 31.72 nmol/μg/min. We also discovered that ebselen, an organoselenium drug, was a selective inhibitor of CpGPI by high-throughput screening of 1200 known drugs. Ebselen acted on CpGPI as an allosteric noncompetitive inhibitor (IC50 = 8.33 μM; Ki = 36.33 μM, while complete inhibition of CpGPI activity was not achieved. Ebselen could also inhibit the growth of C. parvum in vitro (EC50 = 165 μM at concentrations nontoxic to host cells, albeit with a relatively small in vitro safety window of 4.2 (cytotoxicity TC50 on HCT-8 cells = 700 μM. Additionally, ebselen might also target other enzymes in the parasite, leading to the parasite growth reduction. Therefore, although ebselen is useful in studying the inhibition of CpGPI enzyme activity, further proof is needed to chemically and/or genetically validate CpGPI as a drug target. Keywords: Apicomplexan, Cryptosporidium parvum, Glucose-6-phosphate isomerase (GPI, Ebselen

  13. A novel quick transendoscopic enteral tubing in mid-gut: technique and training with video.

    Science.gov (United States)

    Long, Chuyan; Yu, Yan; Cui, Bota; Jagessar, Sabreen Abdul Rahman; Zhang, Jie; Ji, Guozhong; Huang, Guangming; Zhang, Faming

    2018-03-13

    This study aimed to evaluate the feasibility, safety, and value of a quick technique for transendoscopic enteral tubing (TET) through mid-gut. A prospective interventional study was performed in a single center. A TET tube was inserted into mid-gut through the nasal orifice and fixed on the pylorus wall by one tiny titanium endoscopic clip under anesthesia. The feasibility, safety, success rate, and satisfaction with TET placement were evaluated for enteral nutrition or fecal microbiota transplantation. A total of 86 patients underwent mid-gut TET. The success rate of the TET procedure was 98.8% (85/86). Mean tubing time of the TET procedure was 4.2 ± 1.9 min. 10 cases of procedure was enough for training of general endoscopist to shorten the procedure time (7.0 min vs 4.0 min, p tube-related adverse events were observed in 8.1% (7/86) and 7.0% (6/86) of patients respectively. There were no moderate to severe adverse events during tube extubation. TET through mid-gut is a novel, convenient, reliable and safe procedure for mid-gut administration with a high degree of patient satisfaction. This research was retrospectively registered with clinicaltrials.gov. Trial registration date: 29th November 2017. NCT03335982 .

  14. Midgut volvulus: a rare cause of episodes of intestinal obstruction in an adult

    International Nuclear Information System (INIS)

    Palomo, V.; Higuera, A.; Munoz, R.; Sanchez, F.

    2002-01-01

    Midgut volvulus occurs frequently in infants and children, but is uncommon in adults. We present a case of intestinal malrotation complicated by midgut volvulus in a young woman who complained of chronic intermittent abdominal pain of increasing intensity. The radiologies diagnosis was based mainly on upper gastrointestinal barium study, and was confirmed intraoperatively. (Author) 11 refs

  15. Cognitive and Psychiatric Effects of STN versus GPi Deep Brain Stimulation in Parkinson's Disease: A Meta-Analysis of Randomized Controlled Trials.

    Directory of Open Access Journals (Sweden)

    Jia-Wei Wang

    Full Text Available Deep brain stimulation (DBS of either the subthalamic nucleus (STN or the globus pallidus interna (GPi can reduce motor symptoms in patients with Parkinson's disease (PD and improve their quality of life. However, the effects of STN DBS and GPi DBS on cognitive functions and their psychiatric effects remain controversial. The present meta-analysis was therefore performed to clarify these issues.We searched the PUBMED, EMBASE, and the Cochrane Central Register of Controlled Trials databases. Other sources, including internet-based clinical trial registries and grey literature sources, were also searched. After searching the literature, two investigators independently performed literature screens to assess the quality of the included trials and to extract the data. The outcomes included the effects of STN DBS and GPi DBS on multiple cognitive domains, depression, anxiety, and quality of life.Seven articles related to four randomized controlled trials that included 521 participants were incorporated into the present meta-analysis. Compared with GPi DBS, STN DBS was associated with declines in selected cognitive domains after surgery, including attention, working memory and processing speed, phonemic fluency, learning and memory, and global cognition. However, there were no significant differences in terms of quality of life or psychiatric effects, such as depression and anxiety, between the two groups.A selective decline in frontal-subcortical cognitive functions is observed after STN DBS in comparison with GPi DBS, which should not be ignored in the target selection for DBS treatment in PD patients. In addition, compared to GPi DBS, STN DBS does not affect depression, anxiety, and quality of life.

  16. Cognitive and Psychiatric Effects of STN versus GPi Deep Brain Stimulation in Parkinson's Disease: A Meta-Analysis of Randomized Controlled Trials.

    Science.gov (United States)

    Wang, Jia-Wei; Zhang, Yu-Qing; Zhang, Xiao-Hua; Wang, Yun-Peng; Li, Ji-Ping; Li, Yong-Jie

    2016-01-01

    Deep brain stimulation (DBS) of either the subthalamic nucleus (STN) or the globus pallidus interna (GPi) can reduce motor symptoms in patients with Parkinson's disease (PD) and improve their quality of life. However, the effects of STN DBS and GPi DBS on cognitive functions and their psychiatric effects remain controversial. The present meta-analysis was therefore performed to clarify these issues. We searched the PUBMED, EMBASE, and the Cochrane Central Register of Controlled Trials databases. Other sources, including internet-based clinical trial registries and grey literature sources, were also searched. After searching the literature, two investigators independently performed literature screens to assess the quality of the included trials and to extract the data. The outcomes included the effects of STN DBS and GPi DBS on multiple cognitive domains, depression, anxiety, and quality of life. Seven articles related to four randomized controlled trials that included 521 participants were incorporated into the present meta-analysis. Compared with GPi DBS, STN DBS was associated with declines in selected cognitive domains after surgery, including attention, working memory and processing speed, phonemic fluency, learning and memory, and global cognition. However, there were no significant differences in terms of quality of life or psychiatric effects, such as depression and anxiety, between the two groups. A selective decline in frontal-subcortical cognitive functions is observed after STN DBS in comparison with GPi DBS, which should not be ignored in the target selection for DBS treatment in PD patients. In addition, compared to GPi DBS, STN DBS does not affect depression, anxiety, and quality of life.

  17. In Vivo and In Vitro Binding of Vip3Aa to Spodoptera frugiperda Midgut and Characterization of Binding Sites by 125I Radiolabeling

    Science.gov (United States)

    Chakroun, Maissa

    2014-01-01

    Bacillus thuringiensis vegetative insecticidal proteins (Vip3A) have been recently introduced in important crops as a strategy to delay the emerging resistance to the existing Cry toxins. The mode of action of Vip3A proteins has been studied in Spodoptera frugiperda with the aim of characterizing their binding to the insect midgut. Immunofluorescence histological localization of Vip3Aa in the midgut of intoxicated larvae showed that Vip3Aa bound to the brush border membrane along the entire apical surface. The presence of fluorescence in the cytoplasm of epithelial cells seems to suggest internalization of Vip3Aa or a fragment of it. Successful radiolabeling and optimization of the binding protocol for the 125I-Vip3Aa to S. frugiperda brush border membrane vesicles (BBMV) allowed the determination of binding parameters of Vip3A proteins for the first time. Heterologous competition using Vip3Ad, Vip3Ae, and Vip3Af as competitor proteins showed that they share the same binding site with Vip3Aa. In contrast, when using Cry1Ab and Cry1Ac as competitors, no competitive binding was observed, which makes them appropriate candidates to be used in combination with Vip3A proteins in transgenic crops. PMID:25002420

  18. Mechanism of Action of Secreted Newt Anterior Gradient Protein.

    Directory of Open Access Journals (Sweden)

    Kathrin S Grassme

    Full Text Available Anterior gradient (AG proteins have a thioredoxin fold and are targeted to the secretory pathway where they may act in the ER, as well as after secretion into the extracellular space. A newt member of the family (nAG was previously identified as interacting with the GPI-anchored salamander-specific three-finger protein called Prod1. Expression of nAG has been implicated in the nerve dependence of limb regeneration in salamanders, and nAG acted as a growth factor for cultured newt limb blastemal (progenitor cells, but the mechanism of action was not understood. Here we show that addition of a peptide antibody to Prod1 specifically inhibit the proliferation of blastema cells, suggesting that Prod1 acts as a cell surface receptor for secreted nAG, leading to S phase entry. Mutation of the single cysteine residue in the canonical active site of nAG to alanine or serine leads to protein degradation, but addition of residues at the C terminus stabilises the secreted protein. The mutation of the cysteine residue led to no detectable activity on S phase entry in cultured newt limb blastemal cells. In addition, our phylogenetic analyses have identified a new Caudata AG protein called AG4. A comparison of the AG proteins in a cell culture assay indicates that nAG secretion is significantly higher than AGR2 or AG4, suggesting that this property may vary in different members of the family.

  19. Study on Fungal Flora in the Midgut of the Larva and Adult of the Different Populations of the Malaria Vector Anopheles stephensi

    Directory of Open Access Journals (Sweden)

    L Tajedin

    2009-07-01

    Full Text Available Abstract Background: Many microorganisms in midgut of mosquito challenge with their host and also other pathogens pre­sent in midgut. The aim of this study was presence of non-pathogens microorganisms like fungal flora which may be cru­cial on interaction between vectors and pathogens."nMethods: Different populations of Anopheles stephensi were reared in insectary and objected to determine fungal flora in their midguts. The midgut paunch of mosquito adults and larvae as well as breading water and larval food sam­ples transferred on Subaru-dextrose agar, in order to detect the environment fungus."nResults: Although four fungi, Aspergillus, Rhizopus, Geotrichum and Sacharomyces were found in the food and wa­ter, but only Aspiragilus observed in the midgut of larvae. No fungus was found in the midgut of adults. This is the first report on fungal flora in the midgut of the adults and larvae of An. stephensi and possible stadial transmission of fungi from immature stages to adults."nConclusion: The midgut environment of adults is not compatible for survivorship of fungi but the larval midgut may con­tain few fungi as a host or even pathogen.

  20. Response of the common cutworm Spodoptera litura to zinc stress: Zn accumulation, metallothionein and cell ultrastructure of the midgut

    International Nuclear Information System (INIS)

    Shu, Yinghua; Zhang, Guren; Wang, Jianwu

    2012-01-01

    By exposing the common cutworm Spodoptera litura Fabricius larvae to a range of Zinc (Zn) stress, we investigated the effects of dietary Zn on Zn accumulation, metallothionein (MT), and on the ultrastructure of the midgut. The techniques we used were inductively coupled plasma-atomic emission spectrometer (ICP-AES), real-time PCR combined with cadmium-hemoglobin total saturation, and transmission electron microscopy (TEM), respectively. There was a significant dose–response relationship between the Zn accumulations in the midgut of the larvae and the Zn concentrations in the diet. Furthermore, both MT content and MT gene expression in the midgut were significantly induced in the 50–500 mg Zn/kg treatments, and were significantly positively correlated with the Zn accumulations in the midgut. When S. litura larvae were fed with the diet treated with 500 mg Zn/kg, Zn accumulation and MT content in the midgut was 4450.85 mg Zn/kg and 372.77 mg/kg, respectively, thereafter there was a little increase; the level of MT gene expression was maximal, thereafter there was a sharp decrease. TEM showed that numerous electron-dense granules (EDGs) and vacuoles appeared in the cytoplasm of the midgut cells, their number and size being closely correlated with the Zn accumulations in the midgut. Moreover, the nuclei were strongly influenced by Zn stress, evidenced by chromatin condensation and irregular nuclear membranes. Therefore, after being exposed to Zn in the threshold (500 mg Zn/kg) range, S. litura larvae could accumulate Zn in the midgut, which led to the induction of MT and changes in cell ultrastructure (mainly the presence of EDGs). The induction of MT and precipitation of Zn in EDGs may be the effective detoxification mechanisms by which the herbivorous insect S. litura defends itself against heavy metals. -- Graphical abstract: When the herbivorous insect Spodoptera litura Fabricius larvae were fed on the artificial diet with different concentrations of Zn

  1. Investigation of the midgut structure and ultrastructure in Cimex lectularius and Cimex pipistrelli (Hemiptera: Cimicidae).

    Science.gov (United States)

    Rost-Roszkowska, M M; Vilimova, J; Włodarczyk, A; Sonakowska, L; Kamińska, K; Kaszuba, F; Marchewka, A; Sadílek, D

    2017-02-01

    Cimicidae are temporary ectoparasites, which means that they cannot obtain food continuously. Both Cimex species examined here, Cimex lectularius (Linnaeus 1758) and Cimex pipistrelli (Jenyns 1839), can feed on a non-natal host, C. lectularius from humans on bats, C. pipistrelli on humans, but never naturally. The midgut of C. lectularius and C. pipistrelli is composed of three distinct regions-the anterior midgut (AMG), which has a sack-like shape, the long tube-shaped middle midgut (MMG), and the posterior midgut (PMG). The different ultrastructures of the AMG, MMG, and PMG in both of the species examined suggest that these regions must fulfill different functions in the digestive system. Ultrastructural analysis showed that the AMG fulfills the role of storing food and synthesizing and secreting enzymes, while the MMG is the main organ for the synthesis of enzymes, secretion, and the storage of the reserve material. Additionally, both regions, the AMG and MMG, are involved in water absorption in the digestive system of both Cimex species. The PMG is the part of the midgut in which spherites accumulate. The results of our studies confirm the suggestion of former authors that the structure of the digestive tract of insects is not attributed solely to diet but to the basic adaptation of an ancestor.

  2. Does autophagy in the midgut epithelium of centipedes depend on the day/night cycle?

    Science.gov (United States)

    Rost-Roszkowska, M M; Chajec, Ł; Vilimova, J; Tajovský, K; Kszuk-Jendrysik, M

    2015-01-01

    The midgut epithelium of two centipedes, Lithobius forficatus and Scolopendra cingulata, is composed of digestive, secretory and regenerative cells. In L. forficatus, the autophagy occurred only in the cytoplasm of the digestive cells as a sporadic process, while in S. cingulata, it occurred intensively in the digestive, secretory and regenerative cells of the midgut epithelium. In both of the species that were analyzed, this process proceeded in a continuous manner and did not depend on the day/night cycle. Ultrastructural analysis showed that the autophagosomes and autolysosomes were located mainly in the apical and perinuclear cytoplasm of the digestive cells in L. forficatus. However, in S. cingulata, the entire cytoplasm was filled with autophagosomes and autolysosomes. Initially the membranes of phagophores surround organelles during autophagosome formation. Autolysosomes result from the fusion of autophagosomes and lysosomes. Residual bodies which are the last stage of autophagy were released into the midgut lumen due to necrosis. Autophagy in the midgut epithelia that were analyzed was confirmed using acid phosphatase and mono-dansyl-cadaverine stainings. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Transcriptional Signatures in Response to Wheat Germ Agglutinin and Starvation in Drosophila melanogaster Larval Midgut

    Science.gov (United States)

    One function of plant lectins such as wheat germ agglutinin (WGA) is to serve as defenses against herbivorous insects. The midgut is one critical site affected by dietary lectins. We observed marked cellular, structural, and gene expression changes in the midguts of Drosophila melanogaster third-i...

  4. Isolation of the Binding Protein of Periplocoside E from BBMVs in Midgut of the Oriental Amyworm Mythimna separata Walker (Lepidoptera: Noctuidae through Affinity Chromatography

    Directory of Open Access Journals (Sweden)

    Mingxing Feng

    2016-05-01

    Full Text Available Periplocosides, which are insecticidal compounds isolated from the root bark of Periploca sepium Bunge, can affect the digestive system of insects. However, the mechanism though which periplocosides induces a series of symptoms remains unknown. In this study, affinity chromatography was conducted by coupling periplocoside E-semi-succinic acid ester with epoxy amino hexyl (EAH sepharose 4B. Sodium dodecyl sulfonate-polyacrylamide gelelectrophoresis (SDS-PAGE was performed to analyze the fraction eluted by periplocoside E. Eight binding proteins (luciferin 4-monooxygenase, aminopeptidase N, aminopeptidase N3, nicotinamide adenine dinucleotide health (NADH dehydrogenase subunit 5, phosphatidylinositol 3-phosphate 3-phosphatase myotubularin, actin, uncharacterized family 31 glucosidase KIAA1161, and 2OG-Fe(2 oxygenase superfamily protein were obtained and identified through liquid chromatography/quadrupole-time of flight-mass spectrometry (LC/Q-TOF-MS analysis of the midgut epithelium cells of Mythimna separata larvae. Aminopeptidase N and N3 are potential putative targets of periplocosides. This study establishes the foundation for further research on the mechanism of action and target localization of periplocosides in agricultural pests.

  5. Alterations in the Helicoverpa armigera midgut digestive physiology after ingestion of pigeon pea inducible leucine aminopeptidase.

    Directory of Open Access Journals (Sweden)

    Purushottam R Lomate

    Full Text Available Jasmonate inducible plant leucine aminopeptidase (LAP is proposed to serve as direct defense in the insect midgut. However, exact functions of inducible plant LAPs in the insect midgut remain to be estimated. In the present investigation, we report the direct defensive role of pigeon pea inducible LAP in the midgut of Helicoverpa armigera (Lepidoptera: Noctuidae and responses of midgut soluble aminopeptidases and serine proteinases upon LAP ingestion. Larval growth and survival was significantly reduced on the diets supplemented with pigeon pea LAP. Aminopeptidase activities in larvae remain unaltered in presence or absence of inducible LAP in the diet. On the contrary, serine proteinase activities were significantly decreased in the larvae reared on pigeon pea LAP containing diet as compared to larvae fed on diet without LAP. Our data suggest that pigeon pea inducible LAP is responsible for the degradation of midgut serine proteinases upon ingestion. Reduction in the aminopeptidase activity with LpNA in the H. armigera larvae was compensated with an induction of aminopeptidase activity with ApNA. Our findings could be helpful to further dissect the roles of plant inducible LAPs in the direct plant defense against herbivory.

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

    International Nuclear Information System (INIS)

    Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.

    2005-01-01

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

  7. Comparative analysis of Bacillus thuringiensis toxin binding to gypsy moth, browntail moth, and douglas-fir tussock moth midgut tissue sections using fluorescence microscopy

    Science.gov (United States)

    Algimantas P. Valaitis; John D. Podgwaite

    2011-01-01

    Many strains of Bacillus thuringiensis (Bt) produce insecticidal proteins, also referred to as Cry toxins, in crystal inclusions during sporulation. When ingested by insects, the Cry toxins bind to receptors on the brush border midgut epithelial cells and create pores in the epithelial gut membranes resulting in the death of...

  8. The Ogden Anchor.

    Science.gov (United States)

    Knudson, W E; Cerniglia, M W; Carro, A

    1998-06-01

    Many procedures performed by podiatric surgeons today require the use of a soft-tissue anchoring device. In recent years, many new anchoring devices have become available for use in the foot and ankle. The authors introduce a new soft-tissue anchoring device that has yet to be described in the podiatric literature and present two cases in which the new anchor was used.

  9. Response of the common cutworm Spodoptera litura to zinc stress: Zn accumulation, metallothionein and cell ultrastructure of the midgut

    Energy Technology Data Exchange (ETDEWEB)

    Shu, Yinghua [Key Laboratory of Agro-Environments in Tropics, Ministry of Agriculture, South China Agricultural University, Guangzhou 510642 (China); Key Laboratory of Agroecology and Rural Environment of Guangdong Regular Higher Education Institutions, South China Agricultural University, Guangzhou 510642 (China); Institute of Tropical and Subtropical Ecology, South China Agricultural University, Guangzhou 510642 (China); State Key Laboratory of Biological Control and Institute of Entomology, Sun Yat-sen University, Guangzhou 510275 (China); Zhang, Guren [State Key Laboratory of Biological Control and Institute of Entomology, Sun Yat-sen University, Guangzhou 510275 (China); Wang, Jianwu, E-mail: wangjw@scau.edu.cn [Key Laboratory of Agro-Environments in Tropics, Ministry of Agriculture, South China Agricultural University, Guangzhou 510642 (China); Key Laboratory of Agroecology and Rural Environment of Guangdong Regular Higher Education Institutions, South China Agricultural University, Guangzhou 510642 (China); Institute of Tropical and Subtropical Ecology, South China Agricultural University, Guangzhou 510642 (China)

    2012-11-01

    By exposing the common cutworm Spodoptera litura Fabricius larvae to a range of Zinc (Zn) stress, we investigated the effects of dietary Zn on Zn accumulation, metallothionein (MT), and on the ultrastructure of the midgut. The techniques we used were inductively coupled plasma-atomic emission spectrometer (ICP-AES), real-time PCR combined with cadmium-hemoglobin total saturation, and transmission electron microscopy (TEM), respectively. There was a significant dose-response relationship between the Zn accumulations in the midgut of the larvae and the Zn concentrations in the diet. Furthermore, both MT content and MT gene expression in the midgut were significantly induced in the 50-500 mg Zn/kg treatments, and were significantly positively correlated with the Zn accumulations in the midgut. When S. litura larvae were fed with the diet treated with 500 mg Zn/kg, Zn accumulation and MT content in the midgut was 4450.85 mg Zn/kg and 372.77 mg/kg, respectively, thereafter there was a little increase; the level of MT gene expression was maximal, thereafter there was a sharp decrease. TEM showed that numerous electron-dense granules (EDGs) and vacuoles appeared in the cytoplasm of the midgut cells, their number and size being closely correlated with the Zn accumulations in the midgut. Moreover, the nuclei were strongly influenced by Zn stress, evidenced by chromatin condensation and irregular nuclear membranes. Therefore, after being exposed to Zn in the threshold (500 mg Zn/kg) range, S. litura larvae could accumulate Zn in the midgut, which led to the induction of MT and changes in cell ultrastructure (mainly the presence of EDGs). The induction of MT and precipitation of Zn in EDGs may be the effective detoxification mechanisms by which the herbivorous insect S. litura defends itself against heavy metals. -- Graphical abstract: When the herbivorous insect Spodoptera litura Fabricius larvae were fed on the artificial diet with different concentrations of Zn, amounts of

  10. Recurrent intestinal volvulus in midgut malrotation causing acute bowel obstruction: A case report.

    Science.gov (United States)

    Sheikh, Fayed; Balarajah, Vickna; Ayantunde, Abraham Abiodun

    2013-03-27

    Intestinal malrotation occurs when there is a disruption in the normal embryological development of the bowel. The majority of patients present with clinical features in childhood, though rarely a first presentation can take place in adulthood. Recurrent bowel obstruction in patients with previous abdominal operation for midgut malrotation is mostly due to adhesions but very few reported cases have been due to recurrent volvulus. We present the case of a 22-year-old gentleman who had laparotomy in childhood for small bowel volvulus and then presented with acute bowel obstruction. Preoperative computerised tomography scan showed small bowel obstruction and features in keeping with midgut malrotation. Emergency laparotomy findings confirmed midgut malrotation with absent appendix, abnormal location of caecum, ascending colon and small bowel. In addition, there were small bowel volvulus and a segment of terminal ileal stricture. Limited right hemicolectomy was performed with excellent postoperative recovery. This case is presented to illustrate a rare occurrence and raise an awareness of the possibility of dreadful recurrent volvulus even several years following an initial Ladd's procedure for midgut malrotation. Therefore, one will need to exercise a high index of suspicion and this becomes very crucial in order to ensure prompt surgical intervention and thereby preventing an attendant bowel ischaemia with its associated high fatality.

  11. Discovery of ebselen as an inhibitor of Cryptosporidium parvum glucose-6-phosphate isomerase (CpGPI) by high-throughput screening of existing drugs.

    Science.gov (United States)

    Eltahan, Rana; Guo, Fengguang; Zhang, Haili; Xiang, Lixin; Zhu, Guan

    2018-04-01

    Cryptosporidium parvum is a water-borne and food-borne apicomplexan pathogen. It is one of the top four diarrheal-causing pathogens in children under the age of five in developing countries, and an opportunistic pathogen in immunocompromised individuals. Unlike other apicomplexans, C. parvum lacks Kreb's cycle and cytochrome-based respiration, thus relying mainly on glycolysis to produce ATP. In this study, we characterized the primary biochemical features of the C. parvum glucose-6-phosphate isomerase (CpGPI) and determined its Michaelis constant towards fructose-6-phosphate (K m  = 0.309 mM, V max  = 31.72 nmol/μg/min). We also discovered that ebselen, an organoselenium drug, was a selective inhibitor of CpGPI by high-throughput screening of 1200 known drugs. Ebselen acted on CpGPI as an allosteric noncompetitive inhibitor (IC 50  = 8.33 μM; K i  = 36.33 μM), while complete inhibition of CpGPI activity was not achieved. Ebselen could also inhibit the growth of C. parvum in vitro (EC 50  = 165 μM) at concentrations nontoxic to host cells, albeit with a relatively small in vitro safety window of 4.2 (cytotoxicity TC 50 on HCT-8 cells = 700 μM). Additionally, ebselen might also target other enzymes in the parasite, leading to the parasite growth reduction. Therefore, although ebselen is useful in studying the inhibition of CpGPI enzyme activity, further proof is needed to chemically and/or genetically validate CpGPI as a drug target. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Squamocin induce histological and ultrastructural changes in the midgut cells of Anticarsia gemmatalis (Lepidoptera: Noctuidae).

    Science.gov (United States)

    Fiaz, Muhammad; Martínez, Luis Carlos; Costa, Marilza da Silva; Cossolin, Jamile Fernanda Silva; Plata-Rueda, Angelica; Gonçalves, Wagner Gonzaga; Sant'Ana, Antônio Euzébio Goulart; Zanuncio, José Cola; Serrão, José Eduardo

    2018-07-30

    Annonaceous acetogenins (Annona squamosa Linnaeus) comprises of a series of natural products which are extracted from Annonaceae species, squamocin proved to be highly efficient among those agents. Squamocin is mostly referred as a lethal agent for midgut cells of different insects, with toxic effects when tested against larva of some insects. In present study, LC 50 and LC 90 of squamocin for A. gemmatalis Hübner (Lepidoptera: Noctuidae) were calculated using probit analysis. Morphological changes in midgut cells were analyzed under light, fluorescence and transmission electron microscopes when larvae were treated with LC 50 and LC 90 of squamocin for 24, 48 and 72 h. Results revealed that the maximum damage to midgut cells was found under LC 90 where it showed digestive cells with enlarged basal labyrinth, highly vacuolated cytoplasm, damaged apical surface, cell protrusions to the gut lumen, autophagy and cell death. The midgut goblet cells showed a strong disorganization of their microvilli. Likewise, in insects treated with squamocin, mitochondria were not marked with Mitotracker fluorescent probe, suggesting some molecular damage in these organelles, which was reinforced by decrease in the respiration rate in these insects. These results demonstrate that squamocin has potential to induce enough morphological changes in midgut through epithelial cell damage in A. gemmatalis. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. GPi oscillatory activity differentiates tics from the resting state, voluntary movements, and the unmedicated parkinsonian state

    Directory of Open Access Journals (Sweden)

    Joohi Jimenez-Shahed

    2016-09-01

    Full Text Available Background: Deep brain stimulation (DBS is an emerging treatment strategy for severe, medication-refractory Tourette syndrome (TS. Thalamic (Cm-Pf and pallidal (including globus pallidus interna, GPi targets have been the most investigated. While the neurophysiological correlates of Parkinson’s disease (PD in the GPi and subthalamic nucleus (STN are increasingly recognized, these patterns are not well characterized in other disease states. Recent findings indicate that the cross-frequency coupling (CFC between beta band and high frequency oscillations (HFOs within the STN in PD patients is pathologic. Methods: We recorded intraoperative local field potentials (LFPs from the postero-ventrolateral GPi in three adult patients with TS at rest, during voluntary movements, and during tic activity and compared them to the intraoperative GPi-LFP activity recorded from four unmedicated PD patients at rest. Results: In all PD patients, we noted excessive beta band activity (13-30Hz at rest which consistently modulated the amplitude of the co-existent HFOs observed between 200-400Hz, indicating the presence of beta-HFO CFC. In all 3 TS patients at rest, we observed theta band activity (4-7Hz and HFOs. Two patients had beta band activity, though at lower power than theta oscillations. Tic activity was associated with increased high frequency (200-400Hz and gamma band (35-200Hz activity. There was no beta-HFO CFC in TS patients at rest. However, CFC between the phase of 5-10Hz band activity and the amplitude of HFOs was found in two TS patients. During tics, this shifted to CFC between the phase of beta band activity and the amplitude of HFOs in all subjects. Conclusions: To our knowledge this is the first study that shows that beta-HFO CFC exists in the GPi of TS patients during tics and at rest in PD patients, and suggests that this pattern might be specific to pathologic/involuntary movements. Furthermore, our findings suggest that during tics, resting

  14. Microbial population analysis of the midgut of Melophagus ovinus via high-throughput sequencing

    OpenAIRE

    Duan, De-Yong; Liu, Guo-Hua; Cheng, Tian-Yin; Wang, Ya-Qin

    2017-01-01

    Background Melophagus ovinus, one of the most common haematophagous ectoparasites of sheep, can cause anaemia and reductions in weight gain, wool growth and hide value. However, no information is available about the microfloral structure of the midgut of this ectoparasite. In the present study, we investigated the microbial community structure of the midgut contents of fully engorged female and male M. ovinus using Illumina HiSeq. Results The phylum showing the highest abundance was Proteobac...

  15. Effects of STN and GPi deep brain stimulation on impulse control disorders and dopamine dysregulation syndrome.

    Directory of Open Access Journals (Sweden)

    Sarah J Moum

    Full Text Available Impulse control disorders (ICDs and dopamine dysregulation syndrome (DDS are important behavioral problems that affect a subpopulation of patients with Parkinson's disease (PD and typically result in markedly diminished quality of life for patients and their caregivers. We aimed to investigate the effects of subthalamic nucleus (STN and internal globus pallidus (GPi deep brain stimulation (DBS on ICD/DDS frequency and dopaminergic medication usage.A retrospective chart review was performed on 159 individuals who underwent unilateral or bilateral PD DBS surgery in either STN or GPi. According to published criteria, pre- and post-operative records were reviewed to categorize patients both pre- and post-operatively as having ICD, DDS, both ICD and DDS, or neither ICD nor DDS. Group differences in patient demographics, clinical presentations, levodopa equivalent dose (LED, and change in diagnosis following unilateral/bilateral by brain target (STN or GPi DBS placement were examined.28 patients met diagnostic criteria for ICD or DDS pre- or post-operatively. ICD or DDS classification did not differ by GPi or STN target stimulation. There was no change in DDS diagnosis after unilateral or bilateral stimulation. For ICD, diagnosis resolved in 2 of 7 individuals after unilateral or bilateral DBS. Post-operative development of these syndromes was significant; 17 patients developed ICD diagnoses post-operatively with 2 patients with pre-operative ICD developing DDS post-operatively.Unilateral or bilateral DBS did not significantly treat DDS or ICD in our sample, even though a few cases of ICD resolved post-operatively. Rather, our study provides preliminary evidence that DDS and ICD diagnoses may emerge following DBS surgery.

  16. Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus

    Directory of Open Access Journals (Sweden)

    Paula Rego Bittencourt-Cunha

    2013-06-01

    Full Text Available In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL are transferred to the haemolymphatic lipophorin (Lp and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs. 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80% and NL (20%. The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs.

  17. Barber Pole Sign in CT Angiography, Adult Presentation of Midgut Malrotation: A Case Report

    International Nuclear Information System (INIS)

    Garcelan-Trigo, Juan Arsenio; Tello-Moreno, Manuel; Rabaza-Espigares, Manuel Jesus; Talavera-Martinez, Ildefonso

    2015-01-01

    Adult midgut volvulus is a challenging diagnosis because of its low incidence and nonspecific symptoms. Diagnostic delay and long-term complaints are frequent in this clinical scenario. We reported a patient referred to our diagnostic imaging unit with intermittent abdominal pain, bloating and episodic vomiting for several years. He underwent barium gastrointestinal transit and abdominal ultrasound, which revealed severe gastric dilatation, food retention and slow transit until a depressed duodenojejunal flexure, with malrotation of the midgut and jejunal loops being located in the right upper quadrant. Computed tomography angiography was performed, showing rotation of the small intestine around the mesentery root, suggestive of midgut malrotation. In addition, an abnormal twisted disposition of superior mesenteric artery with corkscrew appearance was seen, shaping the pole-barber sign which was evident in volume rendering three-dimensional reconstructions. The patient underwent scheduled surgical treatment without any complication and had good outcome after hospital discharge and follow-up. Computed tomography plays an important role in evaluation of adult midgut volvulus. In addition, angiographic reconstructions can help us to assess the anatomic disposition of mesenteric vascular supply. Both of these assessments are useful in preoperative management

  18. Vascular compromise in chronic volvulus with midgut malrotation

    Energy Technology Data Exchange (ETDEWEB)

    Mori, H.; Hayashi, K.; Futugawa, S.; Uetani, M.; Kurosaki, N.; Yanagi, T.

    1987-05-01

    Three cases of chornic volvulus of the small bowel in midgut malrotation are presented, all of whom manifested similar angiographic findings: proximal occlusion of the superior mesenteric artery and vein and development of collateral vessels. These findings may indicate the pathophysiology of chronic volvulus in midgut malrotation; the volvulus is progressive and eventually results in the twisting of the mesenteric root itself, but because of its chronic nature collateral circulation develops, eliminating bowel necrosis. Computed tomography (CT), performed in two cases, revealed dilated, tortuous vessels in the mesentery in addition to the known CT finding of a whirl-like pattern of the volvulated small bowel loops. Sonography, performed in one case, showed an unique feature of whirling sonolucent layers probably representing the volvulated small bowel loops intermixed with dilated mesenteric collateral vessels. We would like to emphasize the usefulness of CT and sonography in the early diagnosis of those cases with vague and nonspecific clinical manifestations.

  19. Vascular compromise in chronic volvulus with midgut malrotation

    International Nuclear Information System (INIS)

    Mori, H.; Hayashi, K.; Futugawa, S.; Uetani, M.; Kurosaki, N.; Yanagi, T.

    1987-01-01

    Three cases of chornic volvulus of the small bowel in midgut malrotation are presented, all of whom manifested similar angiographic findings: proximal occlusion of the superior mesenteric artery and vein and development of collateral vessels. These findings may indicate the pathophysiology of chronic volvulus in midgut malrotation; the volvulus is progressive and eventually results in the twisting of the mesenteric root itself, but because of its chronic nature collateral circulation develops, eliminating bowel necrosis. Computed tomography (CT), performed in two cases, revealed dilated, tortuous vessels in the mesentery in addition to the known CT finding of a whirl-like pattern of the volvulated small bowel loops. Sonography, performed in one case, showed an unique feature of whirling sonolucent layers probably representing the volvulated small bowel loops intermixed with dilated mesenteric collateral vessels. We would like to emphasize the usefulness of CT and sonography in the early diagnosis of those cases with vague and nonspecific clinical manifestations. (orig.)

  20. Induction of anti-β2 -glycoprotein I autoantibodies in mice by protein H of Streptococcus pyogenes

    NARCIS (Netherlands)

    van O S, G. M. A.; Meijers, J. C. M.; Agar, Ç; Valls Seron, M.; Marquart, J. A.; Åkesson, P.; Urbanus, R. T.; Derksen, R. H. W. M.; Herwald, H.; Mörgelin, M.; D E Groot, P. G.

    2011-01-01

    The antiphospholipid syndrome (APS) is characterized by the persistent presence of anti-β(2) -glycoprotein I (β(2) -GPI) autoantibodies. β(2) -GPI can exist in two conformations. In plasma it is a circular protein, whereas it adopts a fish-hook conformation after binding to phospholipids. Only the

  1. Monoacylated Cellular Prion Proteins Reduce Amyloid-β-Induced Activation of Cytoplasmic Phospholipase A2 and Synapse Damage

    Directory of Open Access Journals (Sweden)

    Ewan West

    2015-06-01

    Full Text Available Alzheimer’s disease (AD is a progressive neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ and the loss of synapses. Aggregation of the cellular prion protein (PrPC by Aβ oligomers induced synapse damage in cultured neurons. PrPC is attached to membranes via a glycosylphosphatidylinositol (GPI anchor, the composition of which affects protein targeting and cell signaling. Monoacylated PrPC incorporated into neurons bound “natural Aβ”, sequestering Aβ outside lipid rafts and preventing its accumulation at synapses. The presence of monoacylated PrPC reduced the Aβ-induced activation of cytoplasmic phospholipase A2 (cPLA2 and Aβ-induced synapse damage. This protective effect was stimulus specific, as treated neurons remained sensitive to α-synuclein, a protein associated with synapse damage in Parkinson’s disease. In synaptosomes, the aggregation of PrPC by Aβ oligomers triggered the formation of a signaling complex containing the cPLA2.a process, disrupted by monoacylated PrPC. We propose that monoacylated PrPC acts as a molecular sponge, binding Aβ oligomers at the neuronal perikarya without activating cPLA2 or triggering synapse damage.

  2. Fine structure of the midgut epithelium in the millipede Telodeinopus aoutii (Myriapoda, Diplopoda) with special emphasis on epithelial regeneration.

    Science.gov (United States)

    Rost-Roszkowska, M M; Kszuk-Jendrysik, M; Marchewka, A; Poprawa, I

    2018-01-01

    The midgut of millipedes is composed of a simple epithelium that rests on a basal lamina, which is surrounded by visceral muscles and hepatic cells. As the material for our studies, we chose Telodeinopus aoutii (Demange, 1971) (Kenyan millipede) (Diplopoda, Spirostreptida), which lives in the rain forests of Central Africa. This commonly reared species is easy to obtain from local breeders and easy to culture in the laboratory. During our studies, we used transmission and scanning electron microscopes and light and fluorescent microscopes. The midgut epithelium of the species examined here shares similarities to the structure of the millipedes analyzed to date. The midgut epithelium is composed of three types of cells-digestive, secretory, and regenerative cells. Evidence of three types of secretion have been observed in the midgut epithelium: merocrine, apocrine, and microapocrine secretion. The regenerative cells of the midgut epithelium in millipedes fulfill the role of midgut stem cells because of their main functions: self-renewal (the ability to divide mitotically and to maintain in an undifferentiated state) and potency (ability to differentiate into digestive cells). We also confirmed that spot desmosomes are common intercellular junctions between the regenerative and digestive cells in millipedes.

  3. Effects of tunicamycin, mannosamine, and other inhibitors of glycoprotein processing on skeletal alkaline phosphatase in human osteoblast-like cells.

    Science.gov (United States)

    Farley, J R; Magnusson, P

    2005-01-01

    Skeletal alkaline phosphatase (sALP) is a glycoprotein- approximately 20% carbohydrate by weight, with five presumptive sites for N-linked glycosylation, as well as a carboxy-terminal site for attachment of the glycolipid structure (glycosylphosphatidylinositol, GPI), which anchors sALP to the outer surface of osteoblasts. The current studies were intended to characterize the effects of inhibiting glycosylation and glycosyl-processing on the synthesis, plasma membrane attachment, cellular-extracellular distribution, and reaction kinetics of sALP in human osteosarcoma (SaOS-2) cells. sALP synthesis, glycosylation, and GPI-anchor attachment were assessed as total protein synthesis/immunospecific sALP synthesis, sialic acid content (i.e., wheat germ agglutinin precipitation), and insolubility (i.e., temperature-dependent phase-separation), respectively. sALP reaction kinetics were characterized by analysis of dose-dependent initial velocity data, with a phosphoryl substrate. The results of these studies revealed that the inhibition of either N-linked glycosylation or oligosaccharide synthesis for GPI-anchor addition could affect the synthesis and the distribution of sALP, but not the kinetics of the phosphatase reaction. Tunicamycin-which blocks N-linked glycosylation by inhibiting core oligosaccharide synthesis-decreased cell layer protein and the total amount of sALP in the cells, while increasing the relative level of sALP in the cell-conditioned culture medium (CM, i.e., the amount of sALP released). These effects were attributed to dose- and time-dependent decreases in sALP synthesis and N-linked glycosylation, and an increase in apoptotic cell death (P sALP specific activity, in the cells and in the CM; and (3) increases in the percentages of both anchorless and wheat germ agglutinin (WGA)-soluble sALP in the medium, but not in the cells (P sALP to the outside of the plasma membrane surface. Neither mannosammine nor tunicamycin had any effect on the reaction

  4. Effects of gamma irradiation on the mid-gut of Hyphantria Cunea

    International Nuclear Information System (INIS)

    Choi, J.H.

    1980-01-01

    In this paper, the author studied the histological changes of the midgut cells of fall webworms (Hyphantria cunea Drury) through 1.75-7 krad of the whole body gamma irradiation according to their metamorphosis by comparing the control group with the irradiated one through an optical microscope. Here the results were as follows: The epithelium of midgut was composed of columnar, goblet and regenerative cells. The effects of gamma irradiation were varied with the dosages and the stages during the metamorphosis. The degree of histological change mode by irradiation was increased with the dosages. Radiosensitivity was the highest in both last-stage larva and 8-day-old pupae. (Author)

  5. Characterization of the Sclerotinia sclerotiorum cell wall proteome.

    Science.gov (United States)

    Liu, Longzhou; Free, Stephen J

    2016-08-01

    We used a proteomic analysis to identify cell wall proteins released from Sclerotinia sclerotiorum hyphal and sclerotial cell walls via a trifluoromethanesulfonic acid (TFMS) digestion. Cell walls from hyphae grown in Vogel's glucose medium (a synthetic medium lacking plant materials), from hyphae grown in potato dextrose broth and from sclerotia produced on potato dextrose agar were used in the analysis. Under the conditions used, TFMS digests the glycosidic linkages in the cell walls to release intact cell wall proteins. The analysis identified 24 glycosylphosphatidylinositol (GPI)-anchored cell wall proteins and 30 non-GPI-anchored cell wall proteins. We found that the cell walls contained an array of cell wall biosynthetic enzymes similar to those found in the cell walls of other fungi. When comparing the proteins in hyphal cell walls grown in potato dextrose broth with those in hyphal cell walls grown in the absence of plant material, it was found that a core group of cell wall biosynthetic proteins and some proteins associated with pathogenicity (secreted cellulases, pectin lyases, glucosidases and proteases) were expressed in both types of hyphae. The hyphae grown in potato dextrose broth contained a number of additional proteins (laccases, oxalate decarboxylase, peroxidase, polysaccharide deacetylase and several proteins unique to Sclerotinia and Botrytis) that might facilitate growth on a plant host. A comparison of the proteins in the sclerotial cell wall with the proteins in the hyphal cell wall demonstrated that sclerotia formation is not marked by a major shift in the composition of cell wall protein. We found that the S. sclerotiorum cell walls contained 11 cell wall proteins that were encoded only in Sclerotinia and Botrytis genomes. © 2015 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.

  6. Fine-structural changes in the midgut of old Drosophila melanogaster

    Science.gov (United States)

    Anton-Erxleben, F.; Miquel, J.; Philpott, D. E.

    1983-01-01

    Senescent fine-structural changes in the midgut of Drosophila melanogaster are investigated. A large number of midgut mitochondria in old flies exhibit nodular cristae and a tubular system located perpendicular to the normal cristae orientation. Anterior intestinal cells show a senescent accumulation of age pigment, either with a surrounding two-unit membrane or without any membrane. The predominant localization of enlarged mitochondria and pigment in the luminal gut region may be related to the polarized metabolism of the intestinal cells. Findings concur with previous observations of dense-body accumulations and support the theory that mitochondria are involved in the aging of fixed post-mitotic cells. Demonstrated by statistical analyses is that mitochondrial size increase is related to mitochondrial variation increase.

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

    International Nuclear Information System (INIS)

    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. - Highlights: • Structures of pestivirus E2 proteins impose constraints on E1, E2 membrane anchors. • Atomic models of the E1 and E2 membrane anchors were generated in silico. • A “snorkeling” arginine completes the short helical hairpin in the E2 membrane anchor. • Roles in pH sensing and E1–E2 disulfide bond formation are proposed for E1 residues. • Implications for E1 ectodomain structure and disulfide bonding pattern are discussed

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

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jimin, E-mail: jimin.wang@yale.edu; Li, Yue; Modis, Yorgo, E-mail: yorgo.modis@yale.edu

    2014-04-15

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

  9. Enhanced CT perfusion cut-off sign in midgut volvulus

    International Nuclear Information System (INIS)

    Henesch, Stephen M.; Jaramillo, Diego M.; Nance, Michael L.

    2006-01-01

    We present a case of malrotation with midgut volvulus in an infant in which we discovered a unique CT sign called the perfusion cut-off sign. We hope this case will help establish this crucial diagnosis in other cases. (orig.)

  10. Enhanced CT perfusion cut-off sign in midgut volvulus

    Energy Technology Data Exchange (ETDEWEB)

    Henesch, Stephen M.; Jaramillo, Diego M. [Children' s Hospital of Philadelphia, Department of Radiology, Philadelphia, PA (United States); Nance, Michael L. [Children' s Hospital of Philadelphia, Department of Surgery, Philadelphia, PA (United States)

    2006-04-15

    We present a case of malrotation with midgut volvulus in an infant in which we discovered a unique CT sign called the perfusion cut-off sign. We hope this case will help establish this crucial diagnosis in other cases. (orig.)

  11. Diaphorina citri Nymphs Are Resistant to Morphological Changes Induced by "Candidatus Liberibacter asiaticus" in Midgut Epithelial Cells.

    Science.gov (United States)

    Mann, Marina; Fattah-Hosseini, Somayeh; Ammar, El-Desouky; Stange, Richard; Warrick, EricaRose; Sturgeon, Kasie; Shatters, Robert; Heck, Michelle

    2018-04-01

    " Candidatus Liberibacter asiaticus" is the causative bacterium associated with citrus greening disease. " Ca Liberibacter asiaticus" is transmitted by Diaphorina citri more efficiently when it is acquired by nymphs rather than adults. Why this occurs is not known. We compared midguts of D. citri insects reared on healthy or " Ca Liberibacter asiaticus"-infected citrus trees using quantitative PCR, confocal microscopy, and mitochondrial superoxide staining for evidence of oxidative stress. Consistent with its classification as propagative, " Ca Liberibacter asiaticus" titers were higher in adults than in nymphs. Our previous work showed that adult D. citri insects have basal levels of karyorrhexis (fragmentation of the nucleus) in midgut epithelial cells, which is increased in severity and frequency in response to " Ca Liberibacter asiaticus." Here, we show that nymphs exhibit lower levels of early-stage karyorrhexis than adults and are refractory to the induction of advanced karyorrhexis by " Ca Liberibacter asiaticus" in the midgut epithelium. MitoSox Red staining showed that guts of infected adults, particularly males, experienced oxidative stress in response to " Ca Liberibacter asiaticus." A positive correlation between the titers of " Ca Liberibacter asiaticus" and the Wolbachia endosymbiont was observed in adult and nymph midguts, suggesting an interplay between these bacteria during development. We hypothesize that the resistance of the nymph midgut to late-stage karyorrhexis through as yet unknown molecular mechanisms benefits " Ca Liberibacter asiaticus" for efficient invasion of midgut epithelial cells, which may be a factor explaining the developmental dependency of " Ca Liberibacter asiaticus" acquisition by the vector.

  12. Arabidopsis COBRA-LIKE 10, a GPI-anchored protein, mediates directional growth of pollen tubes

    NARCIS (Netherlands)

    Li, S.; Ge, F.R.; Xu, M.; Zhao, X.Y.; Huang, G.Q.; Zhou, L.Z.; Wang, J.G.; Kombrink, A.; McCormick, S.; Zhang, X.S.; Zhang, Y.

    2013-01-01

    Successful reproduction of flowering plants requires constant communication between female tissues and growing pollen tubes. Female cells secrete molecules and peptides as nutrients or guidance cues for fast and directional tube growth, which is executed by dynamic changes of intracellular

  13. Ultrastructure of the midgut endocrine cells in Melipona quadrifasciata anthidioides (Hymenoptera, Apidae

    Directory of Open Access Journals (Sweden)

    C. A. Neves

    Full Text Available In this study we describe the ultrastructure of the endocrine cells observed in the midgut of M. quadrifasciata anthidioides. This bee has two types of endocrine cells, which are numerous on the posterior midgut region. Cells of the closed type are smaller and have irregular secretory granules with lower electrondensity than those of the open cell type. The open cell type has elongated mitochondria mainly on the basal area, where most of the secretory granules are also found. Besides the secretion granules and mitochondria, endocrine cells in this species have well-developed autophagic vacuoles and Golgi complex elements.

  14. Effect of Gamma-Irradiation on the Midgut Epithelial Cells of Female Potato Tuber Moth, Phthorimaea operculella, Zeller

    International Nuclear Information System (INIS)

    Haiba, I.M.; Abd El-Meguid, A.A.; Yousef, H.A.A.; Mahmoud, E.A.

    2008-01-01

    The present work deals with the histological studies of the midgut in non- irradiated 4-day-old potato tuber moth, Phthorimaea operculella and after the exposure of 5-day-old pupae to sub sterilizing and sterilizing doses 50 and 150 Gy, respectively. Anatomically, the midgut was a wide straight tube, the proximal part was marked by the connection to the crop and the distal part was determined by the connection of the Malpighian tubules. The arrangement of the Malpighian tubules around the midgut tube took a special pattern symmetrically on each side. Histologically, the midgut was subdivided into three main subdivisions, anterior, middle and posterior regions. The epithelial cells differed in shape and size according to the regions in the midgut. Exposure to gamma-irradiation showed various forms of changes, there was direct relationship between the dose levels and the observed effects. At dose level 150 Gy, the effects were more advanced than those at 50 Gy dose level. The effects of radiation could be summarized as: appearance of vacuoles and vesicles in the cytoplasm, nuclei in some cells were pyknotic or karyolysed, reduction in the cell number, enlargement in nuclei, absence of regenerative cells from some parts and the epithelial cells suffered from hydropic degeneration

  15. Serine protease from midgut of Bombus terrestris males

    Czech Academy of Sciences Publication Activity Database

    Brabcová, Jana; Kindl, Jiří; Valterová, Irena; Pichová, Iva; Zarevúcka, Marie; Brabcová, J.; Jágr, Michal; Mikšík, Ivan

    2013-01-01

    Roč. 82, č. 3 (2013), s. 117-128 ISSN 0739-4462 R&D Projects: GA ČR GA203/09/1446; GA TA ČR TA01020969 Institutional support: RVO:61388963 ; RVO:67985823 Keywords : Bombus terrestris * midgut * serine protease * bumblebee Subject RIV: CE - Biochemistry; CE - Biochemistry (FGU-C) Impact factor: 1.160, year: 2013

  16. Molecular characterization of calreticulin from Anopheles stephensi midgut cells and functional assay of the recombinant calreticulin with Plasmodium berghei ookinetes.

    Science.gov (United States)

    Borhani Dizaji, Nahid; Basseri, Hamid Reza; Naddaf, Saied Reza; Heidari, Mansour

    2014-10-25

    Transmission blocking vaccines (TBVs) that target the antigens on the midgut epithelium of Anopheles mosquitoes are among the promising tools for the elimination of the malaria parasite. Characterization and analysis of effective antigens is the first step to design TBVs. Calreticulin (CRT), a lectin-like protein, from Anopheles albimanus midgut, has shown antigenic features, suggesting a promising and novel TBV target. CRT is a highly conserved protein with similar features in vertebrates and invertebrates including anopheline. We cloned the full-length crt gene from malaria vector, Anopheles stephensi (AsCrt) and explored the interaction of recombinant AsCrt protein, expressed in a prokaryotic system (pGEX-6p-1), with surface proteins of Plasmodium berghei ookinetes by immunofluorescence assay. The cellular localization of AsCrt was determined using the baculovirus expression system. Sequence analysis of the whole cDNA of AsCrt revealed that AsCrt contains an ORF of 1221 bp. The amino acid sequence of AsCrt protein obtained in this study showed 64% homology with similar protein in human. The AsCrt shares the most common features of CRTs from other species. This gene encodes a 406 amino-acid protein with a molecular mass of 46 kDa, which contains a predicted 16 amino-acid signal peptides, conserved cysteine residues, a proline-rich region, and highly acidic C-terminal domain with endoplasmic reticulum retrieval sequence HDEL. The production of GST-AsCrt recombinant protein was confirmed by Western blot analysis using an antibody against the GST protein. The FITC-labeled GST-AsCrt exhibited a significant interaction with P. berghei ookinete surface proteins. Purified recombinant GST-AsCrt, labeled with FITC, displayed specific binding to the surface of P. berghei ookinetes in comparison with control. Moreover, the expression of AsCrt in baculovirus expression system indicated that AsCrt was localized on the surface of Sf9 cells. Our results suggest that AsCrt could

  17. Anchored PKA as a gatekeeper for gap junctions.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  19. Long SAGE analysis of genes differentially expressed in the midgut ...

    African Journals Online (AJOL)

    Long SAGE analysis of genes differentially expressed in the midgut and silk gland between the sexes of the silkwormBombyx mori. Liping Gan, Ying Wang, Jian Xi, Yanshan Niu, Hongyou Qin, Yanghu Sima, Shiqing Xu ...

  20. Distinct pathways mediate the sorting of tail-anchored proteins to the plastid outer envelope.

    Directory of Open Access Journals (Sweden)

    Preetinder K Dhanoa

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

  1. The cell wall-localized atypical β-1,3 glucanase ZERZAUST controls tissue morphogenesis in Arabidopsis thaliana.

    Science.gov (United States)

    Vaddepalli, Prasad; Fulton, Lynette; Wieland, Jennifer; Wassmer, Katrin; Schaeffer, Milena; Ranf, Stefanie; Schneitz, Kay

    2017-06-15

    Orchestration of cellular behavior in plant organogenesis requires integration of intercellular communication and cell wall dynamics. The underlying signaling mechanisms are poorly understood. Tissue morphogenesis in Arabidopsis depends on the receptor-like kinase STRUBBELIG. Mutations in ZERZAUST were previously shown to result in a strubbelig -like mutant phenotype. Here, we report on the molecular identification and functional characterization of ZERZAUST We show that ZERZAUST encodes a putative GPI-anchored β-1,3 glucanase suggested to degrade the cell wall polymer callose. However, a combination of in vitro , cell biological and genetic experiments indicate that ZERZAUST is not involved in the regulation of callose accumulation. Nonetheless, Fourier-transformed infrared-spectroscopy revealed that zerzaust mutants show defects in cell wall composition. Furthermore, the results indicate that ZERZAUST represents a mobile apoplastic protein, and that its carbohydrate-binding module family 43 domain is required for proper subcellular localization and function whereas its GPI anchor is dispensable. Our collective data reveal that the atypical β-1,3 glucanase ZERZAUST acts in a non-cell-autonomous manner and is required for cell wall organization during tissue morphogenesis. © 2017. Published by The Company of Biologists Ltd.

  2. High quality RNA isolation from Aedes aegypti midguts using laser microdissection microscopy

    Directory of Open Access Journals (Sweden)

    Gobert Geoffrey N

    2011-05-01

    Full Text Available Abstract Background Laser microdissection microscopy (LMM has potential as a research tool because it allows precise excision of target tissues or cells from a complex biological specimen, and facilitates tissue-specific sample preparation. However, this method has not been used in mosquito vectors to date. To this end, we have developed an LMM method to isolate midgut RNA using Aedes aegypti. Results Total RNA was isolated from Ae. aegypti midguts that were either fresh-frozen or fixed with histological fixatives. Generally, fresh-frozen tissue sections are a common source of quality LMM-derived RNA; however, our aim was to develop an LMM protocol that could inactivate pathogenic viruses by fixation, while simultaneously preserving RNA from arbovirus-infected mosquitoes. Three groups (10 - 15 mosquitoes per group of female Ae. aegypti at 24 or 48-hours post-blood meal were intrathoracically injected with one of seven common fixatives (Bouin's, Carnoy's, Formoy's, Cal-Rite, 4% formalin, 10% neutral buffered formalin, or zinc formalin to evaluate their effect on RNA quality. Total RNA was isolated from the fixed abdomens using a Trizol® method. The results indicated that RNA from Carnoy's and Bouin's fixative samples was comparable to that of fresh frozen midguts (control in duplicate experiments. When Carnoy's and Bouin's were used to fix the midguts for the LMM procedure, however, Carnoy's-fixed RNA clearly showed much less degradation than Bouin's-fixed RNA. In addition, a sample of 5 randomly chosen transcripts were amplified more efficiently using the Carnoy's treated LMM RNA than Bouin's-fixed RNA in quantitative real-time PCR (qRT-PCR assays, suggesting there were more intact target mRNAs in the Carnoy's fixed RNA. The yields of total RNA ranged from 0.3 to 19.0 ng per ~3.0 × 106 μm2 in the LMM procedure. Conclusions Carnoy's fixative was found to be highly compatible with LMM, producing high quality RNA from Ae. aegypti midguts while

  3. Blood meal-derived heme decreases ROS levels in the midgut of Aedes aegypti and allows proliferation of intestinal microbiota.

    Directory of Open Access Journals (Sweden)

    Jose Henrique M Oliveira

    2011-03-01

    Full Text Available The presence of bacteria in the midgut of mosquitoes antagonizes infectious agents, such as Dengue and Plasmodium, acting as a negative factor in the vectorial competence of the mosquito. Therefore, knowledge of the molecular mechanisms involved in the control of midgut microbiota could help in the development of new tools to reduce transmission. We hypothesized that toxic reactive oxygen species (ROS generated by epithelial cells control bacterial growth in the midgut of Aedes aegypti, the vector of Yellow fever and Dengue viruses. We show that ROS are continuously present in the midgut of sugar-fed (SF mosquitoes and a blood-meal immediately decreased ROS through a mechanism involving heme-mediated activation of PKC. This event occurred in parallel with an expansion of gut bacteria. Treatment of sugar-fed mosquitoes with increased concentrations of heme led to a dose dependent decrease in ROS levels and a consequent increase in midgut endogenous bacteria. In addition, gene silencing of dual oxidase (Duox reduced ROS levels and also increased gut flora. Using a model of bacterial oral infection in the gut, we show that the absence of ROS resulted in decreased mosquito resistance to infection, increased midgut epithelial damage, transcriptional modulation of immune-related genes and mortality. As heme is a pro-oxidant molecule released in large amounts upon hemoglobin degradation, oxidative killing of bacteria in the gut would represent a burden to the insect, thereby creating an extra oxidative challenge to the mosquito. We propose that a controlled decrease in ROS levels in the midgut of Aedes aegypti is an adaptation to compensate for the ingestion of heme.

  4. Major surface glycoproteins of insect forms of Trypanosoma brucei are not essential for cyclical transmission by tsetse.

    Directory of Open Access Journals (Sweden)

    Erik Vassella

    Full Text Available Procyclic forms of Trypanosoma brucei reside in the midgut of tsetse flies where they are covered by several million copies of glycosylphosphatidylinositol-anchored proteins known as procyclins. It has been proposed that procyclins protect parasites against proteases and/or participate in tropism, directing them from the midgut to the salivary glands. There are four different procyclin genes, each subject to elaborate levels of regulation. To determine if procyclins are essential for survival and transmission of T. brucei, all four genes were deleted and parasite fitness was compared in vitro and in vivo. When co-cultured in vitro, the null mutant and wild type trypanosomes (tagged with cyan fluorescent protein maintained a near-constant equilibrium. In contrast, when flies were infected with the same mixture, the null mutant was rapidly overgrown in the midgut, reflecting a reduction in fitness in vivo. Although the null mutant is patently defective in competition with procyclin-positive parasites, on its own it can complete the life cycle and generate infectious metacyclic forms. The procyclic form of T. brucei thus differs strikingly from the bloodstream form, which does not tolerate any perturbation of its variant surface glycoprotein coat, and from other parasites such as Plasmodium berghei, which requires the circumsporozoite protein for successful transmission to a new host.

  5. Anchor Bolt Position in Base Plate In Terms Of T and J Anchor Bolt

    Directory of Open Access Journals (Sweden)

    b Osman Mohamad Hairi

    2017-01-01

    Full Text Available Generally, L anchor bolt system has been used for a long period of time in construction industry as one of the distributing load structures. However, there are some weaknesses in L anchor bolt which may straighten and pullup when charged with tensile load. Current practices prefer to use other types of anchor bolt systems, such as headed studs anchor bolt system to replace the L anchor bolt design. There has been lack of studies to prove that it is more effective in terms of performance. A new T anchor bolt which was basically modified from headed studs anchor bolt was proposed in this study to compare its performance of tensile loading in concrete failure to typical L design. This study aims to determine whether the T anchor bolt system gives better performance as compared to an L anchor bolt system. The performance was rated based on tensile loading on concrete failure pattern. A pullout test was conducted on two different anchor bolt systems, namely L and T. The anchor bolt embedded depth, h in concrete were varied according to their hook or bend radius. Each sample was repeated twice. There were totally eight samples. The hook or bend radius used were 50 mm and 57.5 mm for sample L1 and L2, respectively. 90-degree bend were used on sample T1 and T2. Based on test results, it can be seen that the performance of concrete failure pattern under tensile load on both L and T anchor bolt design samples with 200 mm embedment depth was better than deeper embedment depth of 230 mm. But the L anchor bolt design gives the best results as compared to T design. Although T anchor bolt design shows higher resistance before first bond failure to the concrete sample. T anchor bolt was analysed and needed deeper embedment depth to allow formation of cone pull-out shape to acquire better performance.

  6. The ultrastructure of the midgut epithelium in millipedes (Myriapoda, Diplopoda)

    Czech Academy of Sciences Publication Activity Database

    Sosinka, A.; Rost-Roszkowska, M.M.; Vilímová, J.; Tajovský, Karel; Kszuk-Jendrysik, M.; Chajec, Ł.; Sonakowska, L.; Kamińska, K.; Hyra, M.; Poprawa, I.

    2014-01-01

    Roč. 43, č. 5 (2014), s. 477-492 ISSN 1467-8039 Institutional support: RVO:60077344 Keywords : digestive cells * midgut epithelium * millipedes * regenerative cells * secretory cells * ultrastructure Subject RIV: EG - Zoology Impact factor: 1.650, year: 2014

  7. Citrobacter amalonaticus phytase on the cell surface of Pichia pastoris exhibits high pH stability as a promising potential feed supplement.

    Science.gov (United States)

    Li, Cheng; Lin, Ying; Huang, Yuanyuan; Liu, Xiaoxiao; Liang, Shuli

    2014-01-01

    Phytase expressed and anchored on the cell surface of Pichia pastoris avoids the expensive and time-consuming steps of protein purification and separation. Furthermore, yeast cells with anchored phytase can be used as a whole-cell biocatalyst. In this study, the phytase gene of Citrobacter amalonaticus was fused with the Pichia pastoris glycosylphosphatidylinositol (GPI)-anchored glycoprotein homologue GCW61. Phytase exposed on the cell surface exhibits a high activity of 6413.5 U/g, with an optimal temperature of 60°C. In contrast to secreted phytase, which has an optimal pH of 5.0, phytase presented on the cell surface is characterized by an optimal pH of 3.0. Moreover, our data demonstrate that phytase anchored on the cell surface exhibits higher pH stability than its secreted counterpart. Interestingly, our in vitro digestion experiments demonstrate that phytase attached to the cell surface is a more efficient enzyme than secreted phytase.

  8. Effects of gamma irradiation on the midgut ultrastructure of Glossina palpalis subspecies

    International Nuclear Information System (INIS)

    Stiles, J.K.; Molyneux, D.H.; Wallbanks, K.R.; Van der Vloedt, A.M.

    1989-01-01

    In the sterile insect technique, insects are sterilized prior to release in areas where they are pests. The sterile males compete for and with fertile wild individuals for mates, thus reducing the population's reproductive rate. Tsetse fly (Glossina spp.) populations have been eradicated after release of laboratory-bred flies sterilized by gamma irradiation. However, no studies exist on radiation-induced damage to the midgut morphology and function of the radiation-sterilized insects. After G. palpalis palpalis and G. p. gambiensis were subjected to 130 Gy gamma radiation, their midgut damage and recovery were monitored by electron microscopy. The first sign of damage was atrophy and loss of the microvillous border from epithelial cells. The rate of cell degeneration increased, with young as well as old cells being affected and cellular debris filling the ectoperitrophic space. Muscle cells were destroyed, patches of basal lamina were left bare, intracellular virus- and rickettsia-like organisms became more frequent, and many replacement cells became unusually large. Partial recovery occurred from the 10th day postirradiation. Such changes in midgut ultrastructure and the corresponding inhibition of functions may increase the susceptibility of the fly to trypanosome infection

  9. Contribution of midgut bacteria to blood digestion and egg production in aedes aegypti (diptera: culicidae (L.

    Directory of Open Access Journals (Sweden)

    Pimenta Paulo FP

    2011-06-01

    Full Text Available Abstract Background The insect gut harbors a variety of microorganisms that probably exceed the number of cells in insects themselves. These microorganisms can live and multiply in the insect, contributing to digestion, nutrition, and development of their host. Recent studies have shown that midgut bacteria appear to strengthen the mosquito's immune system and indirectly enhance protection from invading pathogens. Nevertheless, the physiological significance of these bacteria for mosquitoes has not been established to date. In this study, oral administration of antibiotics was employed in order to examine the contribution of gut bacteria to blood digestion and fecundity in Aedes aegypti. Results The antibiotics carbenicillin, tetracycline, spectinomycin, gentamycin and kanamycin, were individually offered to female mosquitoes. Treatment of female mosquitoes with antibiotics affected the lysis of red blood cells (RBCs, retarded the digestion of blood proteins and reduced egg production. In addition, antibiotics did not affect the survival of mosquitoes. Mosquito fertility was restored in the second gonotrophic cycle after suspension of the antibiotic treatment, showing that the negative effects of antibiotics in blood digestion and egg production in the first gonotrophic cycle were reversible. Conclusions The reduction of bacteria affected RBC lysis, subsequently retarded protein digestion, deprived mosquito from essential nutrients and, finally, oocyte maturation was affected, resulting in the production of fewer viable eggs. These results indicate that Ae. aegypti and its midgut bacteria work in synergism to digest a blood meal. Our findings open new possibilities to investigate Ae. aegypti-associated bacteria as targets for mosquito control strategies.

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

    Science.gov (United States)

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

    2015-09-15

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

  11. Superior mesenteric vein rotation: a CT sign of midgut malrotation

    International Nuclear Information System (INIS)

    Nichols, D.M.; Li, D.K.

    1983-01-01

    Computed tomography (CT) of the pancreas, with its excellent display of peripancreatic anatomy, allows visualization of the major vessels entering the mesenteric root. In scans of the normal upper abdomen obtained at or just below the level of the uncinate process of the pancreas, the proximal superior mesenteric vein (SMV) easily can be identified lying on the right ventral aspect of the superior mesenteric artery (SMA). The authors have observed a characteristic abnormality in this normal vascular arrangement on CT scans of the pancreas in three adult patients with suspected chronic pancreatitis who were subsequently proved to have midgut malrotation. They called this the SMV rotation sign and believe that its detection even on CT scans limited to the level of the pancreas should alert the radiologist to the presence of a midgut malrotation that may have been unsuspected

  12. A Proteomics Investigation of Anchored PKA-RI Signaling

    NARCIS (Netherlands)

    Kovanich, D.

    2013-01-01

    Compartmentalization of kinases and phosphatases plays an important role in the specificity of second messenger mediated signaling events. Localization of the cAMP-dependent protein kinase is mediated by interaction of its regulatory subunit (PKA-R) with the versatile family of A-kinase anchoring

  13. Imidacloprid impairs the post-embryonic development of the midgut in the yellow fever mosquito Stegomyia aegypti (=Aedes aegypti).

    Science.gov (United States)

    Fernandes, K M; Gonzaga, W G; Pascini, T V; Miranda, F R; Tomé, H V V; Serrão, J E; Martins, G F

    2015-09-01

    The mosquito Stegomyia aegypti (=Aedes aegypti) (Diptera: Culicidae) is a vector for the dengue and yellow fever viruses. As blood digestion occurs in the midgut, this organ constitutes the route of entry of many pathogens. The effects of the insecticide imidacloprid on the survival of St. aegypti were investigated and the sub-lethal effects of the insecticide on midgut development were determined. Third instar larvae were exposed to different concentrations of imidacloprid (0.15, 1.5, 3.0, 6.0 and 15.0 p.p.m.) and survival was monitored every 24 h for 10 days. Midguts from imidacloprid-treated insects at different stages of development were dissected and processed for analyses by transmission electron microscopy, immunofluorescence microscopy and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assays. Imidacloprid concentrations of 3.0 and 15.0 p.p.m. were found to affect midgut development similarly. Digestive cells of the fourth instar larvae (L4) midgut exposed to imidacloprid had more multilamellar bodies, abundantly found in the cell apex, and more electron-lucent vacuoles in the basal region compared with those from untreated insects. Moreover, imidacloprid interfered with the differentiation of regenerative cells, dramatically reducing the number of digestive and endocrine cells and leading to malformation of the midgut epithelium in adults. The data demonstrate that imidacloprid can reduce the survival of mosquitoes and thus indicate its potentially high efficacy in the control of St. aegypti populations. © 2015 The Royal Entomological Society.

  14. A Vehicle Haptic Steering by Wire System Based on High Gain GPI Observers

    Directory of Open Access Journals (Sweden)

    A. Rodriguez-Angeles

    2014-01-01

    Full Text Available A vehicle steering by wire (SBW haptic system based on high gain generalized proportional integral (GPI observers is introduced. The observers are considered for the estimation of dynamic perturbations that are present at the tire and steering wheel. To ensure efficient tracking between the commanded steering wheel angle and the tire orientation angle, the estimated perturbations are on line canceled. As to provide a haptic interface with the driver, the estimated dynamic effects at the steering rack are fed back to the steering wheel, yielding a master-slave haptic system with bilateral communication. For implementation purposes few sensors and minimum knowledge of the dynamic model are required, which is a major advantage compared to other approaches. Only position tracking errors are fed back, while all other signals are estimated by the high gain GPI observers. The scheme is robust to uncertainty on the input gain and cancels dynamic perturbation effects such as friction and aligning forces on the tire. Experimental results are presented on a prototype platform.

  15. The risk of midgut volvulus in patients with abdominal wall defects: A multi-institutional study.

    Science.gov (United States)

    Fawley, Jason A; Abdelhafeez, Abdelhafeez H; Schultz, Jessica A; Ertl, Allison; Cassidy, Laura D; Peter, Shawn St; Wagner, Amy J

    2017-01-01

    The management of malrotation in patients with congenital abdominal wall defects has varied among surgeons. We were interested in investigating the risk of midgut volvulus in patients with gastroschisis and omphalocele to help determine if these patients may benefit from undergoing a Ladd procedure. A retrospective chart review was performed for all patients managed at three institutions born between 1/1/2000 and 12/31/2008 with a diagnosis of gastroschisis or omphalocele. Patient charts were reviewed through 12/31/2012 for occurrence of midgut volvulus or need for second laparotomy. Of the 414 patients identified with abdominal wall defects, 299 patients (72%) had gastroschisis, and 115 patients (28%) had omphalocele. The mean gestational age at birth was 36.1±2.3weeks, and the mean birth weight was 2.57±0.7kg. There were a total of 8 (1.9%) cases of midgut volvulus: 3 (1.0%) patients with gastroschisis compared to 5 patients (4.4%) with omphalocele (p=0.04). Patients with omphalocele have a greater risk of developing midgut volvulus, and a Ladd procedure should be considered during definitive repair to mitigate these risks. III; retrospective comparative study. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Does transgenic Cry1Ac + CpTI cotton pollen affect hypopharyngeal gland development and midgut proteolytic enzyme activity in the honey bee Apis mellifera L. (Hymenoptera, Apidae)?

    Science.gov (United States)

    Han, Peng; Niu, Chang-Ying; Biondi, Antonio; Desneux, Nicolas

    2012-11-01

    The transgenic Cry1Ac (Bt toxin) + CpTI (Cowpea Trypsin Inhibitor) cotton cultivar CCRI41 is increasingly used in China and potential side effects on the honey bee Apis mellifera L. have been documented recently. Two studies have assessed potential lethal and sublethal effects in young bees fed with CCRI41 cotton pollen but no effect was observed on learning capacities, although lower feeding activity in exposed honey bees was noted (antifeedant effect). The present study aimed at providing further insights into potential side effects of CCRI41 cotton on honey bees. Emerging honey bees were exposed to different pollen diets using no-choice feeding protocols (chronic exposure) in controlled laboratory conditions and we aimed at documenting potential mechanisms underneath the CCRI41 antifeedant effect previously reported. Activity of midgut proteolytic enzyme of young adult honey bees fed on CCRI41 cotton pollen were not significantly affected, i.e. previously observed antifeedant effect was not linked to disturbed activity of the proteolytic enzymes in bees' midgut. Hypopharyngeal gland development was assessed by quantifying total extractable proteins from the glands. Results suggested that CCRI41 cotton pollen carries no risk to hypopharyngeal gland development of young adult honey bees. In the two bioassays, honey bees exposed to 1 % soybean trypsin inhibitor were used as positive controls for both midgut proteolytic enzymes and hypopharyngeal gland proteins quantification, and bees exposed to 48 ppb (part per billion) (i.e. 48 ng g(-1)) imidacloprid were used as controls for exposure to a sublethal concentration of toxic product. The results show that the previously reported antifeedant effect of CCRI41 cotton pollen on honey bees is not linked to effects on their midgut proteolytic enzymes or on the development of their hypopharyngeal glands. The results of the study are discussed in the framework of risk assessment of transgenic crops on honey bees.

  17. Org-1 is required for the diversification of circular visceral muscle founder cells and normal midgut morphogenesis.

    Science.gov (United States)

    Schaub, Christoph; Frasch, Manfred

    2013-04-15

    The T-Box family of transcription factors plays fundamental roles in the generation of appropriate spatial and temporal gene expression profiles during cellular differentiation and organogenesis in animals. In this study we report that the Drosophila Tbx1 orthologue optomotor-blind-related-gene-1 (org-1) exerts a pivotal function in the diversification of circular visceral muscle founder cell identities in Drosophila. In embryos mutant for org-1, the specification of the midgut musculature per se is not affected, but the differentiating midgut fails to form the anterior and central midgut constrictions and lacks the gastric caeca. We demonstrate that this phenotype results from the nearly complete loss of the founder cell specific expression domains of several genes known to regulate midgut morphogenesis, including odd-paired (opa), teashirt (tsh), Ultrabithorax (Ubx), decapentaplegic (dpp) and wingless (wg). To address the mechanisms that mediate the regulatory inputs from org-1 towards Ubx, dpp, and wg in these founder cells we genetically dissected known visceral mesoderm specific cis-regulatory-modules (CRMs) of these genes. The analyses revealed that the activities of the dpp and wg CRMs depend on org-1, the CRMs are bound by Org-1 in vivo and their T-Box binding sites are essential for their activation in the visceral muscle founder cells. We conclude that Org-1 acts within a well-defined signaling and transcriptional network of the trunk visceral mesoderm as a crucial founder cell-specific competence factor, in concert with the general visceral mesodermal factor Biniou. As such, it directly regulates several key genes involved in the establishment of morphogenetic centers along the anteroposterior axis of the visceral mesoderm, which subsequently organize the formation of midgut constrictions and gastric caeca and thereby determine the morphology of the midgut. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Differentially expressed genes in the midgut of Silkworm infected ...

    African Journals Online (AJOL)

    In this report, we employed suppression subtractive hybridization to compare differentially expressed genes in the midguts of CPV-infected and normal silkworm larvae. 36 genes and 20 novel ESTs were obtained from 2 reciprocal subtractive libraries. Three up-regulated genes (ferritin, rpL11 and alkaline nuclease) and 3 ...

  19. Morphological abnormalities and cell death in the Asian citrus psyllid (Diaphorina citri) midgut associated with Candidatus Liberibacter asiaticus.

    Science.gov (United States)

    Ghanim, Murad; Fattah-Hosseini, Somayeh; Levy, Amit; Cilia, Michelle

    2016-09-15

    Candidatus Liberibacter asiaticus (CLas) is a phloem-limited, gram-negative, fastidious bacterium that is associated with the development of citrus greening disease, also known as Huanglongbing (HLB). CLas is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri, in a circulative manner. Two major barriers to transmission within the insect are the midgut and the salivary glands. We performed a thorough microscopic analysis within the insect midgut following exposure to CLas-infected citrus trees. We observed changes in nuclear architecture, including pyknosis and karyorrhexis as well as changes to the actin cytoskeleton in CLas-exposed midgut cells. Further analyses showed that the changes are likely due to the activation of programmed cell death as assessed by Annexin V staining and DNA fragmentation assays. These results suggest that exposure to CLas-infected trees induces apoptotic responses in the psyllid midgut that should be further investigated. Understanding the adaptive significance of the apoptotic response has the potential to create new approaches for controlling HLB.

  20. Anchored LH2 complexes in 2D polarization imaging.

    Science.gov (United States)

    Tubasum, Sumera; Sakai, Shunsuke; Dewa, Takehisa; Sundström, Villy; Scheblykin, Ivan G; Nango, Mamoru; Pullerits, Tõnu

    2013-09-26

    Protein is a soft material with inherently large structural disorder. Consequently, the bulk spectroscopies of photosynthetic pigment protein complexes provide averaged information where many details are lost. Here we report spectroscopy of single light-harvesting complexes where fluorescence excitation and detection polarizations are both independently rotated. Two samples of peripheral antenna (LH2) complexes from Rhodopseudomonas acidophila were studied. In one, the complexes were embedded in polyvinyl alcohol (PVA) film; in the other, they were anchored on the glass surface and covered by the PVA film. LH2 contains two rings of pigment molecules-B800 and B850. The B800 excitation polarization properties of the two samples were found to be very similar, indicating that orientation statistics of LH2s are the same in these two very different preparations. At the same time, we found a significant difference in B850 emission polarization statistics. We conclude that the B850 band of the anchored sample is substantially more disordered. We argue that both B800 excitation and B850 emission polarization properties can be explained by the tilt of the anchored LH2s due to the spin-casting of the PVA film on top of the complexes and related shear forces. Due to the tilt, the orientation statistics of two samples become similar. Anchoring is expected to orient the LH2s so that B850 is closer to the substrate. Consequently, the tilt-related strain leads to larger deformation and disorder in B850 than in B800.

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

    Science.gov (United States)

    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.

  2. Analysis of Anchoring Mechanism of Fully Grouted Prestressed Anchor

    Directory of Open Access Journals (Sweden)

    WEN Zhi-jie

    2014-01-01

    Full Text Available Some researchers have been carried out on analysis of the influence of the full grouted prestressed anchor shape of borehole wall on its carrying capacity. Based on the self-affine fractal feature of anchor borehole wall structural plane, the relation equation among structural plane shear strength, liquid injection pressure, tensile load and structural plane fractal dimension D was built, the instability judgment criterion of anchoring bearing strata and rock structural plane was determined, the solving equations of disintegrated rock support density were derived. Based on the experimental results, the theoretical basis of support design under the disintegrated rock condition was offered.

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

    Directory of Open Access Journals (Sweden)

    Qingguo Ma

    Full Text Available 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.

  4. The netrin protein family.

    Science.gov (United States)

    Rajasekharan, Sathyanath; Kennedy, Timothy E

    2009-01-01

    The name netrin is derived from the Sanskrit Netr, meaning 'guide'. Netrins are a family of extracellular proteins that direct cell and axon migration during embryogenesis. Three secreted netrins (netrins 1, 3 and 4), and two glycosylphosphatidylinositol (GPI)-anchored membrane proteins, netrins G1 and G2, have been identified in mammals. The secreted netrins are bifunctional, acting as attractants for some cell types and repellents for others. Receptors for the secreted netrins include the Deleted in Colorectal Cancer (DCC) family, the Down's syndrome cell adhesion molecule (DSCAM), and the UNC-5 homolog family: Unc5A, B, C and D in mammals. Netrin Gs do not appear to interact with these receptors, but regulate synaptic interactions between neurons by binding to the transmembrane netrin G ligands NGL1 and 2. The chemotropic function of secreted netrins has been best characterized with regard to axon guidance during the development of the nervous system. Extending axons are tipped by a flattened, membranous structure called the growth cone. Multiple extracellular guidance cues direct axonal growth cones to their ultimate targets where synapses form. Such cues can be locally derived (short-range), or can be secreted diffusible cues that allow target cells to signal axons from a distance (long-range). The secreted netrins function as short-range and long-range guidance cues in different circumstances. In addition to directing cell migration, functional roles for netrins have been identified in the regulation of cell adhesion, the maturation of cell morphology, cell survival and tumorigenesis.

  5. Susceptibility to anchoring effects

    Directory of Open Access Journals (Sweden)

    Todd McElroy

    2007-02-01

    Full Text Available Previous research on anchoring has shown this heuristic to be a very robust psychological phenomenon ubiquitous across many domains of human judgment and decision-making. Despite the prevalence of anchoring effects, researchers have only recently begun to investigate the underlying factors responsible for how and in what ways a person is susceptible to them. This paper examines how one such factor, the Big-Five personality trait of openness-to-experience, influences the effect of previously presented anchors on participants' judgments. Our findings indicate that participants high in openness-to-experience were significantly more influenced by anchoring cues relative to participants low in this trait. These findings were consistent across two different types of anchoring tasks providing convergent evidence for our hypothesis.

  6. Apoptosis and necrosis during the circadian cycle in the centipede midgut

    Czech Academy of Sciences Publication Activity Database

    Rost-Roszkowska, M.M.; Chajec, Ł.; Vilímová, J.; Tajovský, Karel

    2016-01-01

    Roč. 253, č. 4 (2016), s. 1051-1061 ISSN 0033-183X Institutional support: RVO:60077344 Keywords : cell death * centipede * digestive cells * midgut epithelium * ultrastructure Subject RIV: EG - Zoology Impact factor: 2.870, year: 2016

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Daniel Aberle

    Full Text Available 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.

  9. Impacts on silkworm larvae midgut proteomics by transgenic Trichoderma strain and analysis of glutathione S-transferase sigma 2 gene essential for anti-stress response of silkworm larvae.

    Science.gov (United States)

    Li, Yingying; Dou, Kai; Gao, Shigang; Sun, Jianan; Wang, Meng; Fu, Kehe; Yu, Chuanjin; Wu, Qiong; Li, Yaqian; Chen, Jie

    2015-08-03

    Lepidoptera is a large order of insects that have major impacts on humans as agriculture pests. The midgut is considered an important target for insect control. In the present study, 10 up-regulated, 18 down-regulated, and one newly emerged protein were identified in the transgenic Trichoderma-treated midgut proteome. Proteins related to stress response, biosynthetic process, and metabolism process were further characterized through quantitative real-time PCR (qPCR). Of all the identified proteins, the glutathione S-transferase sigma 2 (GSTs2) gene displayed enhanced expression when larvae were fed with Trichoderma wild-type or transgenic strains. Down regulation of GSTs2 expression by RNA interference (RNAi) resulted in inhibition of silkworm growth when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. Weight per larva decreased by 18.2%, 11.9%, and 10.7% in the untreated control, ddH2O, and GFP dsRNA groups, respectively, at 24h, while the weight decrease was higher at 42.4%, 28.8% and 32.4% at 72 h after treatment. Expression of glutathione S-transferase omega 2 (GSTo2) was also enhanced when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. These results indicated that there was indeed correlation between enhanced expression of GSTs2 and the anti-stress response of silkworm larvae against Trichoderma. This study represents the first attempt at understanding the effects of transgenic organisms on the midgut proteomic changes in silkworm larvae. Our findings could not only broaden the biological control targets of insect at the molecular level, but also provide a theoretical foundation for biological safety evaluation of the transgenic Trichoderma strain. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Comparison of Suture-Based Anchors and Traditional Bioabsorbable Anchors in Foot and Ankle Surgery.

    Science.gov (United States)

    Hembree, W Chad; Tsai, Michael A; Parks, Brent G; Miller, Stuart D

    We compared the pullout strength of a suture-based anchor versus a bioabsorbable anchor in the distal fibula and calcaneus and evaluated the relationship between bone mineral density and peak load to failure. Eight paired cadaveric specimens underwent a modified Broström procedure and Achilles tendon reattachment. The fibula and calcaneus in the paired specimens received either a suture-based anchor or a bioabsorbable suture anchor. The fibular and calcaneal specimens were loaded to failure, defined as a substantial decrease in the applied load or pullout from the bone. In the fibula, the peak load to failure was significantly greater with the suture-based versus the bioabsorbable anchors (133.3 ± 41.8 N versus 76.8 ± 35.3 N; p = .002). No significant difference in load with 5 mm of displacement was found between the 2 groups. In the calcaneus, no difference in the peak load to failure was found between the 2 groups, and the peak load to failure with 5 mm of displacement was significantly lower with the suture-based than with the bioabsorbable anchors (52.2 ± 9.8 N versus 75.9 ± 12.4 N; p = .003). Bone mineral density and peak load to failure were significantly correlated in the fibula with the suture-based anchor. An innovative suture-based anchor had a greater peak load to failure compared with a bioabsorbable anchor in the fibula. In the calcaneus, the load at 5 mm of displacement was significantly lower in the suture-based than in the bioabsorbable group. The correlation findings might indicate the need for a cortical bone shelf with the suture-based anchor. Suture-based anchors could be a viable alternative to bioabsorbable anchors for certain foot and ankle procedures. Copyright © 2016 American College of Foot and Ankle Surgeons. Published by Elsevier Inc. All rights reserved.

  11. Distribution of Glycan Motifs at the Surface of Midgut Cells in the Cotton Leafworm (Spodoptera littoralis Demonstrated by Lectin Binding

    Directory of Open Access Journals (Sweden)

    Tomasz Walski

    2017-12-01

    Full Text Available Glycans are involved in many biological phenomena, including signal transduction, cell adhesion, immune response or differentiation. Although a few papers have reported on the role of glycans in the development and proper functioning of the insect midgut, no data are available regarding the localization of the glycan structures on the surface of the cells in the gut of insects. In this paper, we analyzed the spatial distribution of glycans present on the surface of the midgut cells in larvae of the cotton leafworm Spodoptera littoralis, an important agricultural pest insect worldwide. For this purpose, we established primary midgut cell cultures, probed these individual cells that are freely suspended in liquid medium with a selection of seven fluorescently labeled lectins covering a range of different carbohydrate binding specificities [mannose oligomers (GNA and HHA, GalNAc/Gal (RSA and SSA, GlcNAc (WGA and Nictaba and Neu5Ac(α-2,6Gal/GalNAc (SNA-I], and visualized the interaction of these lectins with the different zones of the midgut cells using confocal microscopy. Our analysis focused on the typical differentiated columnar cells with a microvillar brush border at their apical side, which are dominantly present in the Lepidopteran midgut and function in food digestion and absorption, and as well as on the undifferentiated stem cells that are important for midgut development and repair. Confocal microscopy analyses showed that the GalNAc/Gal-binding lectins SSA and RSA and the terminal GlcNAc-recognizing WGA bound preferentially to the apical microvillar zone of the differentiated columnar cells as compared to the basolateral pole. The reverse result was observed for the mannose-binding lectins GNA and HHA, as well as Nictaba that binds preferentially to GlcNAc oligomers. Furthermore, differences in lectin binding to the basal and lateral zones of the cell membranes of the columnar cells were apparent. In the midgut stem cells, GNA and

  12. Membrane Curvature and Lipid Composition Synergize To Regulate N-Ras Anchor Recruitment

    DEFF Research Database (Denmark)

    Larsen, Jannik B.; Kennard, Celeste; Pedersen, Søren L.

    2017-01-01

    Proteins anchored to membranes through covalently linked fatty acids and/or isoprenoid groups play crucial roles in all forms of life. Sorting and trafficking of lipidated proteins has traditionally been discussed in the context of partitioning to membrane domains of different lipid composition. We...

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

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    Johnson Keven R

    2012-07-01

    Full Text Available Abstract 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.

  14. Fluorescence Localization and Comparative Ultrastructural Study of Periplocoside NW from Periploca sepium Bunge in the Midgut of the Oriental Amyworm, Mythimna separata Walker (Lepidoptera: Noctuidae

    Directory of Open Access Journals (Sweden)

    Mingxing Feng

    2014-05-01

    Full Text Available Periplocoside NW (PSNW is a novel insecticidal compound isolated from the root bark of Periploca sepium Bunge and has potent stomach toxicity against some insect pests. Previous studies showed that the Mythimna separata larva is sensitive to PSNW, but the Agrotis ispilon larva is insensitive. In this study, preliminary target localization on the midgut of M. separata larvae was conducted via a fluorescence labeling technique. A comparative ultrastructural study on the effects of PSNW on the midguts of M. separata and A. ispilon larvae was performed. Symptom observation results showed that typical stomach toxicity was induced by PSNW in M. separata larvae. Fluorescence localization results showed that PSNW binds to the midgut cells of M. separata larvae. Ultrastructure observations showed destruction of the microvilli, organelle, and cytomembrane in the midgut cells of M. separata larvae, whereas no obvious changes were observed in midgut cells of A. ispilon larvae. These results were consistent with the insecticidal activity of PSNW. Therefore, PSNW might act on the midgut tissues of the insects, and one or more binding sites of PSNW may exist in M. separata larvae midgut cell cytomembranes.

  15. Midgut volvulus following laparoscopic gastric banding--a rare and dangerous situation.

    Science.gov (United States)

    Arbell, Dan; Koplewitz, Benjamin; Zamir, Gideon; Bala, Miklosh

    2007-06-01

    Intestinal malrotation is usually encountered in infants. Its main complication is midgut volvulus, a situation that presents itself with bilious vomiting. This symptom allows for early surgical treatment. A delay in diagnosis and treatment may lead to catastrophic sequelae, such as extensive bowel necrosis and death. This situation is rare but well known in adults. Laparoscopic gastric banding is a popular option for treating morbid obesity. One of the consequences of this procedure may be impaired vomiting when there is an obstruction below the band. In this paper, we present a case in which a patient suffered from midgut volvulus 4 years after a laparoscopic gastric banding. Owing to impaired vomiting, the diagnosis was delayed, therefore, severely endangering the patient. This case prompted us to suggest that malrotation should be actively sought after before or during any bariatric procedure.

  16. A relay network of extracellular heme-binding proteins drives C. albicans iron acquisition from hemoglobin.

    Science.gov (United States)

    Kuznets, Galit; Vigonsky, Elena; Weissman, Ziva; Lalli, Daniela; Gildor, Tsvia; Kauffman, Sarah J; Turano, Paola; Becker, Jeffrey; Lewinson, Oded; Kornitzer, Daniel

    2014-10-01

    Iron scavenging constitutes a crucial challenge for survival of pathogenic microorganisms in the iron-poor host environment. Candida albicans, like many microbial pathogens, is able to utilize iron from hemoglobin, the largest iron pool in the host's body. Rbt5 is an extracellular glycosylphosphatidylinositol (GPI)-anchored heme-binding protein of the CFEM family that facilitates heme-iron uptake by an unknown mechanism. Here, we characterize an additional C. albicans CFEM protein gene, PGA7, deletion of which elicits a more severe heme-iron utilization phenotype than deletion of RBT5. The virulence of the pga7-/- mutant is reduced in a mouse model of systemic infection, consistent with a requirement for heme-iron utilization for C. albicans pathogenicity. The Pga7 and Rbt5 proteins exhibit distinct cell wall attachment, and discrete localization within the cell envelope, with Rbt5 being more exposed than Pga7. Both proteins are shown here to efficiently extract heme from hemoglobin. Surprisingly, while Pga7 has a higher affinity for heme in vitro, we find that heme transfer can occur bi-directionally between Pga7 and Rbt5, supporting a model in which they cooperate in a heme-acquisition relay. Together, our data delineate the roles of Pga7 and Rbt5 in a cell surface protein network that transfers heme from extracellular hemoglobin to the endocytic pathway, and provide a paradigm for how receptors embedded in the cell wall matrix can mediate nutrient uptake across the fungal cell envelope.

  17. De novo transcriptome sequencing and comparative analysis of midgut tissues of four non-model insects pertaining to Hemiptera, Coleoptera, Diptera and Lepidoptera.

    Science.gov (United States)

    Gazara, Rajesh K; Cardoso, Christiane; Bellieny-Rabelo, Daniel; Ferreira, Clélia; Terra, Walter R; Venancio, Thiago M

    2017-09-05

    Despite the great morphological diversity of insects, there is a regularity in their digestive functions, which is apparently related to their physiology. In the present work we report the de novo midgut transcriptomes of four non-model insects from four distinct orders: Spodoptera frugiperda (Lepidoptera), Musca domestica (Diptera), Tenebrio molitor (Coleoptera) and Dysdercus peruvianus (Hemiptera). We employed a computational strategy to merge assemblies obtained with two different algorithms, which substantially increased the quality of the final transcriptomes. Unigenes were annotated and analyzed using the eggNOG database, which allowed us to assign some level of functional and evolutionary information to 79.7% to 93.1% of the transcriptomes. We found interesting transcriptional patterns, such as: i) the intense use of lysozymes in digestive functions of M. domestica larvae, which are streamlined and adapted to feed on bacteria; ii) the up-regulation of orthologous UDP-glycosyl transferase and cytochrome P450 genes in the whole midguts different species, supporting the existence of an ancient defense frontline to counter xenobiotics; iii) evidence supporting roles for juvenile hormone binding proteins in the midgut physiology, probably as a way to activate genes that help fight anti-nutritional substances (e.g. protease inhibitors). The results presented here shed light on the digestive and structural properties of the digestive systems of these distantly related species. Furthermore, the produced datasets will also be useful for scientists studying these insects. Copyright © 2017. Published by Elsevier B.V.

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

    Science.gov (United States)

    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

  19. Diversity of Cultivable Midgut Microbiota at Different Stages of the Asian Tiger Mosquito, Aedes albopictus from Tezpur, India.

    Directory of Open Access Journals (Sweden)

    Kamlesh K Yadav

    Full Text Available Aedes aegypti and Ae. albopictus are among the most important vectors of arboviral diseases, worldwide. Recent studies indicate that diverse midgut microbiota of mosquitoes significantly affect development, digestion, metabolism, and immunity of their hosts. Midgut microbiota has also been suggested to modulate the competency of mosquitoes to transmit arboviruses, malaria parasites etc. Interestingly, the midgut microbial flora is dynamic and the diversity changes with the development of vectors, in addition to other factors such as species, sex, life-stage, feeding behavior and geographical origin. The aim of the present study was to investigate the midgut bacterial diversity among larva, adult male, sugar fed female and blood fed female Ae. albopictus collected from Tezpur, Northeastern India. Based on colony morphological characteristics, we selected 113 cultivable bacterial isolates for 16S rRNA gene sequence based molecular identification. Of the 113 isolates, we could identify 35 bacterial species belonging to 18 distinct genera under four major phyla, namely Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Phyla Proteobacteria and Firmicutes accounted for majority (80% of the species, while phylum Actinobacteria constituted 17% of the species. Bacteroidetes was the least represented phylum, characterized by a single species- Chryseobacterium rhizoplanae, isolated from blood fed individuals. Dissection of midgut microbiota diversity at different developmental stages of Ae. albopictus will be helpful in better understanding mosquito-borne diseases, and for designing effective strategies to manage mosquito-borne diseases.

  20. MDL28170, a calpain inhibitor, affects Trypanosoma cruzi metacyclogenesis, ultrastructure and attachment to Rhodnius prolixus midgut.

    Directory of Open Access Journals (Sweden)

    Vítor Ennes-Vidal

    Full Text Available BACKGROUND: Trypanosoma cruzi is the etiological agent of Chagas' disease. During the parasite life cycle, many molecules are involved in the differentiation process and infectivity. Peptidases are relevant for crucial steps of T. cruzi life cycle; as such, it is conceivable that they may participate in the metacyclogenesis and interaction with the invertebrate host. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we have investigated the effect of the calpain inhibitor MDL28170 on the attachment of T. cruzi epimastigotes to the luminal midgut surface of Rhodnius prolixus, as well as on the metacyclogenesis process and ultrastructure. MDL28170 treatment was capable of significantly reducing the number of bound epimastigotes to the luminal surface midgut of the insect. Once the cross-reactivity of the anti-Dm-calpain was assessed, it was possible to block calpain molecules by the antibody, leading to a significant reduction in the capacity of adhesion to the insect guts by T. cruzi. However, the antibodies were unable to interfere in metacyclogenesis, which was impaired by the calpain inhibitor presenting a significant reduction in the number of metacyclic trypomastigotes. The calpain inhibitor also promoted a direct effect against bloodstream trypomastigotes. Ultrastructural analysis of epimastigotes treated with the calpain inhibitor revealed disorganization in the reservosomes, Golgi and plasma membrane disruption. CONCLUSIONS/SIGNIFICANCE: The presence of calpain and calpain-like molecules in a wide range of organisms suggests that these proteins could be necessary for basic cellular functions. Herein, we demonstrated the effects of MDL28170 in crucial steps of the T. cruzi life cycle, such as attachment to the insect midgut and metacyclogenesis, as well as in parasite viability and morphology. Together with our previous findings, these results help to shed some light on the functions of T. cruzi calpains. Considering the potential roles of

  1. Properties Of A Midgut Trypanolysin From The Tsetse Fly Glossina Morsitans Morsitans

    Directory of Open Access Journals (Sweden)

    Mahamat H.Abakar

    2015-08-01

    Full Text Available The properties of a bloodmeal-induced trypanolysin from the midgut of the tsetse G. m. morsitans was studied in vitro. The semi-purified trypanolysin from twice-fed tsetse had the highest trypanolysin activity against bloodstream trypanosomes followed by those once-fed and the unfed flies. Serum found to display trypanolysin activity. The trypanolysin had no trypsin activity nor even affected by the enzyme. In addition trypanolysin was not affected by protease inhibitors such as soy bean trypsin inhibitor STI N-a-p-Tosyl-L-lysine chromethyl ketone TLCK phenylmethyl sulphonyl fluoride PMSF diisopropyl fluoro-phosphate DFP and tosylamide-2-phenylethyl chloromethyl ketone TPCK. However the activity was completely inhibited by diethyl pyrocarbonate DEPC and partially by aprotinin. The induction of trypanolysin activity by bloodmeal increased gradually reaching a peak at 72-120 h after the bloodmeal and then decreased rapidly with only 25 of the peak activity remaining after 192 h. The trypanolysin was inactivated during storage at 27amp8451 and 4amp8451 after 15 and 32 days respectively. Similarly heating the midguts trypanolysin to 60 - 80amp8451 led to loss of activity. On the other hand 50amp8451 was found to be the optimum temperature for trypanolysin activity. The activity was also unstable by freeze-thaw at 80amp8451 -70amp8451 -20amp8451 and 0amp8451 after 33 41 55 and 63 days respectively. Trypanolysin caused lyses of bloodstream-form T. b. brucei while the procyclic trypanosomes were unaffected. The highest trypanolysin activity in different tsetse species was found with Glossina longipennis followed by Glossina pallidipes Glossina morsitans centralis Glossina fuscipes fuscipes and G. m. morsitans. When the midgut homogenate was separated by anion-exchange chromatography the trypanolysin activity was recovered in the bound fraction. These results suggest that the midgut trypanolysin plays an important role in the establishment of

  2. Complex Spiral Structure in the HD 100546 Transitional Disk as Revealed by GPI and MagAO

    Energy Technology Data Exchange (ETDEWEB)

    Follette, Katherine B.; Macintosh, Bruce; Mullen, Wyatt; Bailey, Vanessa P. [Kavli Institute for Particle Astrophysics and Cosmology, Department of Physics, Stanford University, Stanford, CA, 94305 (United States); Rameau, Julien [Institut de Recherche sur les Exoplanètes, Départment de Physique, Université de Montréal, Montréal QC H3C 3J7 (Canada); Dong, Ruobing; Close, Laird M.; Males, Jared R.; Morzinski, Katie M. [Steward Observatory, University of Arizona, Tucson, AZ 85721 (United States); Pueyo, Laurent; Perrin, Marshall [Space Telescope Science Institute, Baltimore, MD 21218 (United States); Duchêne, Gaspard; Fung, Jeffrey; Wang, Jason [Astronomy Department, University of California, Berkeley, Berkeley CA 94720 (United States); Leonard, Clare; Spiro, Elijah [Physics and Astronomy Department, Amherst College, 21 Merrill Science Drive, Amherst, MA 01002 (United States); Marois, Christian [National Research Council of Canada Herzberg, 5071 West Saanich Road, Victoria, BC V9E 2E7 (Canada); Millar-Blanchaer, Maxwell A. [Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA 91125 (United States); Ammons, S. Mark [Lawrence Livermore National Laboratory, Livermore, CA 94551 (United States); Barman, Travis [Lunar and Planetary Laboratory, University of Arizona, Tucson, AZ 85721 (United States); and others

    2017-06-01

    We present optical and near-infrared high-contrast images of the transitional disk HD 100546 taken with the Magellan Adaptive Optics system (MagAO) and the Gemini Planet Imager (GPI). GPI data include both polarized intensity and total intensity imagery, and MagAO data are taken in Simultaneous Differential Imaging mode at H α . The new GPI H -band total intensity data represent a significant enhancement in sensitivity and field rotation compared to previous data sets and enable a detailed exploration of substructure in the disk. The data are processed with a variety of differential imaging techniques (polarized, angular, reference, and simultaneous differential imaging) in an attempt to identify the disk structures that are most consistent across wavelengths, processing techniques, and algorithmic parameters. The inner disk cavity at 15 au is clearly resolved in multiple data sets, as are a variety of spiral features. While the cavity and spiral structures are identified at levels significantly distinct from the neighboring regions of the disk under several algorithms and with a range of algorithmic parameters, emission at the location of HD 100546 “ c ” varies from point-like under aggressive algorithmic parameters to a smooth continuous structure with conservative parameters, and is consistent with disk emission. Features identified in the HD 100546 disk bear qualitative similarity to computational models of a moderately inclined two-armed spiral disk, where projection effects and wrapping of the spiral arms around the star result in a number of truncated spiral features in forward-modeled images.

  3. Defining the conformational features of anchorless, poorly neuroinvasive prions.

    Directory of Open Access Journals (Sweden)

    Cyrus Bett

    Full Text Available Infectious prions cause diverse clinical signs and form an extraordinary range of structures, from amorphous aggregates to fibrils. How the conformation of a prion dictates the disease phenotype remains unclear. Mice expressing GPI-anchorless or GPI-anchored prion protein exposed to the same infectious prion develop fibrillar or nonfibrillar aggregates, respectively, and show a striking divergence in the disease pathogenesis. To better understand how a prion's physical properties govern the pathogenesis, infectious anchorless prions were passaged in mice expressing anchorless prion protein and the resulting prions were biochemically characterized. Serial passage of anchorless prions led to a significant decrease in the incubation period to terminal disease and altered the biochemical properties, consistent with a transmission barrier effect. After an intraperitoneal exposure, anchorless prions were only weakly neuroinvasive, as prion plaques rarely occurred in the brain yet were abundant in extracerebral sites such as heart and adipose tissue. Anchorless prions consistently showed very high stability in chaotropes or when heated in SDS, and were highly resistant to enzyme digestion. Consistent with the results in mice, anchorless prions from a human patient were also highly stable in chaotropes. These findings reveal that anchorless prions consist of fibrillar and highly stable conformers. The additional finding from our group and others that both anchorless and anchored prion fibrils are poorly neuroinvasive strengthens the hypothesis that a fibrillar prion structure impedes efficient CNS invasion.

  4. Histopathological and ultrastructural effects of delta-endotoxins of Bacillus thuringiensis serovar israelensis in the midgut of Simulium pertinax larvae (Diptera, Simuliidae

    Directory of Open Access Journals (Sweden)

    CFG Cavados

    2004-08-01

    Full Text Available The bacterium Bacillus thuringiensis (Bt produces parasporal crystals containing delta-endotoxins responsible for selective insecticidal activity on larvae. Upon ingestion, these crystals are solubilized in the midgut lumen and converted into active toxins that bind to receptors present on the microvilli causing serious damage to the epithelial columnar cells. We investigated the effect of these endotoxins on larvae of the Simulium pertinax, a common black fly in Brazil, using several concentrations during 4 h of the serovar israelensis strain IPS-82 (LFB-FIOCRUZ 584, serotype H-14 type strain of the Institute Pasteur, Paris. Light and electron microscope observations revealed, by time and endotoxin concentration, increasing damages of the larvae midgut epithelium. The most characteristic effects were midgut columnar cell vacuolization, microvilli damages, epithelium cell contents passing into the midgut lumen and finally the cell death. This article is the first report of the histopathological effects of the Bti endotoxins in the midgut of S. pertinax larvae and the data obtained may contribute to a better understanding of the mode of action of this bacterial strain used as bioinsecticide against black fly larvae.

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

    Science.gov (United States)

    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.

  6. Experimental Research on Destruction Mode and Anchoring Performance of Carbon Fiber Phyllostachys pubescens Anchor Rod with Different Forms

    Directory of Open Access Journals (Sweden)

    Wang Yulan

    2018-01-01

    Full Text Available The anchoring technology is extensively applied in reinforcing protection of the earth relics. Now that no specification is available for different new anchor rods in earth relics protection due to diversified destruction modes of earth relics and complexity of engineering technology conditions, it is urgent to guide reinforcing design and construction with a complete detailed anchor rod research document. With the new carbon fiber Phyllostachys pubescens anchor rod as the research object, six lots of in situ tests are designed to, respectively, study the destruction mode and anchoring performance of the carbon fiber Phyllostachys pubescens anchor rod under different anchor length L, anchor rod diameter D, bore diameter H, grouting material S, rib spacing R, and inclination angle A in this paper. By studying load shift curve experiment in drawing of the anchor rod, the destruction mode and ultimate bearing capacity of the carbon fiber Phyllostachys pubescens anchor rod in different experiment lots are obtained, and the concept of permitted application value N in anchor rod design is proposed. By studying strain distribution characteristics of anchor rods in experimental lots along the length direction under action of the permitted application value N and combining the existing destruction mode and ultimate bearing capacity, this paper analyzes influences of L, D, H, S, R, and A on anchoring effect of the carbon fiber Phyllostachys pubescens anchor rod; gives the reasonable value range of L, D, H, and R when the carbon fiber Phyllostachys pubescens anchor rod is used for reinforcing design of the earth relics; and provides favorable experiment basis for reinforcing design of the earth relics based on the carbon fiber Phyllostachys pubescens anchor rod.

  7. A Lectin from Dioclea violacea Interacts with Midgut Surface of Lutzomyia migonei, Unlike Its Homologues, Cratylia floribunda Lectin and Canavalia gladiata Lectin

    Directory of Open Access Journals (Sweden)

    Juliana Montezuma Barbosa Monteiro Tínel

    2014-01-01

    Full Text Available Leishmaniasis is a vector-borne disease transmitted by phlebotomine sand fly. Susceptibility and refractoriness to Leishmania depend on the outcome of multiple interactions that take place within the sand fly gut. Promastigote attachment to sand fly midgut epithelium is essential to avoid being excreted together with the digested blood meal. Promastigote and gut sand fly surface glycans are important ligands in this attachment. The purpose of the present study was to evaluate the interaction of three lectins isolated from leguminous seeds (Diocleinae subtribe, D-glucose and D-mannose-binding, with glycans on Lutzomyia migonei midgut. To study this interaction the lectins were labeled with FITC and a fluorescence assay was performed. The results showed that only Dioclea violacea lectin (DVL was able to interact with midgut glycans, unlike Cratylia floribunda lectin (CFL and Canavalia gladiata lectin (CGL. Furthermore, when DVL was blocked with D-mannose the interaction was inhibited. Differences of spatial arrangement of residues and volume of carbohydrate recognition domain (CRD may be the cause of the fine specificity of DVL for glycans in the surface on Lu. migonei midgut. The findings in this study showed the presence of glycans in the midgut with glucose/mannose residues in its composition and these residues may be important in interaction between Lu. migonei midgut and Leishmania.

  8. Identification of irradiated insects: changes in the midgut of the confused flour beetle, Tribolium confusum Duv., induced by gamma radiation

    International Nuclear Information System (INIS)

    Szczepanik, M.

    1994-01-01

    The radiation doses applied for quarantine treatment does not generally cause immediate death of insects and mites. Alive pests may be present in the agricultural commodities for several days. This can be a disadvantage if the product is for immediate export and there is a nil insect requirement. A simple test is needed to ensure the quarantine personnel that a pest of quarantine importance has been irradiated and it does not pose a quarantine risk. Development of a practical technique for identification of irradiated pests was recommended by the ICGFI Task Force Meeting on Irradiation as Quarantine Treatment. The midgut, metabolically very active tissue, is the main site for digestion and absorption of the products of digestion. The old epithelium is replaced by new cells produced by the regenerative nidi. Regenerative cells are very sensitive to radiation. Its damage results in loss of the midgut epithelium in irradiated insects. The following changes in the midgut structure of the confused flour beetle were observed: 1. destruction of the regenerative nidi; 2. elongation and enlargement of epithelial cells; 3. vacuolization of the epithelial cells; 4. fading of cells boundaries in the epithelium; 5. damage of the nuclei (chromatin grains scattered throughout the cytoplasm of epithelial cells); 6. loss of the brush border; 7. disintegration and further loss of epithelium. The most expressed effect of the irradiation treatment was the destruction of regenerative cells of the midgut. Their destruction prevented the replacement of the secretory cells of the epithelium. As a result the epithelium disappeared and the gut lumen enlarged. Causes other than ionizing radiation resulting in disintegration of the midgut epithelium of insects are unknown. Since the degenerative changes in the midgut are positively correlated with both dose and time elapsed after irradiation exposure, a pathological syndrome of irradiation effects on the midgut may be used for a rapid and

  9. PI3KC2{alpha}, a class II PI3K, is required for dynamin-independent internalization pathways

    DEFF Research Database (Denmark)

    Krag, Claudia; Malmberg, Emily Kim; Salcini, Anna Elisabetta

    2010-01-01

    as fluid-phase endocytosis. Our data suggest a general role for PI3KC2a in regulating physiologically relevant dynamin-independent internalization pathways by recruiting early endosome antigen 1 (EEA1) to vesicular compartments, a step required for the intracellular trafficking of vesicles generated...... screen using a cell line expressing a diphtheria toxin receptor (DTR, officially known as HBEGF) anchored to GPI (DTR-GPI), which internalizes diphtheria toxin (DT, officially known as DTX) in a dynamin-independent manner, identified PI3KC2a, a class II phosphoinositide 3-kinase (PI3K), as a specific...... regulator of dynamin-independent DT internalization. We found that the internalization of several proteins that enter the cell through dynamin-independent pathways led to a relocalization of PI3KC2a to cargo-positive vesicles. Furthermore, downregulation of PI3KC2a impaired internalization of CD59 as well...

  10. Comparative Proteomic Analysis of the Effect of Periplocoside P from Periploca sepium on Brush Border Membrane Vesicles in Midgut Epithelium of Mythimna separata Larvae

    Directory of Open Access Journals (Sweden)

    Mingxing Feng

    2017-12-01

    Full Text Available Periplocoside P (PSP, a novel compound isolated from Periploca sepium Bunge, possesses insecticidal activity against some lepidopterans, such as Mythimna separata. In M. separata, the brush border membrane vesicles of the midgut epithelium are the initial site of action of periplocosides. We conducted two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry analysis to analyze differentially expressed proteins (DEPs from periplocoside P (PSP-treated M. separata. We successfully isolated seven up-regulated and three down-regulated DEPs that have been previously identified, as well as a novel DEP. The DEPs are implicated in protein degradation, transporter, folding, and synthesis, and in juvenile hormone biosynthesis. DEPs involved in the oxidative phosphorylation energy metabolism pathway are enriched. Through real-time polymerase chain reaction assay, we confirmed that vma1 expression is significantly up-regulated expression levels in PSP-treated M. separata larvae. Enzymology validation further indicated that PSP can significantly inhibit V-type ATPase activity in a concentration-dependent manner. Given these results, we speculate that in M. separata, the V-type ATPase A subunit in the midgut epithelium is the putative target binding site of periplocosides. This finding provides preliminary evidence for the mode of action of periplocosides.

  11. Immuno-histochemical localization of cholesterol binding proteins in ...

    African Journals Online (AJOL)

    This manuscript aims to investigate immunocytochemical localization of cholesterol binding proteins (CBPs) in semi-thin sections of midgut of Schistocerca gregaria (Forskal). For this purpose ... Further, same protein was also localized in other tissues like fat body, testis, and ovary of male and female insects of S. gregaria.

  12. Experimental testing of anchoring devices for bottom rails in partially anchored timber frame shear walls

    OpenAIRE

    Caprolu, Giuseppe

    2011-01-01

    Källsner and Girhammar have presented a new plastic design method of wood-framed shear walls at ultimate limit state. This method allows the designer to calculate the load-carrying capacity of shear walls partially anchored, where the leading stud is not anchored against the uplift.The anchorage system of shear walls is provided from anchor bolts and hold downs. Anchor bolts provide horizontal shear continuity between the bottom rail and the foundation. Hold downs are directly connected from ...

  13. The relationship between autophagy and apoptosis in the midgut epithelium of Myriapoda

    Czech Academy of Sciences Publication Activity Database

    Rost-Roszkowska, M.M.; Vilímová, J.; Tajovský, Karel; Płachno, B.J.; Pavlíček, T.; Sosinka, A.; Ostróžka, A.; Kaszuba, F.; Chajec, Ł.; Włodarczyk, A.; Marchewka, A.

    -, Suppl. 5 (2017), s. 24 ISSN 1513-9700. [International Congress of Myriapodology /17./. 23.07.2017-26.07.2017, Krabi] Institutional support: RVO:60077344 Keywords : millipedes * centipedes * midgut ultrastructure * autophagy * apoptosis Subject RIV: EG - Zoology OBOR OECD: Zoology

  14. Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

    Science.gov (United States)

    Richards, Mark J; Hsia, Chih-Yun; Singh, Rohit R; Haider, Huma; Kumpf, Julia; Kawate, Toshimitsu; Daniel, Susan

    2016-03-29

    Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions

  15. Transforming p21 ras protein: flexibility in the major variable region linking the catalytic and membrane-anchoring domains

    DEFF Research Database (Denmark)

    Willumsen, B M; Papageorge, A G; Hubbert, N

    1985-01-01

    or increasing it to 50 amino acids has relatively little effect on the capacity of the gene to induce morphological transformation of NIH 3T3 cells. Assays of GTP binding, GTPase and autophosphorylating activities of such mutant v-rasH-encoded proteins synthesized in bacteria indicated that the sequences...... that is required for post-translational processing, membrane localization and transforming activity of the proteins. We have now used the viral oncogene (v-rasH) of Harvey sarcoma virus to study the major variable region by deleting or duplicating parts of the gene. Reducing this region to five amino acids...... that encode these biochemical activities are located upstream from the major variable region. In the context of transformation, we propose that the region of sequence heterogeneity serves principally to connect the N-terminal catalytic domain with amino acids at the C terminus that are required to anchor...

  16. Biogenesis of the mitochondrial TOM complex: Mim1 promotes insertion and assembly of signal-anchored receptors.

    Science.gov (United States)

    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. A novel biosurfactant produced by Aureobasidium pullulans L3-GPY from a tiger lily wild flower, Lilium lancifolium Thunb.

    Science.gov (United States)

    Kim, Jong Shik; Lee, In Kyoung; Yun, Bong Sik

    2015-01-01

    Yeast biosurfactants are important biotechnological products in the food industry, and they have medical and cosmeceutical applications owing to their specific modes of action, low toxicity, and applicability. Thus, we have isolated and examined biosurfactant-producing yeast for various industrial and medical applications. A rapid and simple method was developed to screen biosurfactant-producing yeasts for high production of eco-friendly biosurfactants. Using this method, several potential niches of biosurfactant-producing yeasts, such as wild flowers, were investigated. We successfully selected a yeast strain, L3-GPY, with potent surfactant activity from a tiger lily, Lilium lancifolium Thunb. Here, we report the first identification of strain L3-GPY as the black yeast Aureobasidium pullulans. In addition, we isolated a new low-surface-tension chemical, designated glycerol-liamocin, from the culture supernatant of strain L3-GPY through consecutive chromatography steps, involving an ODS column, solvent partition, silica gel, Sephadex LH-20, and an ODS Sep-Pak cartridge column. The chemical structure of glycerol-liamocin, determined by mass spectrometry and nuclear magnetic resonance spectroscopy, indicates that it is a novel compound with the molecular formula C33H62O12. Furthermore, glycerol-liamocin exhibited potent biosurfactant activity (31 mN/m). These results suggest that glycerol-liamocin is a potential novel biosurfactantfor use in various industrial applications.

  18. Phylogenetically Diverse Burkholderia Associated with Midgut Crypts of Spurge Bugs, Dicranocephalus spp. (Heteroptera: Stenocephalidae).

    Science.gov (United States)

    Kuechler, Stefan Martin; Matsuura, Yu; Dettner, Konrad; Kikuchi, Yoshitomo

    2016-06-25

    Diverse phytophagous heteropteran insects, commonly known as stinkbugs, are associated with specific gut symbiotic bacteria, which have been found in midgut cryptic spaces. Recent studies have revealed that members of the stinkbug families Coreidae and Alydidae of the superfamily Coreoidea are consistently associated with a specific group of the betaproteobacterial genus Burkholderia, called the "stinkbug-associated beneficial and environmental (SBE)" group, and horizontally acquire specific symbionts from the environment every generation. However, the symbiotic system of another coreoid family, Stenocephalidae remains undetermined. We herein investigated four species of the stenocephalid genus Dicranocephalus. Examinations via fluorescence in situ hybridization (FISH) and transmission electron microscopy (TEM) revealed the typical arrangement and ultrastructures of midgut crypts and gut symbionts. Cloning and molecular phylogenetic analyses of bacterial genes showed that the midgut crypts of all species are colonized by Burkholderia strains, which were further assigned to different subgroups of the genus Burkholderia. In addition to the SBE-group Burkholderia, a number of stenocephalid symbionts belonged to a novel clade containing B. sordidicola and B. udeis, suggesting a specific symbiont clade for the Stenocephalidae. The symbiotic systems of stenocephalid bugs may provide a unique opportunity to study the ongoing evolution of symbiont associations in the stinkbug-Burkholderia interaction.

  19. Female-Specific Specialization of a Posterior End Region of the Midgut Symbiotic Organ in Plautia splendens and Allied Stinkbugs

    Science.gov (United States)

    Hayashi, Toshinari; Hosokawa, Takahiro; Meng, Xian-Ying; Koga, Ryuichi

    2015-01-01

    Many stinkbugs (Insecta: Hemiptera: Heteroptera) are associated with bacterial symbionts in a posterior region of the midgut. In these stinkbugs, adult females excrete symbiont-containing materials from the anus for transmission of the beneficial symbionts to their offspring. For ensuring the vertical symbiont transmission, a variety of female-specific elaborate traits at the cellular, morphological, developmental, and behavioral levels have been reported from diverse stinkbugs of the families Plataspidae, Urostylididae, Parastrachiidae, etc. Meanwhile, such elaborate female-specific traits for vertical symbiont transmission have been poorly characterized for the largest and economically important stinkbug family Pentatomidae. Here, we investigated the midgut symbiotic system of a pentatomid stinkbug, Plautia splendens. A specific gammaproteobacterial symbiont was consistently present extracellularly in the cavity of numerous crypts arranged in four rows on the midgut fourth section. The symbiont was smeared on the egg surface upon oviposition by adult females, orally acquired by newborn nymphs, and thereby transmitted vertically to the next generation and important for growth and survival of the host insects. We found that, specifically in adult females, several rows of crypts at the posterior end region of the symbiotic midgut were morphologically differentiated and conspicuously enlarged, often discharging the symbiotic bacteria from the crypt cavity to the main tract of the symbiotic midgut. The female-specific enlarged end crypts were also found in other pentatomid stinkbugs Plautia stali and Carbula crassiventris. These results suggest that the enlarged end crypts represent a female-specific specialized morphological trait for vertical symbiont transmission commonly found among stinkbugs of the family Pentatomidae. PMID:25636847

  20. Phagocytosis of gram-negative bacteria by a unique CD14-dependent mechanism.

    Science.gov (United States)

    Schiff, D E; Kline, L; Soldau, K; Lee, J D; Pugin, J; Tobias, P S; Ulevitch, R J

    1997-12-01

    THP-1-derived cell lines were stably transfected with constructs encoding glycophosphatidylinositol (GPI)-anchored or transmembrane forms of human CD14. CD14 expression was associated with enhanced phagocytosis of serum (heat-inactivated)-opsonized Escherichia coli (opEc). Both the GPI-anchored and transmembrane forms of CD14 supported phagocytosis of opEc equally well. Lipopolysaccharide-binding protein (LBP) played a role in CD14-dependent phagocytosis as evidenced by inhibition of CD14-dependent phagocytosis of opEc with anti-LBP monoclonal antibody (mAb) and by enhanced phagocytosis of E. coli opsonized with purified LBP. CD14-dependent phagocytosis was inhibited by a phosphatidylinositol (PI) 3-kinase inhibitor (wortmannin) and a protein tyrosine kinase inhibitor (tyrphostin 23) but not a protein kinase C inhibitor (bisindolyl-maleimide) or a divalent cation chelator (ethylenediaminetetraacetate). Anti-LBP mAb 18G4 and anti-CD14 mAb 18E12 were used to differentiate between the pathways involved in CD14-dependent phagocytosis and CD14-dependent cell activation. F(ab')2 fragments of 18G4, a mAb to LBP that does not block cell activation, inhibited ingestion of opEc by THP1-wtCD14 cells. 18E12 (an anti-CD14 mAb that does not block LPS binding to CD14 but does inhibit CD14-dependent cell activation) did not inhibit phagocytosis of LBP-opEc by THP1-wtCD14 cells. Furthermore, CD14-dependent phagocytosis was not inhibited by anti-CD18 (CR3 and CR4 beta-chain) or anti-Fcgamma receptor mAb.

  1. Acute exposure of mercury chloride stimulates the tissue regeneration program and reactive oxygen species production in the Drosophila midgut.

    Science.gov (United States)

    Chen, Zhi; Wu, Xiaochun; Luo, Hongjie; Zhao, Lingling; Ji, Xin; Qiao, Xianfeng; Jin, Yaping; Liu, Wei

    2016-01-01

    We used Drosophila as an animal model to study the digestive tract in response to the exposure of inorganic mercury (HgCl2). We found that after oral administration, mercury was mainly sequestered within the midgut. This resulted in increased cell death, which in turn stimulated the tissue regeneration program, including accelerated proliferation and differentiation of the intestinal stem cells (ISCs). We further demonstrated that these injuries correlate closely with the excessive production of the reactive oxygen species (ROS), as vitamin E, an antioxidant reagent, efficiently suppressed the HgCl2-induced phenotypes of midgut and improved the viability. We propose that the Drosophila midgut could serve as a suitable model to study the treatment of acute hydrargyrism on the digestive systems. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Investigation of the effect of heated ethanol fuel on combustion and emissions of an ethanol direct injection plus gasoline port injection (EDI + GPI) engine

    International Nuclear Information System (INIS)

    Huang, Yuhan; Hong, Guang

    2016-01-01

    Highlights: • Effect of EDI heating on the EDI + GPI engine performance was investigated. • CO and HC were significantly reduced and NO was slightly increased by EDI heating. • IMEP and combustion speed were slightly reduced by EDI heating. • EDI heating is effective to address the evaporation and over-cooling issues of EDI + GPI engine. - Abstract: Ethanol direct injection plus gasoline port injection (EDI + GPI) is a new technology to utilise ethanol fuel more efficiently and flexibly in spark ignition engines. One issue needs to be addressed in the development of EDI + GPI is the ethanol fuel’s low vapour pressure and large latent heat which slow down the ethanol’s evaporation and result in the mixture unready for combustion by the time of spark ignition and the consequent increase of CO and HC emissions. Heating the ethanol fuel to be directly injected (EDI heating) has been proposed to address this issue. This paper reports the investigation of the effect of EDI heating on the combustion and emissions of a research engine equipped with EDI + GPI. The results showed that EDI heating effectively reduced the CO and HC emissions of the engine due to the increase of evaporation rate and reduced fuel impingement and local over-cooling. The reduction of CO and HC became more significant with the increase of ethanol ratio. When the temperature of the ethanol fuel was increased by 40 °C, the CO and HC were reduced by as much as 43% and 51% respectively in EDI only condition at the original spark timing of 15 CAD BTDC, and 15% and 47% respectively at the minimum spark advance for best torque (MBT) timing of 19 CAD BTDC. On the other hand, the NO emission was slightly increased, but still much smaller than that in GPI only condition due to the strong cooling effect and low combustion temperature of EDI. The IMEP and combustion speed were slightly reduced by EDI heating due to the decrease of injector fuel flow rate and spray collapse of flash-boiling. The

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

    Science.gov (United States)

    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.

  4. Not all Anchors Weigh Equally.

    Science.gov (United States)

    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.

  5. Dengue virus serotype 2 infection alters midgut and carcass gene expression in the Asian tiger mosquito, Aedes albopictus.

    Directory of Open Access Journals (Sweden)

    Hitoshi Tsujimoto

    Full Text Available The Asian tiger mosquito, Aedes albopictus is currently an important vector for dengue, chikungunya and Zika virus, and its role in transmission of arthropod-borne viruses (arboviruses may increase in the future due to its ability to colonize temperate regions. In contrast to Aedes aegypti, the dominant vector of dengue, chikungunya and Zika virus, genetic responses of Ae. albopictus upon infection with an arbovirus are not well characterized. Here we present a study of the changes in transcript expression in Ae. albopictus exposed to dengue virus serotype 2 via feeding on an artificial bloodmeal.We isolated midguts and midgut-free carcasses of Ae. albopictus fed on bloodmeals containing dengue virus as well as controls fed on virus-free control meals at day 1 and day 5 post-feeding. We confirmed infection of midguts from mosquitoes sampled on day 5 post-feeding via RT-PCR. RNAseq analysis revealed dynamic modulation of the expression of several putative immunity and dengue virus-responsive genes, some of whose expression was verified by qRT-PCR. For example, a serine protease gene was up-regulated in the midgut at 1 day post infection, which may potentially enhance mosquito susceptibility to dengue infection, while 14 leucine-rich repeat genes, previously shown to be involved in mosquito antiviral defenses, were down-regulated in the carcass at 5 days post infection. The number of significantly modulated genes decreased over time in midguts and increased in carcasses.Dengue virus exposure results in the modulation of genes in a time- and site-specific manner. Previous literature on the interaction between mosquitoes and mosquito-borne pathogens suggests that most of the changes that occurred in Ae. albopictus exposed to DENV would favor virus infection. Many genes identified in this study warrant further characterization to understand their role in viral manipulation of and antiviral response of Ae. albopictus.

  6. The transverse colon cancer with the reversed rotation of the midgut treated with single incision laparoscopic colectomy.

    Science.gov (United States)

    Hirano, Yasumitsu; Hattori, Masakazu; Fujita, Manami; Nishida, Youji; Douden, Kenji; Hashizume, Yasuo

    2013-06-01

    Reversed rotation of the midgut is a rare type of intestinal malrotation. Moreover, synchronous colon cancer has rarely been reported. Preliminary experience with single-incision laparoscopic colectomy (SILC) for colon cancer with reversed rotation of the midgut is reported. An 82-year-old woman was admitted because of a fecal occult blood. A colonoscopy revealed transverse colon cancer. An air-barium contrast enema showed the right-sided sigmoid colon and the left-sided cecum. A computed tomography revealed that the duodenum and the transverse colon were situated at the ventral side of the superior mesenteric artery, and a preoperative diagnosis of suspicion of reversed rotation of the midgut was made. First, a lap protector was inserted through a 4.0 cm transumbilical incision. Four 5 mm ports were placed in the lap protector. On the observation of laparoscopy, the cecum and the ascending colon were not fixed with the retroperitoneum and situated on the left, and the sigmoid colon was situated on the right. We successfully mobilized the transverse colon using a single-incision laparoscopic approach. Resection was achieved following extracorporealization, and the anastomosis was performed extracorporeally using staplers. The patient was discharged on the thirteenth postoperative day. Postoperative follow-up did not reveal any umbilical wound complications. SILC for colon cancer associated with malrotation of the midgut is feasible and a promising alternative method because of its less invasiveness and its adaptability to the malrotation without extending the skin incision.

  7. Specificity of Bacillus thuringiensis endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts

    International Nuclear Information System (INIS)

    Hofmann, C.; Vanderbruggen, H.; Hoefte, H.; Van Rie, J.; Jansens, S.; Van Mellaert, H.

    1988-01-01

    Binding studies were performed with two 125 I-labeled Bacillus thuringiensis δ-endotoxins on brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm Manduca sexta or the cabbage butterfly Pieris brassicae. One δ-endotoxin, Bt2-protoxin, is a 130-kDa recombinant crystalline protein from B. thuringiensis subsp. berliner. It kills larvae of both insect species. The active Bt2-toxin is a 60-kDa proteolytic fragment of the Bt2-protoxin. It binds saturably and with high affinity to brush border membrane vesicles from the midgut of both species. The other δ-endotoxin, Bt4412-protoxin, is a 136-kDa crystalline protein from B. thuringiensis subsp. thuringiensis, which is highly toxic for P. brassicae, but not for M. sexta larvae. Bt4412-toxin, obtained after proteolytic activation of Bt4412-protoxin, shows high-affinity saturable binding to P. brassicae vesicles but not to M. sexta vesicles. The correlation between toxicity and specific binding is further strengthened by competition studies. Other B. thuringiensis δ-endotoxins active against M. sexta compete for binding of 125 I-labeled Bt2-toxin to M. sexta vesicles, whereas toxins active against dipteran or coleopteran larvae do not compete. Bt2-toxin and Bt4412-toxin bind to different sites on P. brassicae vesicles

  8. Speed Controls in Translating Secretory Proteins in Eukaryotes - an Evolutionary Perspective

    Science.gov (United States)

    Mahlab, Shelly; Linial, Michal

    2014-01-01

    Protein translation is the most expensive operation in dividing cells from bacteria to humans. Therefore, managing the speed and allocation of resources is subject to tight control. From bacteria to humans, clusters of relatively rare tRNA codons at the N′-terminal of mRNAs have been implicated in attenuating the process of ribosome allocation, and consequently the translation rate in a broad range of organisms. The current interpretation of “slow” tRNA codons does not distinguish between protein translations mediated by free- or endoplasmic reticulum (ER)-bound ribosomes. We demonstrate that proteins translated by free- or ER-bound ribosomes exhibit different overall properties in terms of their translation efficiency and speed in yeast, fly, plant, worm, bovine and human. We note that only secreted or membranous proteins with a Signal peptide (SP) are specified by segments of “slow” tRNA at the N′-terminal, followed by abundant codons that are considered “fast.” Such profiles apply to 3100 proteins of the human proteome that are composed of secreted and signal peptide (SP)-assisted membranous proteins. Remarkably, the bulks of the proteins (12,000), or membranous proteins lacking SP (3400), do not have such a pattern. Alternation of “fast” and “slow” codons was found also in proteins that translocate to mitochondria through transit peptides (TP). The differential clusters of tRNA adapted codons is not restricted to the N′-terminal of transcripts. Specifically, Glycosylphosphatidylinositol (GPI)-anchored proteins are unified by clusters of low adapted tRNAs codons at the C′-termini. Furthermore, selection of amino acids types and specific codons was shown as the driving force which establishes the translation demands for the secretory proteome. We postulate that “hard-coded” signals within the secretory proteome assist the steps of protein maturation and folding. Specifically, “speed control” signals for delaying the translation

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

    Science.gov (United States)

    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.

  10. Exosome Secretion by the Parasitic Protozoan Leishmania within the Sand Fly Midgut

    Directory of Open Access Journals (Sweden)

    Vanessa Diniz Atayde

    2015-11-01

    Full Text Available Despite several studies describing the secretion of exosomes by Leishmania in vitro, observation of their formation and release in vivo has remained a major challenge. Herein, we show that Leishmania constitutively secretes exosomes within the lumen of the sand fly midgut through a mechanism homologous to the mammalian pathway. Through egestion experiments, we demonstrate that Leishmania exosomes are part of the sand fly inoculum and are co-egested with the parasite during the insect’s bite, possibly influencing the host infectious process. Indeed, co-inoculation of mice footpads with L. major plus midgut-isolated or in-vitro-isolated L. major exosomes resulted in a significant increase in footpad swelling. Notably, co-injections produced exacerbated lesions through overinduction of inflammatory cytokines, in particular IL-17a. Our data indicate that Leishmania exosomes are an integral part of the parasite’s infectious life cycle, and we propose to add these vesicles to the repertoire of virulence factors associated with vector-transmitted infections.

  11. Does autophagy in the midgut epithelium of centipedes depend on the day/night cycle?

    Czech Academy of Sciences Publication Activity Database

    Rost-Roszkowska, M.M.; Chajec, Ł.; Vilímová, J.; Tajovský, Karel; Kszuk-Jendrysik, M.

    2015-01-01

    Roč. 68, January (2015), s. 130-139 ISSN 0968-4328 Institutional support: RVO:60077344 Keywords : centipede * midgut epithelium * digestive cells * ultrastructure * autophagy Subject RIV: EG - Zoology Impact factor: 1.838, year: 2015

  12. Pre-sorting endosomal transport of the GPI-anchored protein, CD59, is regulated by EHD1

    Czech Academy of Sciences Publication Activity Database

    Cai, B.; Katafiasz, D.; Hořejší, Václav; Naslavsky, N.

    2011-01-01

    Roč. 12, č. 1 (2011), s. 102-120 ISSN 1398-9219 Institutional research plan: CEZ:AV0Z50520514 Keywords : canine kidney cells * recycling compartment * receptor Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.919, year: 2011

  13. The antibacterial activity of peptides derived from human beta-2 glycoprotein I is inhibited by protein H and M1 protein from Streptococcus pyogenes

    NARCIS (Netherlands)

    Nilsson, Maria; Wasylik, Sylwia; Mörgelin, Matthias; Olin, Anders I.; Meijers, Joost C. M.; Derksen, Ronald H. W. M.; de Groot, Philip G.; Herwald, Heiko

    2008-01-01

    During the last years, the importance of antibacterial peptides has attracted considerable attention. We report here that peptides derived from the fifth domain of beta-2 glycoprotein I (beta(2)GPI), a human heparin binding plasma protein, have antibacterial activities against Gram-positive and

  14. Gene expression profiling provides insights into the immune mechanism of Plutella xylostella midgut to microbial infection.

    Science.gov (United States)

    Lin, Junhan; Xia, Xiaofeng; Yu, Xiao-Qiang; Shen, Jinhong; Li, Yong; Lin, Hailan; Tang, Shanshan; Vasseur, Liette; You, Minsheng

    2018-03-20

    Insect gut immunity plays a key role in defense against microorganism infection. The knowledge of insect gut immunity has been obtained mostly from Drosophila melanogaster. Little is known about gut immunity in the diamondback moth, Plutella xylostella (L.), a pest destroying cruciferous crops worldwide. In this study, expressions of the immune-related genes in the midgut of P. xylostella orally infected with Staphylococcus aureus, Escherichia coli and Pichia pastoris were profiled by RNA-seq and qRT-PCR approaches. The results revealed that the Toll, IMD, JNK and JAK-STAT pathways and possibly the prophenoloxidase activation system in P. xylostella could be activated by oral infections, and moricins, gloverins and lysozyme2 might act as important effectors against microorganisms. Subsequent knock-down of IMD showed that this gene was involved in regulating the expression of down-stream genes in the IMD pathway. Our work indicates that the Toll, IMD, JNK and JAK-STAT pathways may synergistically modulate immune responses in the P. xylostella midgut, implying a complex and diverse immune system in the midgut of insects. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Uncovering molecular structural mechanisms of signaling mediated by the prion protein

    International Nuclear Information System (INIS)

    Romano, Sebastian A.; Linden, Rafael; Silva, Jerson L.; Foguel, Debora

    2009-01-01

    The glycosyl phosphatidylinositol (GPI) - anchored prion protein (PrP c ), usually associated with neurodegenerative diseases, modulates various cellular responses and may scaffold multiprotein cell surface signaling complexes. Engagement of PrP c with the secretable cochaperone hop/STI 1 induces neurotrophic transmembrane signals through unknown molecular mechanisms. We addressed whether interaction of Pr P c and hop STI 1 entails structural rearrangements relevant for signaling. Circular dichroism and fluorescence spectroscopy showed that PrP c :hop/STI 1 interaction triggers loss of PrP helical structures, involving at least a perturbation of the Pr P c 143-153 beta-helix. Novel SAXS models revealed a significant C-terminal compaction of hop/STI 1 when bound to PrP c . Differing from a recent dimeric model of human hop/STI 1, both size exclusion chromatography and SAXS data support a monomeric form of free murine hop/STI 1. Changes in the Pr P c 143-153 beta-helix may engage the transmembrane signaling protein laminin receptor precursor and neural cell adhesion molecule, both of which bind that domain of Pr P c , and further ligands may be engaged by the tertiary structural changes of hop/STI 1. These reciprocal structural modifications indicate a versatile mechanism for signaling mediated by Pr P c :hop/STI 1 interaction, consistent with the hypothesis that Pr P c scaffolds multiprotein signaling complexes at the cell surface. (author)

  16. Uncovering molecular structural mechanisms of signaling mediated by the prion protein

    Energy Technology Data Exchange (ETDEWEB)

    Romano, Sebastian A.; Linden, Rafael [Universidade Federal do Rio de Janeiro (IBCCF/UFRl), RJ (Brazil). Inst. de Biofisica Carlos Chagas Filho; Cordeiro, Yraima; Rocha e Lima, Luis M.T. da [Universidade Federal do Rio de Janeiro (FF/UFRl), RJ (Brazil). Fac. de Farmacia; Lopes, Marilene H. [Instituto Ludwig de Pesquisa de Cancer, Sao Paulo, SP (Brazil); Silva, Jerson L.; Foguel, Debora [Universidade Federal do Rio de Janeiro (IBqM/UFRl), RJ (Brazil). Inst. de Bioquimica Medica

    2009-07-01

    The glycosyl phosphatidylinositol (GPI) - anchored prion protein (PrP{sup c}), usually associated with neurodegenerative diseases, modulates various cellular responses and may scaffold multiprotein cell surface signaling complexes. Engagement of PrP{sup c} with the secretable cochaperone hop/STI 1 induces neurotrophic transmembrane signals through unknown molecular mechanisms. We addressed whether interaction of Pr P{sup c} and hop STI 1 entails structural rearrangements relevant for signaling. Circular dichroism and fluorescence spectroscopy showed that PrP{sup c}:hop/STI 1 interaction triggers loss of PrP helical structures, involving at least a perturbation of the Pr P{sup c}{sub 143-153} beta-helix. Novel SAXS models revealed a significant C-terminal compaction of hop/STI 1 when bound to PrP{sup c}. Differing from a recent dimeric model of human hop/STI 1, both size exclusion chromatography and SAXS data support a monomeric form of free murine hop/STI 1. Changes in the Pr P{sup c}{sub 143-153} beta-helix may engage the transmembrane signaling protein laminin receptor precursor and neural cell adhesion molecule, both of which bind that domain of Pr P{sup c}, and further ligands may be engaged by the tertiary structural changes of hop/STI 1. These reciprocal structural modifications indicate a versatile mechanism for signaling mediated by Pr P{sup c}:hop/STI 1 interaction, consistent with the hypothesis that Pr P{sup c} scaffolds multiprotein signaling complexes at the cell surface. (author)

  17. The Distinct Transcriptional Response of the Midgut of Amblyomma sculptum and Amblyomma aureolatum Ticks to Rickettsia rickettsii Correlates to Their Differences in Susceptibility to Infection

    Directory of Open Access Journals (Sweden)

    Andréa C. Fogaça

    2017-04-01

    Full Text Available Rickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes Rocky Mountain Spotted Fever (RMSF. In Brazil, two species of ticks in the genus Amblyomma, A. sculptum and A. aureolatum, are incriminated as vectors of this bacterium. Importantly, these two species present remarkable differences in susceptibility to R. rickettsii infection, where A. aureolatum is more susceptible than A. sculptum. In the current study, A. aureolatum and A. sculptum ticks were fed on suitable hosts previously inoculated with R. rickettsii, mimicking a natural infection. As control, ticks were fed on non-infected animals. Both midgut and salivary glands of all positively infected ticks were colonized by R. rickettsii. We did not observe ticks with infection restricted to midgut, suggesting that important factors for controlling rickettsial colonization were produced in this organ. In order to identify such factors, the total RNA extracted from the midgut (MG was submitted to next generation RNA sequencing (RNA-seq. The majority of the coding sequences (CDSs of A. sculptum differentially expressed by infection were upregulated, whereas most of modulated CDSs of A. aureolatum were downregulated. The functional categories that comprise upregulated CDSs of A. sculptum, for instance, metabolism, signal transduction, protein modification, extracellular matrix, and immunity also include CDSs of A. aureolatum that were downregulated by infection. This is the first study that reports the effects of an experimental infection with the highly virulent R. rickettsii on the gene expression of two natural tick vectors. The distinct transcriptional profiles of MG of A. sculptum and A. aureolatum upon infection stimulus strongly suggest that molecular factors in this organ are responsible for delineating the susceptibility to R. rickettsii. Functional studies to determine the role played by proteins encoded by differentially expressed CDSs in the acquisition of R

  18. Structure, diversity and evolution of protein toxins from spore-forming entomopathogenic bacteria

    NARCIS (Netherlands)

    Maagd, de R.A.; Bravo, A.; Berry, C.; Crickmore, N.; Schnepf, H.E.

    2003-01-01

    Gram-positive spore-forming entomopathogenic bacteria can utilize a large variety of protein toxins to help them invade, infect, and finally kill their hosts, through their action on the insect midgut. These toxins belong to a number of homology groups containing a diversity of protein structures

  19. Active site characterization and molecular cloning of Tenebrio molitor midgut trehalase and comments on their insect homologs.

    Science.gov (United States)

    Gomez, Ana; Cardoso, Christiane; Genta, Fernando A; Terra, Walter R; Ferreira, Clélia

    2013-08-01

    The soluble midgut trehalase from Tenebrio molitor (TmTre1) was purified after several chromatographic steps, resulting in an enzyme with 58 kDa and pH optimum 5.3 (ionizing active groups in the free enzyme: pK(e1) = 3.8 ± 0.2 pK(e2) = 7.4 ± 0.2). The purified enzyme corresponds to the deduced amino acid sequence of a cloned cDNA (TmTre1-cDNA), because a single cDNA coding a soluble trehalase was found in the T. molitor midgut transcriptome. Furthermore, the mass of the protein predicted to be coded by TmTre1-cDNA agrees with that of the purified enzyme. TmTre1 has the essential catalytic groups Asp 315 and Glu 513 and the essential Arg residues R164, R217, R282. Carbodiimide inactivation of the purified enzyme at different pH values reveals an essential carboxyl group with pKa = 3.5 ± 0.3. Phenylglyoxal modified a single Arg residue with pKa = 7.5 ± 0.2, as observed in the soluble trehalase from Spodoptera frugiperda (SfTre1). Diethylpyrocarbonate modified a His residue that resulted in a less active enzyme with pK(e1) changed to 4.8 ± 0.2. In TmTre1 the modified His residue (putatively His 336) is more exposed than the His modified in SfTre1 (putatively His 210) and that affects the ionization of an Arg residue. The architecture of the active site of TmTre1 and SfTre1 is different, as shown by multiple inhibition analysis, the meaning of which demands further research. Trehalase sequences obtained from midgut transcriptomes (pyrosequencing and Illumina data) from 8 insects pertaining to 5 different orders were used in a cladogram, together with other representative sequences. The data suggest that the trehalase gene went duplication and divergence prior to the separation of the paraneopteran and holometabolan orders and that the soluble trehalase derived from the membrane-bound one by losing the C-terminal transmembrane loop. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Enhancement of the Immunogenicity and Protective Efficacy of a Mucosal Influenza Subunit Vaccine by the Saponin Adjuvant GPI-0100

    NARCIS (Netherlands)

    Liu, Heng; Patil, Harshad P.; de Vries-Idema, Jacqueline; Wilschut, Jan; Huckriede, Anke

    2012-01-01

    Identification of safe and effective adjuvants remains an urgent need for the development of inactivated influenza vaccines for mucosal administration. Here, we used a murine challenge model to evaluate the adjuvant activity of GPI-0100, a saponin-derived adjuvant, on influenza subunit vaccine

  1. Imaging findings of midgut volvuIus associated with a large small-bowel diverticulum in an aduIt patient: case report

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jee Young; Rha, Sung Eun; Oh, Soon Nam; Bo, Seal Hwang; Byun, Jae Young [College of Medicine, The Catholic Univ. of Korea, Seoul (Korea, Republic of)

    2004-05-01

    Although most patients with jejunoileal diverticulum are asymptomatic, a large, small-bowel diverticulum can be associated with midgut volvulus in an adult. We present a rare case of midgut volvulus that was associated with a large, small-bowel diverticulum in a 77-year-old woman presenting with chronic recurrent abdominal pain. The CT showed the characteristic whirl sign of twisted mesentery, the small bowel loops along the superior mesenteric artery and a large sac-like small-bowel diverticulum. A small bowel series also demonstrated a corkscrew appearance of proximal jejunum, a finding suggestive of midgut volvulus, and a large jejunal diverticulum. During the laparotomy, the small bowel was seen twisted counterclockwise 270 .deg.. The mesenteric root was very shortened. A 4 cm sized diverticulum was seen on the mesenteric border of jejunum, on the portion about 40 cm distal from the Treitz ligament.

  2. Imaging findings of midgut volvuIus associated with a large small-bowel diverticulum in an aduIt patient: case report

    International Nuclear Information System (INIS)

    Kim, Jee Young; Rha, Sung Eun; Oh, Soon Nam; Bo, Seal Hwang; Byun, Jae Young

    2004-01-01

    Although most patients with jejunoileal diverticulum are asymptomatic, a large, small-bowel diverticulum can be associated with midgut volvulus in an adult. We present a rare case of midgut volvulus that was associated with a large, small-bowel diverticulum in a 77-year-old woman presenting with chronic recurrent abdominal pain. The CT showed the characteristic whirl sign of twisted mesentery, the small bowel loops along the superior mesenteric artery and a large sac-like small-bowel diverticulum. A small bowel series also demonstrated a corkscrew appearance of proximal jejunum, a finding suggestive of midgut volvulus, and a large jejunal diverticulum. During the laparotomy, the small bowel was seen twisted counterclockwise 270 .deg.. The mesenteric root was very shortened. A 4 cm sized diverticulum was seen on the mesenteric border of jejunum, on the portion about 40 cm distal from the Treitz ligament

  3. 20-hydroxyecdysone positively regulates the transcription of the antimicrobial peptide, lebocin, via BmEts and BmBR-C Z4 in the midgut of Bombyx mori during metamorphosis.

    Science.gov (United States)

    Mai, Taoyi; Chen, Shuna; Lin, Xianyu; Zhang, Xiaojuan; Zou, Xiaopeng; Feng, Qili; Zheng, Sichun

    2017-09-01

    Metamorphosis is an essential physiological process in insects. This process is triggered by 20-hydroxyecydsone (20E). Lebocin, an antimicrobial peptide of Lepidoptera insects, was significantly up-regulated in the midgut, but not in the fat body of Bombyx mori during metamorphosis. In this study, the expression regulation of lebocin in B. mori midgut was studied. The results showed that B. mori lebocin and its activator BmEts were not responsive to bacterial infection in the midgut, instead, the expression of both genes was up-regulated by 20E treatment. The transcription factor BR-C Z4 in the 20E signal pathway enhanced lebocin promoter activity by directly binding to an upstream cis-response element of the promoter. In the fat body, the mRNA level of B. mori lebocin was decreased when the insect transformed from larval to pupal stage and was increased by immune challenge. The expression profiles of lebocin in Lepidopteran Spodoptera litura was also analyzed and the similar results were observed, S. litura lebocin was significantly up-regulated during midgut regeneration and mainly present in the new-formed intestinal cells of the midgut. All results together suggest that during metamorphosis 20E may activate lebocin expression via BmBR-C Z4 and BmEts in the midgut, where the antimicrobial peptide was produced to protect the midgut from infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Biomechanical comparison of traditional anchors to all-suture anchors in a double-row rotator cuff repair cadaver model.

    Science.gov (United States)

    Goschka, Andrew M; Hafer, Jason S; Reynolds, Kirk A; Aberle, Nicholas S; Baldini, Todd H; Hawkins, Monica J; McCarty, Eric C

    2015-10-01

    To further reduce the invasiveness of arthroscopic rotator cuff repair surgery the all-suture anchor has been developed. The all-suture anchor requires less bone removal and reduces the potential of loose body complications. The all-suture anchor must also have adequate biomechanical strength for the repair to heal. The hypothesis is there is no significant difference in the biomechanical performance of supraspinatus repairs using an all-suture anchor when compared to traditional solid-body suture anchors. Using nine shoulders per group, the supraspinatus tendon was dissected from the greater tuberosity. The four different double row repairs tested were (medial row/lateral row): A: ICONIX2/ICONIX2; B: ICONIX2/Stryker ReelX 3.9mm; C: ICONIX2/Stryker ReelX 4.5mm; D: Arthrex BioComposite CorkScrew FT 4.5mm/Arthrex BioComposite SwiveLock 4.75mm. The ICONIX2 was the only all-suture anchor tested. Tendons underwent cyclic loading from 10 to 100N for 500 cycles, followed by load-to-failure. Data was collected at cycles 5, 100, 200, 300, 400, and 500. One-way ANOVA analysis was used to assess significance (P≤0.05). The anchor combinations tested did not differ significantly in anterior (P>0.4) or posterior (P>0.3) gap formation, construct stiffness (P>0.7), ultimate load (P=0.06), or load to 5mm gap formation (P=0.84). The all-suture anchor demonstrated comparable biomechanical performance in multiple double-row anchor combinations to a combination of traditional solid-body anchors. Thus it may be an attractive option to further reduce the invasiveness of rotator cuff repairs. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    OpenAIRE

    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 root...

  6. An atypical clinical presentation of acute appendicitis in a young man with midgut malrotation

    International Nuclear Information System (INIS)

    Pinto, Antonio; Di Raimondo, Domenico; Tuttolomondo, Antonino; Fernandez, Paola; Caronia, Aurelio; Lagalla, Roberto; Arnao, Valentina; Law, Robert L.; Licata, Giuseppe

    2007-01-01

    Midgut malrotation occurs as a result of failure in normal intestinal rotation and fixation during early pregnancy. Pathological conditions reported in the literature involving midgut malrotation predominantly relate to infants and children. In adults malrotation is often revealed as an incidental finding on computed tomography (CT), or the associated altered anatomy can be the cause of atypical clinical symptoms of relatively common intestinal disorders. An unusual presentation of acute appendicitis, with fever and recurrent pain in left iliac fossa is reported. Underlying intestinal malrotation delayed the correct clinical diagnosis of acute appendicitis. It was not until a CT scan was performed that a malrotation was identified. The predominant appearances of malrotation are the siting of the ascending colon, caecum (and appendix) in the left side of the abdomen and the right-sided placement of the duodenojejunal junction

  7. Effects of tannic acid on trypsin and leucine aminopeptidase activities in gypsy moth larval midgut

    Directory of Open Access Journals (Sweden)

    Mrdaković Marija

    2013-01-01

    Full Text Available The effects of allelochemical stress on genetic variations in the specific activities of gypsy moth digestive enzymes (trypsin and leucine aminopeptidase and relative midgut mass (indirect measure of food consumption, as well as variability in their plasticity, were investigated in fifth instar gypsy moths originating from two populations with different trophic adaptations (oak and locust-tree forests. Thirty-two full-sib families from the Quercus population and twenty-six full-sib families from the Robinia population were reared on an artificial diet with or without supplementation with tannic acid. Between population differences were observed as higher average specific activity of trypsin and relative midgut mass in larvae from the Robinia population. Significant broad-sense heritabilities were observed for the specific activity of trypsin in the control state, and for specific activity of leucine aminopeptidase in a stressful environment. Significantly lower heritability for relative midgut mass was recorded in larvae from the Robinia population reared under stressful conditions. Significant variability of trypsin plasticity in larvae from both populations and significant variability of leucine aminopeptidase plasticity in larvae from the Robinia population point to the potential for the evolution of enzyme adaptive plastic responses to the presence of stressor. Non-significant across-environment genetic correlations do not represent a constraint for the evolution of enzyme plasticity. [Projekat Ministarstva nauke Republike Srbije, br. 173027

  8. Individual co-variation between viral RNA load and gene expression reveals novel host factors during early dengue virus infection of the Aedes aegypti midgut.

    Directory of Open Access Journals (Sweden)

    Vincent Raquin

    2017-12-01

    Full Text Available Dengue virus (DENV causes more human infections than any other mosquito-borne virus. The current lack of antiviral strategies has prompted genome-wide screens for host genes that are required for DENV infectivity. Earlier transcriptomic studies that identified DENV host factors in the primary vector Aedes aegypti used inbred laboratory colonies and/or pools of mosquitoes that erase individual variation. Here, we performed transcriptome sequencing on individual midguts in a field-derived Ae. aegypti population to identify new candidate host factors modulating DENV replication. We analyzed the transcriptomic data using an approach that accounts for individual co-variation between viral RNA load and gene expression. This approach generates a prediction about the agonist or antagonist effect of candidate genes on DENV replication based on the sign of the correlation between gene expression and viral RNA load. Using this method, we identified 39 candidate genes that went undetected by conventional pairwise comparison of gene expression levels between DENV-infected midguts and uninfected controls. Only four candidate genes were detected by both methods, emphasizing their complementarity. We demonstrated the value of our approach by functional validation of a candidate agonist gene encoding a sterol regulatory element-binding protein (SREBP, which was identified by correlation analysis but not by pairwise comparison. We confirmed that SREBP promotes DENV infection in the midgut by RNAi-mediated gene knockdown in vivo. We suggest that our approach for transcriptomic analysis can empower genome-wide screens for potential agonist or antagonist factors by leveraging inter-individual variation in gene expression. More generally, this method is applicable to a wide range of phenotypic traits displaying inter-individual variation.

  9. Microgravity Drill and Anchor System

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  11. Tissue-specific transcriptome profiling of Plutella xylostella third instar larval midgut.

    Science.gov (United States)

    Xie, Wen; Lei, Yanyuan; Fu, Wei; Yang, Zhongxia; Zhu, Xun; Guo, Zhaojiang; Wu, Qingjun; Wang, Shaoli; Xu, Baoyun; Zhou, Xuguo; Zhang, Youjun

    2012-01-01

    The larval midgut of diamondback moth, Plutella xylostella, is a dynamic tissue that interfaces with a diverse array of physiological and toxicological processes, including nutrient digestion and allocation, xenobiotic detoxification, innate and adaptive immune response, and pathogen defense. Despite its enormous agricultural importance, the genomic resources for P. xylostella are surprisingly scarce. In this study, a Bt resistant P. xylostella strain was subjected to the in-depth transcriptome analysis to identify genes and gene networks putatively involved in various physiological and toxicological processes in the P. xylostella larval midgut. Using Illumina deep sequencing, we obtained roughly 40 million reads containing approximately 3.6 gigabases of sequence data. De novo assembly generated 63,312 ESTs with an average read length of 416 bp, and approximately half of the P. xylostella sequences (45.4%, 28,768) showed similarity to the non-redundant database in GenBank with a cut-off E-value below 10(-5). Among them, 11,092 unigenes were assigned to one or multiple GO terms and 16,732 unigenes were assigned to 226 specific pathways. In-depth analysis identified genes putatively involved in insecticide resistance, nutrient digestion, and innate immune defense. Besides conventional detoxification enzymes and insecticide targets, novel genes, including 28 chymotrypsins and 53 ABC transporters, have been uncovered in the P. xylostella larval midgut transcriptome; which are potentially linked to the Bt toxicity and resistance. Furthermore, an unexpectedly high number of ESTs, including 46 serpins and 7 lysozymes, were predicted to be involved in the immune defense.As the first tissue-specific transcriptome analysis of P. xylostella, this study sheds light on the molecular understanding of insecticide resistance, especially Bt resistance in an agriculturally important insect pest, and lays the foundation for future functional genomics research. In addition, current

  12. Tissue-Specific Transcriptome Profiling of Plutella Xylostella Third Instar Larval Midgut

    Science.gov (United States)

    Xie, Wen; Lei, Yanyuan; Fu, Wei; Yang, Zhongxia; Zhu, Xun; Guo, Zhaojiang; Wu, Qingjun; Wang, Shaoli; Xu, Baoyun; Zhou, Xuguo; Zhang, Youjun

    2012-01-01

    The larval midgut of diamondback moth, Plutella xylostella, is a dynamic tissue that interfaces with a diverse array of physiological and toxicological processes, including nutrient digestion and allocation, xenobiotic detoxification, innate and adaptive immune response, and pathogen defense. Despite its enormous agricultural importance, the genomic resources for P. xylostella are surprisingly scarce. In this study, a Bt resistant P. xylostella strain was subjected to the in-depth transcriptome analysis to identify genes and gene networks putatively involved in various physiological and toxicological processes in the P. xylostella larval midgut. Using Illumina deep sequencing, we obtained roughly 40 million reads containing approximately 3.6 gigabases of sequence data. De novo assembly generated 63,312 ESTs with an average read length of 416bp, and approximately half of the P. xylostella sequences (45.4%, 28,768) showed similarity to the non-redundant database in GenBank with a cut-off E-value below 10-5. Among them, 11,092 unigenes were assigned to one or multiple GO terms and 16,732 unigenes were assigned to 226 specific pathways. In-depth analysis indentified genes putatively involved in insecticide resistance, nutrient digestion, and innate immune defense. Besides conventional detoxification enzymes and insecticide targets, novel genes, including 28 chymotrypsins and 53 ABC transporters, have been uncovered in the P. xylostella larval midgut transcriptome; which are potentially linked to the Bt toxicity and resistance. Furthermore, an unexpectedly high number of ESTs, including 46 serpins and 7 lysozymes, were predicted to be involved in the immune defense. As the first tissue-specific transcriptome analysis of P. xylostella, this study sheds light on the molecular understanding of insecticide resistance, especially Bt resistance in an agriculturally important insect pest, and lays the foundation for future functional genomics research. In addition, current

  13. Insect midgut α-mannosidases from family 38 and 47 with emphasis on those of Tenebrio molitor.

    Science.gov (United States)

    Moreira, Nathalia R; Cardoso, Christiane; Ribeiro, Alberto F; Ferreira, Clelia; Terra, Walter R

    2015-12-01

    α-Mannosidases are enzymes which remove non-reducing terminal residues from glycoconjugates. Data on both GH47 and GH38 (Golgi and lysosomal) enzymes are available. Data on insect midgut α-mannosidases acting in digestion are preliminary and do not include enzyme sequences. Tenebrio molitor midgut α-mannosidases were separated by chromatography into two activity peaks: a major (Man1) and a minor (Man2). An antibody generated against a synthetic peptide corresponding to a sequence of α-mannosidase fragment recognizes Man2 but not Man1. That fragment was later found to correspond to TmMan2 (GenBank access KP892646), showing that the cDNA coding for Man2 is actually TmMan2. TmMan2 codes for a mature α-mannosidase with 107.5 kDa. Purified Man2 originates after SDS-PAGE one band of about 72 kDa and another of 51 kDa, which sums 123 kDa, in agreement with gel filtration (123 kDa) data. These results suggest that Man2 is processed into peptides that remain noncovalently linked within the functional enzyme. The physical and kinetical properties of purified Man1 and Man2 are similar. They have a molecular mass of 123 kDa (gel filtration), pH optimum (5.6) and response to inhibitors like swainsonine (Man1 Ki, 68 nM; Man2 Ki, 63 nM) and deoxymannojirimycin (Man1 Ki, 0.12 mM; Man2 Ki, 0.15 mM). Their substrate specificities are a little different as Man2 hydrolyzes α-1,3 and α-1,6 bonds better than α-1,2, whereas the contrary is true for Man1. Thus, they pertain to Class II (GH38 α-mannosidases), that are catabolic α-mannosidases similar to lysosomal α-mannosidase. However, Man2, in contrast to true lysosomal α-mannosidase, is secreted (immunocytolocalization data) into the midgut contents. There, Man2 may participate in digestion of fungal cell walls, known to have α-mannosides in their outermost layer. The amount of family 38 α-mannosidase sequences found in the transcriptome (454 pyrosequencing) of the midgut of 9 insects pertaining to 5 orders is

  14. The Holding Power of Anchors

    Indian Academy of Sciences (India)

    The efficiency of an anchor may be expressed as the ratio (holding force + weight of anchor). In dry sand .... the market at the beginning of the coming season in three sizes, namely 20, 35 and. 60 lb. These are ... Taylor frozen-flow hypothesis.

  15. Epididymosomes: transfer of fertility-modulating proteins to the sperm surface

    OpenAIRE

    Patricia A Martin-DeLeon

    2015-01-01

    A variety of glycosylphosphatidylinositol (GPI)-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP) which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the ...

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

    Science.gov (United States)

    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.

  17. InterProScan Result: FS774270 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available FS774270 FS774270_5_ORF1 FB109746C115A036 PFAM PF04114 Gaa1 NA ? IPR007246 unintegrated Cellular... Component: integral to membrane (GO:0016021)|Cellular Component: GPI-anchor transamidase complex (GO:0042765) ...

  18. InterProScan Result: CK559994 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available CK559994 CK559994_4_ORF2 020DFCD801EB69C2 PFAM PF04114 Gaa1 NA ? IPR007246 unintegrated Cellular... Component: integral to membrane (GO:0016021)|Cellular Component: GPI-anchor transamidase complex (GO:0042765) ...

  19. Anchoring effects on early autobiographical memories.

    Science.gov (United States)

    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. Glycosylphosphatidylinositol-Anchored Anti-HIV scFv Efficiently Protects CD4 T Cells from HIV-1 Infection and Deletion in hu-PBL Mice

    Science.gov (United States)

    Ye, Chaobaihui; Wang, Weiming; Cheng, Liang; Li, Guangming; Wen, Michael; Wang, Qi; Zhang, Qing; Li, Dan

    2016-01-01

    ABSTRACT Despite success in viral inhibition and CD4 T cell recovery by highly active antiretroviral treatment (HAART), HIV-1 is still not curable due to the persistence of the HIV-1 reservoir during treatment. One patient with acute myeloid leukemia who received allogeneic hematopoietic stem cell transplantation from a homozygous CCR5 Δ32 donor has had no detectable viremia for 9 years after HAART cessation. This case has inspired a field of HIV-1 cure research focusing on engineering HIV-1 resistance in permissive cells. Here, we employed a glycosylphosphatidylinositol (GPI)-scFv X5 approach to confer resistance of human primary CD4 T cells to HIV-1. We showed that primary CD4 T cells expressing GPI-scFv X5 were resistant to CCR5 (R5)-, CXCR4 (X4)-, and dual-tropic HIV-1 and had a survival advantage compared to control cells ex vivo. In a hu-PBL mouse study, GPI-scFv X5-transduced CD4 T cells were selected in peripheral blood and lymphoid tissues upon HIV-1 infection. Finally, GPI-scFv X5-transduced CD4 T cells, after being cotransfused with HIV-infected cells, showed significantly reduced viral loads and viral RNA copy numbers relative to CD4 cells in hu-PBL mice compared to mice with GPI-scFv AB65-transduced CD4 T cells. We conclude that GPI-scFv X5-modified CD4 T cells could potentially be used as a genetic intervention against both R5- and X4-tropic HIV-1 infections. IMPORTANCE Blocking of HIV-1 entry is one of most promising approaches for therapy. Genetic disruption of the HIV-1 coreceptor CCR5 by nucleases in T cells is under 2 clinical trials and leads to reduced viremia in patients. However, the emergence of viruses using the CXCR4 coreceptor is a concern for therapies applying single-coreceptor disruption. Here, we report that HIV-1-permissive CD4 T cells engineered with GPI-scFv X5 are resistant to R5-, X4-, or dual-tropic virus infection ex vivo. In a preclinical study using hu-PBL mice, we show that CD4 T cells were protected and that GPI-scFv X5

  1. Preclinical evaluation of the saponin derivative GPI-0100 as an immunostimulating and dose-sparing adjuvant for pandemic influenza vaccines

    NARCIS (Netherlands)

    Liu, Heng; Bungener, Laura; ter Veer, Wouter; Coller, Beth-Ann; Wilschut, Jan; Huckriede, Anke

    2011-01-01

    With the current global influenza vaccine production capacity the large demand for vaccines in case of a pandemic can only be fulfilled when antigen dose sparing strategies are employed. Here we used a murine challenge model to evaluate the potential of GPI-0100, a semi-synthetic saponin derivative,

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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...

  4. Semisynthetic prion protein (PrP) variants carrying glycan mimics at position 181 and 197 do not form fibrils.

    Science.gov (United States)

    Araman, Can; Thompson, Robert E; Wang, Siyao; Hackl, Stefanie; Payne, Richard J; Becker, Christian F W

    2017-09-01

    The prion protein (PrP) is an N -glycosylated protein attached to the outer leaflet of eukaryotic cell membranes via a glycosylphosphatidylinositol (GPI) anchor. Different prion strains have distinct glycosylation patterns and the extent of glycosylation of potentially pathogenic misfolded prion protein (PrP Sc ) has a major impact on several prion-related diseases (transmissible spongiform encephalopathies, TSEs). Based on these findings it is hypothesized that posttranslational modifications (PTMs) of PrP influence conversion of cellular prion protein (PrP C ) into PrP Sc and, as such, modified PrP variants are critical tools needed to investigate the impact of PTMs on the pathogenesis of TSEs. Here we report a semisynthetic approach to generate PrP variants modified with monodisperse polyethyleneglycol (PEG) units as mimics of N-glycans. Incorporating PEG at glycosylation sites 181 and 197 in PrP induced only small changes to the secondary structure when compared to unmodified, wildtype PrP. More importantly, in vitro aggregation was abrogated for all PEGylated PrP variants under conditions at which wildtype PrP aggregated. Furthermore, the addition of PEGylated PrP as low as 10 mol% to wildtype PrP completely blocked aggregation. A similar effect was observed for synthetic PEGylated PrP segments comprising amino acids 179-231 alone if these were added to wildtype PrP in aggregation assays. This behavior raises the question if large N-glycans interfere with aggregation in vivo and if PEGylated PrP peptides could serve as potential therapeutics.

  5. TAT improves in vitro transportation of fortilin through midgut and into hemocytes of white shrimp Litopenaeus vannamei

    Science.gov (United States)

    Zhou, Yi; Zhang, Wenbing; Mai, Kangsen; Xu, Wei; Zhang, Yanjiao; Ai, Qinghui; Wang, Xiaojie

    2012-06-01

    Fortilin is a multifunctional protein implicated in many important cellular processes. Since injection of Pm-fortilin reduces shrimp mortality caused by white spot syndrome virus (WSSV), there is potential application of fortilin in shrimp culture. In the present study, in order to improve trans-membrane transportation efficiency, the protein transduction domain of the transactivator of transcription (TAT) peptide was fused to fortilin. The Pichia pastoris yeast expression system, which is widely accepted in animal feeds, was used for production of recombinant fusion protein. Green fluorescence protein (GFP) was selected as a reporter because of its intrinsic visible fluorescence. The fortilin, TAT and GFP fusion protein were constructed. Their trans-membrane transportation efficiency and effects on immune response of shrimp were analyzed in vitro. Results showed that TAT peptide improved in vitro uptake of fortilin into the hemocytes and midgut of Litopenaeus vannamei. The phenoloxidase (PO) activity of hemocytes incubated with GFP-Fortilin or GFP-Fortilin-TAT was significantly increased compared with that in the control without expressed fortilin. The PO activity of hemocytes incubated with 200 μg mL-1 GFP-Fortilin-TAT was significantly higher than that in the group with the same concentration of GFP-Fortilin. Hemocytes incubated with GFP-Fortilin-TAT at all concentrations showed significantly higher nitric oxide synthase (NOS) activity than those in the control or in the GFP-Fortilin treatment. The present in vitro study indicated that TAT fusion protein improved the immune effect of fortilin.

  6. A MIDGUT DIGESTIVE PHOSPHOLIPASE A2 IN LARVAL MOSQUITOES, AEDES ALBOPICTUS AND CULEX QUINQUEFASCIATUS

    Science.gov (United States)

    Phospholipase A2 (PLA2) is a secretory digestive enzyme that hydrolyzes ester bond at sn-2 position of dietary phospholipids, creating free fatty acid and lysophopholipid. The free fatty acids (arachidonic acid) are absorbed into midgut cells. Aedes albopictus and Culex quinquefasciatus digestive PL...

  7. Desenvolvimento pós-embrionário do intestino médio de Dermatobia hominis (Linnaeus Jr. (Diptera, Cuterebridae Post-embryonic development of Dermatobia hominis (Linnaeus Jr. (Diptera, Cuterebridae midgut

    Directory of Open Access Journals (Sweden)

    Edy de Lello

    2001-03-01

    Full Text Available Dermatobia hominis (Linnaeus, 1781 midgut is internally lined by an epithelium of polytenic cells, some low others prismatic with well developed brush border. Their apical portion are enlarged by secretory vesicles, forming button-like structures that are pinched off to the lumen, some accompained by the nucleus characterizing apocrine and holocrine secretions. This epithelium is gradually renewed by small, non polytenic regenerative cells, found scattered at its basal portion. At the end of the third instar the metamorphosis begins. The epithelial cells present signs of degeneration and at the first day of pupation the regenerative cells increase in number. By the 5th day of pupation these regenerative cells, besides being increased in number, differentiate themselves into two layers: one similar to the dense conective tissue that sustainning the larval epithelium is pinched off to the midgut lumen forming the "yellow bodies"; the other, develops right under it as the imaginal epitelium. The disorganized muscles bundles of the midgut wall, are invaded by phagocytes. At the end of pupation the midgut has a low prismatic epithelium with brush-border. In the adult, the torax portion of the midgut has prismatic homogeneously basophilic epithelium while in the abdominal portion the epithelium is made of high prismatic cells full of small vacuoles. The larval midgut epithelium suffers programmed cell death non compatible with apoptose. During the metamorphosis the midgut lenght diminishes from 31mm in the larva to 14mm in the adult.

  8. Exploring the Strengths and Limits of Strong and Weak Sustainability Indicators: A Case Study of the Assessment of China’s Megacities with EF and GPI

    Directory of Open Access Journals (Sweden)

    Lu Huang

    2018-01-01

    Full Text Available The perspective of strong/weak sustainability has a great impact on sustainability assessment. In this study, two most widely used indices, Ecological Footprint (EF and Genuine Progress Indicator (GPI for strong and weak sustainability assessment, were employed to evaluate the sustainability of China’s ten megacities between 1978 and 2015. The results showed that the ecological footprint had been enlarged in the past twenty years; while the genuine economic welfare started to increase since 2005. The cities of Xi’an, Chengdu, Chongqing, and Shanghai met the threshold of below 2.5 global hectares for EF/capita, and over 3000 dollars/capita (in 2010 US$ for GPI/capita. By analyzing and comparing the characteristics, the processes and results, and the complementary features of evaluation methods of EF and GPI, the research suggested that: (1 Strong and weak sustainability indicators, with their own pros/cons in sustainability assessment, should be used carefully; (2 Weak sustainability indicators could be analyzed from the perspective of strong sustainability; (3 Strong sustainability indicators need to be developed urgently. The results in this study could guide the selection of sustainability indicators, and help interpret the results of sustainability assessment.

  9. Alteration of carbohydrates metabolism and midgut glucose absorption in Gromphadorhina portentosa after subchronic exposure to imidacloprid and fenitrothion.

    Science.gov (United States)

    Sawczyn, Tomasz; Dolezych, Bogdan; Klosok, Marcin; Augustyniak, Maria; Stygar, Dominika; Buldak, Rafal J; Kukla, Michal; Michalczyk, Katarzyna; Karcz-Socha, Iwona; Zwirska-Korczala, Krystyna

    2012-01-01

    This study was undertaken to test the hypothesis that following exposure to insecticides, changes take place in the metabolism of carbohydrates and absorption in the midgut of insects. The Madagascar hissing cockroach (Gromphadorhina portentosa) was chosen for the experiment as a model organism, due to it being easy to breed and its relatively large alimentary tract, which was important when preparing the microperfusion midgut bioassay. In each group of cockroaches treated with imidacloprid and fenitrothion, absorption of glucose, expressed as the area under the curve (AUC), was elevated compared to the control group. Glucose in the hemolymph of the examined insects was present in a vestigial amount, often below the threshold of determination, so the determinable carbohydrate indices were: hemolymph trehalose concentration and fat body glycogen content. The level of trehalose found in the hemolymph of insects when exposed to fenitrothion, and irrespective of the level of concentration mixed into food, were significantly lower when comparing to the control samples. Imidacloprid acted analogically with one exception at the concentration of 10 mg·kg(-1) dry food where trehalose concentration did not differ from the control values. Coupling with fat body glycogen concentration was less visible and appeared only at the concentrations of 5 and 10 mg imidacloprid·kg(-1) dry food. As described in this study changes in the sugar distribution and midgut glucose absorption indicate that insects cover the increased energy needs induced by insecticides; also at the gastrointestinal tract level. The result indicates that the midgut glucose absorption parameters could be considered as a non-specific biomarker of insecticide toxicity.

  10. Differential Midgut Attachment of Leishmania (Viannia braziliensis in the Sand Flies Lutzomyia (Nyssomyia whitmani and Lutzomyia (Nyssomyia intermedia

    Directory of Open Access Journals (Sweden)

    Rodrigo P. Soares

    2010-01-01

    Full Text Available The interaction between Leishmania and sand flies has been demonstrated in many Old and New World species. Besides the morphological differentiation from procyclic to infective metacyclic promastigotes, the parasite undergoes biochemical transformations in its major surface lipophosphoglycan (LPG. An upregulation of β-glucose residues was previously shown in the LPG repeat units from procyclic to metacyclic phase in Leishmania (Viannia braziliensis, which has not been reported in any Leishmania species. LPG has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the Subgenus Leishmania. These adaptations were explored for the first time in a species from the Subgenus Viannia, L. (V. braziliensis with its natural vectors Lutzomyia (Nyssomyia intermedia and Lutzomyia (Nyssomyia whitmani. Using two in vitro binding techniques, phosphoglycans (PGs derived from procyclic and metacyclic parasites were able to bind to the insect midgut and inhibit L. braziliensis attachment. Interestingly, L. braziliensis procyclic parasite attachment was ∼11-fold greater in the midgut of L. whitmani than in L. intermedia. The epidemiological relevance of L. whitmani as a vector of American Cutaneous Leishmaniasis (ACL in Brazil is discussed.

  11. Improving performance by anchoring movement and "nerves".

    Science.gov (United States)

    Iso-Ahola, Seppo E; Dotson, Charles O; Jagodinsky, Adam E; Clark, Lily C; Smallwood, Lorraine L; Wilburn, Christopher; Weimar, Wendi H; Miller, Matthew W

    2016-10-01

    Golf's governing bodies' recent decision to ban all putting styles "anchoring one end of the club against the body" bridges an important practical problem with psychological theory. We report the first experiment testing whether anchoring provides technical and/or psychological advantage in competitive performance. Many "greats" of professional golf from Arnold Palmer and Jack Nicklaus to Tiger Woods have argued against anchoring, believing that it takes "nerves" out of competitive performance and therefore artificially levels the playing field. To shed more light on the issue, we tested participants' performance with anchored and unanchored putters under low and high pressure when controlling for the putter length. We found no statistically significant evidence for a technical advantage due to anchoring but a clear psychological advantage: participants who anchored their putters significantly outperformed unanchored counterparts under high, but not low, pressure. Results provide tentative evidence for the ban's justification from a competitive standpoint. However, before any definite conclusions can be made, more research is needed when using high-level golfers. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Testing and modeling of cyclically loaded rock anchors

    Directory of Open Access Journals (Sweden)

    Joar Tistel

    2017-12-01

    Full Text Available The Norwegian Public Roads Administration (NPRA is planning for an upgrade of the E39 highway route at the westcoast of Norway. Fixed links shall replace ferries at seven fjord crossings. Wide spans and large depths at the crossings combined with challenging subsea topography and environmental loads call for an extension of existing practice. A variety of bridge concepts are evaluated in the feasibility study. The structures will experience significant loads from deadweight, traffic and environment. Anchoring of these forces is thus one of the challenges met in the project. Large-size subsea rock anchors are considered a viable alternative. These can be used for anchoring of floating structures but also with the purpose of increasing capacity of fixed structures. This paper presents first a thorough study of factors affecting rock anchor bond capacity. Laboratory testing of rock anchors subjected to cyclic loading is thereafter presented. Finally, the paper presents a model predicting the capacity of a rock anchor segment, in terms of a ribbed bar, subjected to a cyclic load history. The research assumes a failure mode occurring in the interface between the rock anchor and the surrounding grout. The constitutive behavior of the bonding interface is investigated for anchors subjected to cyclic one-way tensile loads. The model utilizes the static bond capacity curve as a basis, defining the ultimate bond τbu and the slip s1 at τbu. A limited number of input parameters are required to apply the model. The model defines the bond-slip behavior with the belonging rock anchor capacity depending on the cyclic load level (τmax cy/τbu, the cyclic load ratio (R = τmin cy/τmax cy, and the number of load cycles (N. The constitutive model is intended to model short anchor lengths representing an incremental length of a complete rock anchor.

  13. Baculoviral mid-gut gland necrosis (BMN) of kuruma shrimp (Penaeus japonicus) larvae in Japanese intensive culture systems

    Science.gov (United States)

    Sano, T.; Nishimura, T.; Fukuda, H.; Hayashida, T.; Momoyama, K.

    1984-03-01

    In many shrimp farms in the Kyushu and Chugoku areas of Japan, the so-called mid-gut gland cloudy disease of kuruma shrimp larvae (Penaeus japonicus) has occurred since 1971. The pathological changes associated with this baculoviral mid-gut gland necrosis (BMN) are extensive cellular necrosis, collapse of mid-gut gland cells, nuclear hypertrophy and finally karyorrhexis. Electron microscopic examination revealed the presence of virions and virogenic stages in the affected nuclei. Average length and diameter of the virions detected was 310 and 72 nm, respectively; nucleocapsids were 250 nm in size. Virions enclosing 2 nucleocapsids within a single envelope were rarely found. The spirally arranged capsomeres were at an angle of 37 to 38° to a horizontal line meeting at right angles with the long axis of the virion. Infectivity trials resulted in high mortality of healthy mysis and juveniles (2nd post-larval stage). Juveniles at the 9th post-larval stage showed no mortality, although they could be infected easily by the agent. Hypertrophied nuclei in squashed and stained preparations of the affected gland cells can be considered to be of reliable presumptive diagnostic character, and fluorescent antibody staining can be employed to confirm the diagnosis of BMN.

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

    Science.gov (United States)

    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.

  15. Requirement of matrix metalloproteinase-1 for intestinal homeostasis in the adult Drosophila midgut

    International Nuclear Information System (INIS)

    Lee, Shin-Hae; Park, Joung-Sun; Kim, Young-Shin; Chung, Hae-Young; Yoo, Mi-Ae

    2012-01-01

    Stem cells are tightly regulated by both intrinsic and extrinsic signals as well as the extracellular matrix (ECM) for tissue homeostasis and regenerative capacity. Matrix metalloproteinases (MMPs), proteolytic enzymes, modulate the turnover of numerous substrates, including cytokine precursors, growth factors, and ECM molecules. However, the roles of MMPs in the regulation of adult stem cells are poorly understood. In the present study, we utilize the Drosophila midgut, which is an excellent model system for studying stem cell biology, to show that Mmp1 is involved in the regulation of intestinal stem cells (ISCs). The results showed that Mmp1 is expressed in the adult midgut and that its expression increases with age and with exposure to oxidative stress. Mmp1 knockdown or Timp-overexpressing flies and flies heterozygous for a viable, hypomorphic Mmp1 allele increased ISC proliferation in the gut, as shown by staining with an anti-phospho-histone H3 antibody and BrdU incorporation assays. Reduced Mmp1 levels induced intestinal hyperplasia, and the Mmp1depletion-induced ISC proliferation was rescued by the suppression of the EGFR signaling pathway, suggesting that Mmp1 regulates ISC proliferation through the EGFR signaling pathway. Furthermore, adult gut-specific knockdown and whole-animal heterozygotes of Mmp1 increased additively sensitivity to paraquat-induced oxidative stress and shortened lifespan. Our data suggest that Drosophila Mmp1 is involved in the regulation of ISC proliferation for maintenance of gut homeostasis. -- Highlights: ► Mmp1 is expressed in the adult midgut. ► Mmp1 is involved in the regulation of ISC proliferation activity. ► Mmp1-related ISC proliferation is associated with EGFR signaling. ► Mmp1 in the gut is required for the intestinal homeostasis and longevity.

  16. Requirement of matrix metalloproteinase-1 for intestinal homeostasis in the adult Drosophila midgut

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Shin-Hae; Park, Joung-Sun [Department of Molecular Biology, College of Natural Science, Pusan National University, Busan 609-735 (Korea, Republic of); Kim, Young-Shin [Research Institute of Genetic Engineering, Pusan National University, Busan 609-735 (Korea, Republic of); Chung, Hae-Young [Molecular Inflammation Research Center for Aging Intervention (MRCA), College of Pharmacy, Pusan National University, Busan 609-735 (Korea, Republic of); Yoo, Mi-Ae, E-mail: mayoo@pusan.ac.kr [Department of Molecular Biology, College of Natural Science, Pusan National University, Busan 609-735 (Korea, Republic of)

    2012-03-10

    Stem cells are tightly regulated by both intrinsic and extrinsic signals as well as the extracellular matrix (ECM) for tissue homeostasis and regenerative capacity. Matrix metalloproteinases (MMPs), proteolytic enzymes, modulate the turnover of numerous substrates, including cytokine precursors, growth factors, and ECM molecules. However, the roles of MMPs in the regulation of adult stem cells are poorly understood. In the present study, we utilize the Drosophila midgut, which is an excellent model system for studying stem cell biology, to show that Mmp1 is involved in the regulation of intestinal stem cells (ISCs). The results showed that Mmp1 is expressed in the adult midgut and that its expression increases with age and with exposure to oxidative stress. Mmp1 knockdown or Timp-overexpressing flies and flies heterozygous for a viable, hypomorphic Mmp1 allele increased ISC proliferation in the gut, as shown by staining with an anti-phospho-histone H3 antibody and BrdU incorporation assays. Reduced Mmp1 levels induced intestinal hyperplasia, and the Mmp1depletion-induced ISC proliferation was rescued by the suppression of the EGFR signaling pathway, suggesting that Mmp1 regulates ISC proliferation through the EGFR signaling pathway. Furthermore, adult gut-specific knockdown and whole-animal heterozygotes of Mmp1 increased additively sensitivity to paraquat-induced oxidative stress and shortened lifespan. Our data suggest that Drosophila Mmp1 is involved in the regulation of ISC proliferation for maintenance of gut homeostasis. -- Highlights: Black-Right-Pointing-Pointer Mmp1 is expressed in the adult midgut. Black-Right-Pointing-Pointer Mmp1 is involved in the regulation of ISC proliferation activity. Black-Right-Pointing-Pointer Mmp1-related ISC proliferation is associated with EGFR signaling. Black-Right-Pointing-Pointer Mmp1 in the gut is required for the intestinal homeostasis and longevity.

  17. Wood cell-wall structure requires local 2D-microtubule disassembly by a novel plasma membrane-anchored protein.

    Science.gov (United States)

    Oda, Yoshihisa; Iida, Yuki; Kondo, Yuki; Fukuda, Hiroo

    2010-07-13

    Plant cells have evolved cortical microtubules, in a two-dimensional space beneath the plasma membrane, that regulate patterning of cellulose deposition. Although recent studies have revealed that several microtubule-associated proteins facilitate self-organization of transverse cortical microtubules, it is still unknown how diverse patterns of cortical microtubules are organized in different xylem cells, which are the major components of wood. Using our newly established in vitro xylem cell differentiation system, we found that a novel microtubule end-tracking protein, microtubule depletion domain 1 (MIDD1), was anchored to distinct plasma membrane domains and promoted local microtubule disassembly, resulting in pits on xylem cell walls. The introduction of RNA interference for MIDD1 resulted in the failure of local microtubule depletion and the formation of secondary walls without pits. Conversely, the overexpression of MIDD1 reduced microtubule density. MIDD1 has two coiled-coil domains for the binding to microtubules and for the anchorage to plasma membrane domains, respectively. Combination of the two coils caused end tracking of microtubules during shrinkage and suppressed their rescue events. Our results indicate that MIDD1 integrates spatial information in the plasma membrane with cortical microtubule dynamics for determining xylem cell wall pattern. Copyright 2010 Elsevier Ltd. All rights reserved.

  18. Comparative analysis of carbohydrate residues in the midgut of phlebotomines (Diptera: Psychodidae) from colony and field populations from Amazon, Brazil.

    Science.gov (United States)

    de Oliveira, Davi Marcos Souza; da Silva, Bruno José Martins; de Sena, Chubert Bernardo Castro; Lima, José Aprígio Nunes; Vasconcelos Dos Santos, Thiago; Silveira, Fernando Tobias; Silva, Edilene Oliveira

    2016-09-01

    Leishmaniasis are worldwide diseases that occur in 98 countries including Brazil, transmitted by the bite of female phlebotomines during blood feeding. In Brazil it is known that some species of sand flies as Lutzomyia longipalpis sensun latum (vector of Leishmania infantum chagasi), Lutzomyia flaviscutellata (vector of Leishmania (Leishmania) amazonensis) and Lutzomyia antunesi [suspected vector of Leishmania (Viannia) lindenbergi] are incriminated of transmitting the parasite Leishmania for the vertebrate host. The phlebotomine-parasite is mediated by the attachment of the promastigote lipophosphoglycan (LPG) to the midgut epithelium. However, another mechanism that is LPG-independent and mediated by N-acetyl-galactosamine (GalNAc) seems to occur in some species of phlebotomines that are classified as permissive. The aim of this study was to characterize the carbohydrate residues that, probably, play a role in parasite attachment to the midgut of phlebotomine from colony and field populations from the Brazilian Amazonian region. We observed the presence of GalNAc, mannose, galactose and GlcNAc in all phlebotomine species. A binding assay between L. (L.) amazonensis and L. i.chagasi to the midguts of different species of phlebotomines was performed. The attachment of both Leishmania and vector species suggests the presence of GalNAc on the midgut surfaces. Thus, these results suggested that GalNAc is a possible binding sites of Leishmania in sand flies from the Brazilian Amazonian region. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Cell wall structure suitable for surface display of proteins in Saccharomyces cerevisiae.

    Science.gov (United States)

    Matsuoka, Hiroyuki; Hashimoto, Kazuya; Saijo, Aki; Takada, Yuki; Kondo, Akihiko; Ueda, Mitsuyoshi; Ooshima, Hiroshi; Tachibana, Taro; Azuma, Masayuki

    2014-02-01

    A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β-Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9-fold higher than that of the wild-type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild-type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild-type strain. The relative value (mnn2 deletion mutant/wild-type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β-glucosidase activity using p-nitrophenyl-β-glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high-molecular-weight substrates to the active sites of the displayed enzymes. Copyright © 2014 John Wiley & Sons, Ltd.

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

    Science.gov (United States)

    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.

  1. Raft-based sphingomyelin interactions revealed by new fluorescent sphingomyelin analogs

    Science.gov (United States)

    Kinoshita, Masanao; Suzuki, Kenichi G.N.; Takada, Misa; Ano, Hikaru; Abe, Mitsuhiro; Makino, Asami; Kobayashi, Toshihide; Hirosawa, Koichiro M.; Fujiwara, Takahiro K.; Murata, Michio

    2017-01-01

    Sphingomyelin (SM) has been proposed to form cholesterol-dependent raft domains and sphingolipid domains in the plasma membrane (PM). How SM contributes to the formation and function of these domains remains unknown, primarily because of the scarcity of suitable fluorescent SM analogs. We developed new fluorescent SM analogs by conjugating a hydrophilic fluorophore to the SM choline headgroup without eliminating its positive charge, via a hydrophilic nonaethylene glycol linker. The new analogs behaved similarly to the native SM in terms of their partitioning behaviors in artificial liquid order-disorder phase-separated membranes and detergent-resistant PM preparations. Single fluorescent molecule tracking in the live-cell PM revealed that they indirectly interact with each other in cholesterol- and sphingosine backbone–dependent manners, and that, for ∼10–50 ms, they undergo transient colocalization-codiffusion with a glycosylphosphatidylinositol (GPI)-anchored protein, CD59 (in monomers, transient-dimer rafts, and clusters), in CD59-oligomer size–, cholesterol-, and GPI anchoring–dependent manners. These results suggest that SM continually and rapidly exchanges between CD59-associated raft domains and the bulk PM. PMID:28330937

  2. Differential trypanosome surface coat regulation by a CCCH protein that co-associates with procyclin mRNA cis-elements.

    Directory of Open Access Journals (Sweden)

    Pegine Walrad

    2009-02-01

    Full Text Available The genome of Trypanosoma brucei is unusual in being regulated almost entirely at the post-transcriptional level. In terms of regulation, the best-studied genes are procyclins, which encode a family of major surface GPI-anchored glycoproteins (EP1, EP2, EP3, GPEET that show differential expression in the parasite's tsetse-fly vector. Although procyclin mRNA cis-regulatory sequences have provided the paradigm for post-transcriptional control in kinetoplastid parasites, trans-acting regulators of procyclin mRNAs are unidentified, despite intensive effort over 15 years. Here we identify the developmental regulator, TbZFP3, a CCCH-class predicted RNA binding protein, as an isoform-specific regulator of Procyclin surface coat expression in trypanosomes. We demonstrate (i that endogenous TbZFP3 shows sequence-specific co-precipitation of EP1 and GPEET, but not EP2 and EP3, procyclin mRNA isoforms, (ii that ectopic overexpression of TbZFP3 does not perturb the mRNA abundance of procyclin transcripts, but rather that (iii their protein expression is regulated in an isoform-specific manner, as evidenced by mass spectrometric analysis of the Procyclin expression signature in the transgenic cell lines. The TbZFP3 mRNA-protein complex (TbZFP3mRNP is identified as a trans-regulator of differential surface protein expression in trypanosomes. Moreover, its sequence-specific interactions with procyclin mRNAs are compatible with long-established predictions for Procyclin regulation. Combined with the known association of TbZFP3 with the translational apparatus, this study provides a long-sought missing link between surface protein cis-regulatory signals and the gene expression machinery in trypanosomes.

  3. Ringstone anchors from Gujarat, west coast of India

    Digital Repository Service at National Institute of Oceanography (India)

    Gaur, A.S.; Sundaresh; Tripati, S.; Bandodkar, S.N.

    of Dwarka and Somanath have yielded several ringstone anchors along with other stone anchors such as triangular and grapnel types. The raw material used for these ring stones comprises basalt, sandstone and limestone. Earlier, these anchors were identified...

  4. Spin selection at organic spinterface by anchoring group

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Zhao; Qiu, Shuai; Miao, Yuan-yuan; Ren, Jun-feng; Wang, Chuan-kui [School of Physics and Electronics, Shandong Normal University, Jinan 250014 (China); Hu, Gui-chao, E-mail: hgc@sdnu.edu.cn [School of Physics and Electronics, Shandong Normal University, Jinan 250014 (China); Institute of Theoretical Physics, Technische Universität Dresden, 01062 Dresden (Germany)

    2017-07-01

    Highlights: • The sign of interfacial spin polarization can be selected by using different anchoring groups. • A sp{sup 3}-d or sp-d hybridization may occur and induce spin polarization when the anchoring group changes. • Interfacial spin polarization depends on both the type of the outer orbital of the anchoring atom as well as its energy. - Abstract: Control of organic interfacial spin polarization is crucial in organic spintronics. Based on ab initio theory, here we proposed a spin selection at organic interface via anchoring group by adsorbing an organic molecule onto Ni(111) surface. The results demonstrate that either a positive or negative interfacial spin polarization may be obtained by choosing different anchoring groups. The orbital analysis via the projected density of states shows that the interfacial spin polarization is sensitive to the hybridization of the outer orbital of the anchoring atom as well as its energy relative to the d orbital of the ferromagnetic atom. The work indicates a feasible way to realize spin selection at the organic spinterface by anchoring group.

  5. QUASIMODO, a novel GPI-anchored zona pellucida protein involved in light input to the Drosophila circadian clock

    Czech Academy of Sciences Publication Activity Database

    Chen, K. F.; Peschel, N.; Závodská, Radka; Sehadová, Hana; Stanewsky, R.

    2011-01-01

    Roč. 21, č. 9 (2011), s. 719-729 ISSN 0960-9822 R&D Projects: GA MŠk LC07032 Institutional research plan: CEZ:AV0Z50070508 Keywords : QUASIMODO * Drosophila * circadian clock Subject RIV: ED - Physiology Impact factor: 9.647, year: 2011

  6. High-Resolution FRET Microsopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Mambranes

    Czech Academy of Sciences Publication Activity Database

    Kenworthy, A. K.; Brdičková, Naděžda; Edidin, M.

    2000-01-01

    Roč. 11, č. 5 (2000), s. 1645-1655 ISSN 0248-4900 R&D Projects: GA AV ČR IAA7052904 Institutional research plan: CEZ:AV0Z5052915 Keywords : High-Resolution * Cholera Toxin * Cell Plasma Membrane s Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.670, year: 2000

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

    Directory of Open Access Journals (Sweden)

    Yuan Zhuang

    Full Text Available 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.

  8. Proteomic Identification of Dengue Virus Binding Proteins in Aedes aegypti Mosquitoes and Aedes albopictus Cells

    Directory of Open Access Journals (Sweden)

    Maria de Lourdes Muñoz

    2013-01-01

    Full Text Available The main vector of dengue in America is the mosquito Aedes aegypti, which is infected by dengue virus (DENV through receptors of midgut epithelial cells. The envelope protein (E of dengue virus binds to receptors present on the host cells through its domain III that has been primarily recognized to bind cell receptors. In order to identify potential receptors, proteins from mosquito midgut tissue and C6/36 cells were purified by affinity using columns with the recombinant E protein domain III (rE-DIII or DENV particles bound covalently to Sepharose 4B to compare and evaluate their performance to bind proteins including putative receptors from female mosquitoes of Ae. aegypti. To determine their identity mass spectrometric analysis of purified proteins separated by polyacrylamide gel electrophoresis was performed. Our results indicate that both viral particles and rE-DIII bound proteins with the same apparent molecular weights of 57 and 67 kDa. In addition, viral particles bound high molecular weight proteins. Purified proteins identified were enolase, beta-adrenergic receptor kinase (beta-ARK, translation elongation factor EF-1 alpha/Tu, and cadherin.

  9. Genome-wide analysis of cell wall-related genes in Tuber melanosporum.

    Science.gov (United States)

    Balestrini, Raffaella; Sillo, Fabiano; Kohler, Annegret; Schneider, Georg; Faccio, Antonella; Tisserant, Emilie; Martin, Francis; Bonfante, Paola

    2012-06-01

    A genome-wide inventory of proteins involved in cell wall synthesis and remodeling has been obtained by taking advantage of the recently released genome sequence of the ectomycorrhizal Tuber melanosporum black truffle. Genes that encode cell wall biosynthetic enzymes, enzymes involved in cell wall polysaccharide synthesis or modification, GPI-anchored proteins and other cell wall proteins were identified in the black truffle genome. As a second step, array data were validated and the symbiotic stage was chosen as the main focus. Quantitative RT-PCR experiments were performed on 29 selected genes to verify their expression during ectomycorrhizal formation. The results confirmed the array data, and this suggests that cell wall-related genes are required for morphogenetic transition from mycelium growth to the ectomycorrhizal branched hyphae. Labeling experiments were also performed on T. melanosporum mycelium and ectomycorrhizae to localize cell wall components.

  10. Preparation and demonstration of poly(dopamine)-triggered attapulgite-anchored polyurethane as a high-performance rod-like elastomer to reinforce soy protein-isolated composites

    Science.gov (United States)

    Zhao, Shujun; Wen, Yingying; Wang, Zhong; Kang, Haijiao; Li, Jianzhang; Zhang, Shifeng; Ji, Yong

    2018-06-01

    Nanophase modification is an effective path to improve composite properties, however, it remains a great challenge to increase the mechanical strength of the modified materials without sacrificing elongation and toughness. This study presents a novel and efficient design for interface anchoring of a waterborne polyurethane (WPU) elastomer with attapulgite (ATP) triggered by poly(dopamine) (PDA) formation due to self-polymerization of the dopamine moieties. The WPU-PDA-ATP (WDA) rod-like elastomer served as an active enhancer for a soy protein isolate (SPI)-based composite to facilitate multiple interactions between SPI and the elastomer. As expected, the PDA layer was coated onto ATP, inducing the nanofiller to successfully anchor onto the WPU elastomer, as confirmed by solid-state 13C NMR, XPS, and ATR-FTIR results. Compared with the control SPI-based film, the tensile strength and toughness increased by 145.6% and 118.3% respectively by introducing WDA rod-like elastomer. The water resistance and thermal stability of the prepared SPI composites were also favorable. The proposed approach represents an efficient way to utilize high-performance elastomer in biobased materials to concurrently enhance strength and toughness.

  11. Pyrosequencing the Midgut Transcriptome of the Banana Weevil Cosmopolites sordidus (Germar (Coleoptera: Curculionidae Reveals Multiple Protease-Like Transcripts.

    Directory of Open Access Journals (Sweden)

    Arnubio Valencia

    Full Text Available The banana weevil Cosmopolites sordidus is an important and serious insect pest in most banana and plantain-growing areas of the world. In spite of the economic importance of this insect pest very little genomic and transcriptomic information exists for this species. In the present study, we characterized the midgut transcriptome of C. sordidus using massive 454-pyrosequencing. We generated over 590,000 sequencing reads that assembled into 30,840 contigs with more than 400 bp, representing a significant expansion of existing sequences available for this insect pest. Among them, 16,427 contigs contained one or more GO terms. In addition, 15,263 contigs were assigned an EC number. In-depth transcriptome analysis identified genes potentially involved in insecticide resistance, peritrophic membrane biosynthesis, immunity-related function and defense against pathogens, and Bacillus thuringiensis toxins binding proteins as well as multiple enzymes involved with protein digestion. This transcriptome will provide a valuable resource for understanding larval physiology and for identifying novel target sites and management approaches for this important insect pest.

  12. Pyrosequencing the Midgut Transcriptome of the Banana Weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) Reveals Multiple Protease-Like Transcripts.

    Science.gov (United States)

    Valencia, Arnubio; Wang, Haichuan; Soto, Alberto; Aristizabal, Manuel; Arboleda, Jorge W; Eyun, Seong-Il; Noriega, Daniel D; Siegfried, Blair

    2016-01-01

    The banana weevil Cosmopolites sordidus is an important and serious insect pest in most banana and plantain-growing areas of the world. In spite of the economic importance of this insect pest very little genomic and transcriptomic information exists for this species. In the present study, we characterized the midgut transcriptome of C. sordidus using massive 454-pyrosequencing. We generated over 590,000 sequencing reads that assembled into 30,840 contigs with more than 400 bp, representing a significant expansion of existing sequences available for this insect pest. Among them, 16,427 contigs contained one or more GO terms. In addition, 15,263 contigs were assigned an EC number. In-depth transcriptome analysis identified genes potentially involved in insecticide resistance, peritrophic membrane biosynthesis, immunity-related function and defense against pathogens, and Bacillus thuringiensis toxins binding proteins as well as multiple enzymes involved with protein digestion. This transcriptome will provide a valuable resource for understanding larval physiology and for identifying novel target sites and management approaches for this important insect pest.

  13. Glycosylphosphatidylinositol-anchored CD4 supports human immunodeficiency virus type 1 replication, but not cytopathic effect, in T-cell transfectants.

    OpenAIRE

    Marshall, W L; Mittler, E S; Avery, P; Lawrence, J P; Finberg, R W

    1994-01-01

    Despite equivalent p24 antigen production, HSB-2 T cells expressing glycosylphosphatidylinositol (GPi)-linked CD4 were productively infected without cell death or syncytium formation, unlike HSB-2 transfectants expressing wild-type CD4 (wtCD4). HSB-2 transfectants dually expressing wtCD4 and GPi-linked CD4 formed syncytia and died. Thus, wtCD4 expression is critical for human immunodeficiency virus cytopathic effect in HSB-2 transfectants.

  14. Characterization of the Mamestra configurata (Lepidoptera: Noctuidae) larval midgut protease complement and adaptation to feeding on artificial diet, Brassica species, and protease inhibitor.

    Science.gov (United States)

    Erlandson, Martin A; Hegedus, Dwayne D; Baldwin, Douglas; Noakes, Amy; Toprak, Umut

    2010-10-01

    The midgut protease profiles from 5th instar Mamestra configurata larvae fed various diets (standard artificial diet, low protein diet, low protein diet with soybean trypsin inhibitor [SBTI], or Brassica napus) were characterized by one-dimensional enzymography in gelatin gels. The gut protease profile of larvae fed B. napus possessed protease activities of molecular masses of approximately 33 and 55 kDa, which were not present in the guts of larvae fed artificial diet. Similarly, larvae fed artificial diet had protease activities of molecular masses of approximately 21, 30, and 100 kDa that were absent in larvae fed B. napus. Protease profiles changed within 12 to 24 h after switching larvae from artificial diet to plant diet and vice versa. The gut protease profiles from larvae fed various other brassicaceous species and lines having different secondary metabolite profiles did not differ despite significant differences in larval growth rates on the different host plants. Genes encoding putative digestive proteolytic enzymes, including four carboxypeptidases, five aminopeptidases, and 48 serine proteases, were identified in cDNA libraries from 4th instar M. configurata midgut tissue. Many of the protease-encoding genes were expressed at similar levels on all diets; however, three chymoptrypsin-like genes (McSP23, McSP27, and McSP37) were expressed at much higher levels on standard artificial diet and diet containing SBTI as was the trypsin-like gene McSP34. The expression of the trypsin-like gene McSP50 was highest on B. napus. The adaptation of M. configurata digestive biochemistry to different diets is discussed in the context of the flexibility of polyphagous insects to changing diet sources.

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

    Science.gov (United States)

    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…

  16. High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation.

    Science.gov (United States)

    Anandapadamanaban, Madhanagopal; Andresen, Cecilia; Helander, Sara; Ohyama, Yoshifumi; Siponen, Marina I; Lundström, Patrik; Kokubo, Tetsuro; Ikura, Mitsuhiko; Moche, Martin; Sunnerhagen, Maria

    2013-08-01

    The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 Å) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 bound to yeast TBP, together with mutational data. We find that yeast TAF1-TAND1, which in itself acts as a transcriptional activator, binds TBP's concave DNA-binding surface by presenting similar anchor residues to TBP as does Mot1 but from a distinct structural scaffold. Furthermore, we show how TAF1-TAND2 uses an aromatic and acidic anchoring pattern to bind a conserved TBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides insight into the competitive multiprotein TBP interplay critical to transcriptional regulation.

  17. Effects of accuracy motivation and anchoring on metacomprehension judgment and accuracy.

    Science.gov (United States)

    Zhao, Qin

    2012-01-01

    The current research investigates how accuracy motivation impacts anchoring and adjustment in metacomprehension judgment and how accuracy motivation and anchoring affect metacomprehension accuracy. Participants were randomly assigned to one of six conditions produced by the between-subjects factorial design involving accuracy motivation (incentive or no) and peer performance anchor (95%, 55%, or no). Two studies showed that accuracy motivation did not impact anchoring bias, but the adjustment-from-anchor process occurred. Accuracy incentive increased anchor-judgment gap for the 95% anchor but not for the 55% anchor, which induced less certainty about the direction of adjustment. The findings offer support to the integrative theory of anchoring. Additionally, the two studies revealed a "power struggle" between accuracy motivation and anchoring in influencing metacomprehension accuracy. Accuracy motivation could improve metacomprehension accuracy in spite of anchoring effect, but if anchoring effect is too strong, it could overpower the motivation effect. The implications of the findings were discussed.

  18. Dynamic performance of concrete undercut anchors for Nuclear Power Plants

    Energy Technology Data Exchange (ETDEWEB)

    Mahrenholtz, Christoph, E-mail: christoph@mahrenholtz.net; Eligehausen, Rolf

    2013-12-15

    Graphical abstract: - Highlights: • Behavior of undercut anchors under dynamic actions simulating earthquakes. • First high frequency load and crack cycling tests on installed concrete anchors ever. • Comprehensive review of anchor qualification for Nuclear Power Plants. - Abstract: Post-installed anchors are widely used for structural and nonstructural connections to concrete. In many countries, concrete anchors used for Nuclear Power Plants have to be qualified to ensure reliable behavior even under extreme conditions. The tests required for qualification of concrete anchors are carried out at quasi-static loading rates well below the rates to be expected for dynamic actions deriving from earthquakes, airplane impacts or explosions. To investigate potentially beneficial effects of high loading rates and cycling frequencies, performance tests on installed undercut anchors were conducted. After introductory notes on anchor technology and a comprehensive literature review, this paper discusses the qualification of anchors for Nuclear Power Plants and the testing carried out to quantify experimentally the effects of dynamic actions on the load–displacement behavior of undercut anchors.

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

    Science.gov (United States)

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

    2015-03-16

    A-Kinase Anchoring Proteins (AKAPs) coordinate the specificity of protein kinase A signaling by localizing the kinase to subcellular sites. The 1936G (V646) AKAP10 allele has been associated in adults with low cholinergic/vagus nerve sensitivity, shortened PR intervals in ECG recording and in newborns with increased blood pressure and higher cholesterol cord blood concentration. The aim of the study was to answer the question of whether 1936A > G AKAP10 polymorphism is associated with the newborn electrocardiographic variables. Electrocardiograms were recorded from 114 consecutive healthy Polish newborns (55 females, 59 males), born after 37 gestational weeks to healthy women with uncomplicated pregnancies. All recordings were made between 3(rd) and 7(th) day of life to avoid QT variability. The heart rate per minute and duration of PR, QRS, RR and QT intervals were usually measured. The ECGs were evaluated independently by three observers. At birth, cord blood of neonates was obtained for isolation of genomic DNA. The distribution of anthropometric and electrocardiographic variables in our cohort approached normality (skewness G variant and QTc interval in Polish newborns.

  20. GPIHBP1 and Plasma Triglyceride Metabolism.

    Science.gov (United States)

    Fong, Loren G; Young, Stephen G; Beigneux, Anne P; Bensadoun, André; Oberer, Monika; Jiang, Haibo; Ploug, Michael

    2016-07-01

    GPIHBP1, a GPI-anchored protein in capillary endothelial cells, is crucial for the lipolytic processing of triglyceride-rich lipoproteins (TRLs). GPIHBP1 shuttles lipoprotein lipase (LPL) to its site of action in the capillary lumen and is essential for the margination of TRLs along capillaries - such that lipolytic processing can proceed. GPIHBP1 also reduces the unfolding of the LPL catalytic domain, thereby stabilizing LPL catalytic activity. Many different GPIHBP1 mutations have been identified in patients with severe hypertriglyceridemia (chylomicronemia), the majority of which interfere with folding of the protein and abolish its capacity to bind and transport LPL. The discovery of GPIHBP1 has substantially revised our understanding of intravascular triglyceride metabolism but has also raised many new questions for future research. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Midgut volvulus: a rare cause of episodes of intestinal obstruction in an adult; Volvulo de intestino medio: una rara causa de crisis oclusivas en el adulto

    Energy Technology Data Exchange (ETDEWEB)

    Palomo, V.; Higuera, A.; Munoz, R.; Sanchez, F. [Hospital Alto Guadalquivir. Andujar. Jaen (Spain)

    2002-07-01

    Midgut volvulus occurs frequently in infants and children, but is uncommon in adults. We present a case of intestinal malrotation complicated by midgut volvulus in a young woman who complained of chronic intermittent abdominal pain of increasing intensity. The radiologies diagnosis was based mainly on upper gastrointestinal barium study, and was confirmed intraoperatively. (Author) 11 refs.

  2. End-anchored polymers in good solvents from the single chain limit to high anchoring densities.

    Science.gov (United States)

    Whitmore, Mark D; Grest, Gary S; Douglas, Jack F; Kent, Michael S; Suo, Tongchuan

    2016-11-07

    An increasing number of applications utilize grafted polymer layers to alter the interfacial properties of solid substrates, motivating refinement in our theoretical understanding of such layers. To assess existing theoretical models of them, we have investigated end-anchored polymer layers over a wide range of grafting densities, σ, ranging from a single chain to high anchoring density limits, chain lengths ranging over two orders of magnitude, for very good and marginally good solvent conditions. We compare Monte Carlo and molecular dynamics simulations, numerical self-consistent field calculations, and experimental measurements of the average layer thickness, h, with renormalization group theory, the Alexander-de Gennes mushroom theory, and the classical brush theory. Our simulations clearly indicate that appreciable inter-chain interactions exist at all simulated areal anchoring densities so that there is no mushroom regime in which the layer thickness is independent of σ. Moreover, we find that there is no high coverage regime in which h follows the predicted scaling, h ∼ Nσ 1/3 , for classical polymer brushes either. Given that no completely adequate analytic theory seems to exist that spans wide ranges of N and σ, we applied scaling arguments for h as a function of a suitably defined reduced anchoring density, defined in terms of the solution radius of gyration of the polymer chains and N. We find that such a scaling approach enables a smooth, unified description of h in very good solvents over the full range of anchoring density and chain lengths, although this type of data reduction does not apply to marginal solvent quality conditions.

  3. Studies on the mechanical behavior of rock anchors. ; Results of in-situ pull-out tests. Rock anchor no rikigaku kyodo ni kansuru kenkyu. ; Gen prime ichi shiken ni okeru anchor no kyodo

    Energy Technology Data Exchange (ETDEWEB)

    Arimoto, K.; Ebisu, S.; Nakagawa, M.; Usui, M.; Someya, T.; Machida, N. (Okumura Corp., Tokyo (Japan))

    1991-10-31

    The rock anchor method is planned to apply to some permanent structures but since this method was developed for temporary structures, the clarification of the transferring mechanism of force from an anchor to a rockmass, the fracture mechanism and the development of the dynamic model have not been established. This paper arranged the data obtained by a full-scale, in-situ pulling out test of a rock anchor as the first step to understand the dynamic behavior and analyzed by paying attetion to the modulus of deformation of the rockmass where the anchor was embedded to elucidate the affecting degree of rockmass modulus of deformation, the embedded length and the tendon diameter on the dynamic behavior of the anchor. The rock anchor behavior could be expressed accurately by applying a theoretical solution derived by the balancing condition of forces at the boundary face. Especially, when the rockmass is uniform and the fracture occurrs at the interface between the tendon and grout, this approach can express the fracture with the accuracy similar to that made by the finite element method. 6 refs., 11 figs.,1 tab.

  4. Containment liner plate anchors and steel embedments test results

    International Nuclear Information System (INIS)

    Chang-Lo, P.L.; Johnson, T.E.; Pfeifer, B.W.

    1977-01-01

    This paper summarizes test data on shear load and deformation capabilities for liner plate line anchors and structural steel embedments in reinforced and prestressed concrete nuclear containments. Reinforced and prestressed nuclear containments designed and constructed in the United States are lined with a minimum of 0.64 cm steel plate. The liner plates are anchored by the use of either studs or structural members (line anchors) which usually run in the vertical direction. This paper will only address line anchors. Static load versus displacement test data is necessary to assure that the design is adequate for the maximum loads. The test program for the liner anchors had the following major objectives: determine load versus displacement data for a variety of anchors considering structural tees and small beams with different weld configurations, from the preceding tests, determine which anchors would lead to an economical and extremely safe design and test these anchors for cyclic loads resulting from thermal fluctuations. Various concrete embeds in the containment and other structures are subjected to loads such as pipe rupture which results in shear. Since many of the loads are transient by nature, it is necessary to know the load-displacement relationship so that the energy absorption can be determined. The test program for the embeds had the following objectives: determine load-displacement relationship for various size anchors from 6.5 cm 2 to 26 cm 2 with maximum capacities of approximately 650 kN; determine the effect of various anchor width-to-thickness ratios for the same shear area

  5. Field-Evolved Mode 1 Resistance of the Fall Armyworm to Transgenic Cry1Fa-Expressing Corn Associated with Reduced Cry1Fa Toxin Binding and Midgut Alkaline Phosphatase Expression

    Science.gov (United States)

    Jakka, Siva R. K.; Gong, Liang; Hasler, James; Banerjee, Rahul; Sheets, Joel J.; Narva, Kenneth; Blanco, Carlos A.

    2015-01-01

    Insecticidal protein genes from the bacterium Bacillus thuringiensis (Bt) are expressed by transgenic Bt crops (Bt crops) for effective and environmentally safe pest control. The development of resistance to these insecticidal proteins is considered the most serious threat to the sustainability of Bt crops. Resistance in fall armyworm (Spodoptera frugiperda) populations from Puerto Rico to transgenic corn producing the Cry1Fa insecticidal protein resulted, for the first time in the United States, in practical resistance, and Bt corn was withdrawn from the local market. In this study, we used a field-collected Cry1Fa corn-resistant strain (456) of S. frugiperda to identify the mechanism responsible for field-evolved resistance. Binding assays detected reduced Cry1Fa, Cry1Ab, and Cry1Ac but not Cry1Ca toxin binding to midgut brush border membrane vesicles (BBMV) from the larvae of strain 456 compared to that from the larvae of a susceptible (Ben) strain. This binding phenotype is descriptive of the mode 1 type of resistance to Bt toxins. A comparison of the transcript levels for putative Cry1 toxin receptor genes identified a significant downregulation (>90%) of a membrane-bound alkaline phosphatase (ALP), which translated to reduced ALP protein levels and a 75% reduction in ALP activity in BBMV from 456 compared to that of Ben larvae. We cloned and heterologously expressed this ALP from susceptible S. frugiperda larvae and demonstrated that it specifically binds with Cry1Fa toxin. This study provides a thorough mechanistic description of field-evolved resistance to a transgenic Bt crop and supports an association between resistance and reduced Cry1Fa toxin binding and levels of a putative Cry1Fa toxin receptor, ALP, in the midguts of S. frugiperda larvae. PMID:26637593

  6. Modified Kidner procedure utilizing a Mitek bone anchor.

    Science.gov (United States)

    Dawson, D M; Julsrud, M E; Erdmann, B B; Jacobs, P M; Ringstrom, J B

    1998-01-01

    The recent development of small bone suture anchors has created several potential applications in reconstructive surgery of the foot. Mitek bone anchors are simple to insert, require less aggressive dissection and surgical time than reefing of the redundant posterior tibial tendon, and are a reliable method of tendon-to-bone fixation. Mitek bone anchors are an excellent technique for the treatment of redundant tibialis posterior tendon following a modified Kidner procedure. In modified Kidner procedures involving an excessively large os tibiale externum, Mitek anchoring of the redundant tibialis posterior tendon to the navicular bone is an excellent means for secure plication of the posterior tibial tendon in cases involving intraoperative tendon laxity. A description of the Mitek Anchor System and technique of application in a modified Kinder procedure is presented. The purpose of this study was to describe patient satisfaction and long-term clinical outcomes of the modified Kinder procedure with and without the Mitek bone anchoring system. A retrospective study of the modified Kinder procedure was performed with 13 patients being evaluated, seven with Mitek anchoring and six without. The University of Maryland 100-point Painful Foot Center Scoring System was modified to be more specific to the modified Kinder procedure for assessment of subjective long-term results. Patient overall satisfaction was rated good to excellent by 85.6% of patients in the Mitek group and by 100% of patients in the non-Mitek group. Use of the Mitek anchor allowed for quicker postoperative recovery to resumption of ambulation without assistive devices (average of 3 weeks vs. 4.42 weeks) and a quicker return to pain-free ambulation in normal shoegear (average of 4 weeks vs. 6 weeks). Mitek anchoring of the tibialis posterior tendon, theoretically, increases medial arch support as evidenced by 14% of the Mitek group and 67% of the non-Mitek group requiring postoperative orthotics.

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

    Science.gov (United States)

    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.

  8. Amount and metal composition of midgut gland metallothionein in shore crabs (Carcinus maenas) after exposure to cadmium in the food

    International Nuclear Information System (INIS)

    Pedersen, Knud Ladegaard; Bach, Louise Thornhøj; Bjerregaard, Poul

    2014-01-01

    Highlights: • Crabs were fed with Cd in concentrations of 1.1–5.1 μg g −1 food. • Metallothionein concentrations only increased at 5.1 μg g −1 . • Cd contents of metallothionein increased linearly with exposure. • A marked influence by the variable Cu contents on metal composition was recorded. • Digestive gland metallothionein is a poor biomarker for Cd exposure. - Abstract: Accumulation of cadmium in aquatic invertebrates may compromise human food safety and anthropogenic additions of cadmium to coastal areas cause concern. Induction of crustacean metallothionein has been suggested as a useful biomarker for contamination of the aquatic environment with cadmium. We investigated how exposure to low concentrations of cadmium in the food affects the subcellular binding of cadmium with the shore crab Carcinus maenas as model organism. Approximately 80% of the assimilated cadmium was bound in the soluble fraction of the midgut gland and of this, 82% was found in the metallothionein fraction. Metallothionein synthesis was only induced at the highest exposure level. However, the number of cadmium atoms bound per molecule of metallothionein increased linearly with exposure, from approximately 0.18 in the control group to 1.4 in a group administered food containing 5.1 μg Cd g −1 . We noted a marked interaction between the presence of copper and zinc in the midgut gland and the binding of cadmium. The usefulness of crustacean midgut gland metallothionein as a biomarker for cadmium exposure at modest levels was questioned since exposures at levels producing significant increases in the tissue contents of the metal did not result in elevated concentrations of metallothionein in the midgut gland

  9. Amount and metal composition of midgut gland metallothionein in shore crabs (Carcinus maenas) after exposure to cadmium in the food

    Energy Technology Data Exchange (ETDEWEB)

    Pedersen, Knud Ladegaard; Bach, Louise Thornhøj; Bjerregaard, Poul, E-mail: poul@biology.sdu.dk

    2014-05-01

    Highlights: • Crabs were fed with Cd in concentrations of 1.1–5.1 μg g⁻¹ food. • Metallothionein concentrations only increased at 5.1 μg g⁻¹. • Cd contents of metallothionein increased linearly with exposure. • A marked influence by the variable Cu contents on metal composition was recorded. • Digestive gland metallothionein is a poor biomarker for Cd exposure. - Abstract: Accumulation of cadmium in aquatic invertebrates may compromise human food safety and anthropogenic additions of cadmium to coastal areas cause concern. Induction of crustacean metallothionein has been suggested as a useful biomarker for contamination of the aquatic environment with cadmium. We investigated how exposure to low concentrations of cadmium in the food affects the subcellular binding of cadmium with the shore crab Carcinus maenas as model organism. Approximately 80% of the assimilated cadmium was bound in the soluble fraction of the midgut gland and of this, 82% was found in the metallothionein fraction. Metallothionein synthesis was only induced at the highest exposure level. However, the number of cadmium atoms bound per molecule of metallothionein increased linearly with exposure, from approximately 0.18 in the control group to 1.4 in a group administered food containing 5.1 μg Cd g⁻¹. We noted a marked interaction between the presence of copper and zinc in the midgut gland and the binding of cadmium. The usefulness of crustacean midgut gland metallothionein as a biomarker for cadmium exposure at modest levels was questioned since exposures at levels producing significant increases in the tissue contents of the metal did not result in elevated concentrations of metallothionein in the midgut gland.

  10. Adsorption phenomena and anchoring energy in nematic liquid crystals

    CERN Document Server

    Barbero, Giovanni

    2005-01-01

    Despite the large quantity of phenomenological information concerning the bulk properties of nematic phase liquid crystals, little is understood about the origin of the surface energy, particularly the surface, interfacial, and anchoring properties of liquid crystals that affect the performance of liquid crystal devices. Self-contained and unique, Adsorption Phenomena and Anchoring Energy in Nematic Liquid Crystals provides an account of new and established results spanning three decades of research into the problems of anchoring energy and adsorption phenomena in liquid crystals.The book contains a detailed discussion of the origin and possible sources of anchoring energy in nematic liquid crystals, emphasizing the dielectric contribution to the anchoring energy in particular. Beginning with fundamental surface and anchoring properties of liquid crystals and the definition of the nematic phase, the authors explain how selective ion adsorption, dielectric energy density, thickness dependence, and bias voltage...

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

  12. Insights into the Evolution of Hydroxyproline-Rich Glycoproteins from 1000 Plant Transcriptomes.

    Science.gov (United States)

    Johnson, Kim L; Cassin, Andrew M; Lonsdale, Andrew; Wong, Gane Ka-Shu; Soltis, Douglas E; Miles, Nicholas W; Melkonian, Michael; Melkonian, Barbara; Deyholos, Michael K; Leebens-Mack, James; Rothfels, Carl J; Stevenson, Dennis W; Graham, Sean W; Wang, Xumin; Wu, Shuangxiu; Pires, J Chris; Edger, Patrick P; Carpenter, Eric J; Bacic, Antony; Doblin, Monika S; Schultz, Carolyn J

    2017-06-01

    The carbohydrate-rich cell walls of land plants and algae have been the focus of much interest given the value of cell wall-based products to our current and future economies. Hydroxyproline-rich glycoproteins (HRGPs), a major group of wall glycoproteins, play important roles in plant growth and development, yet little is known about how they have evolved in parallel with the polysaccharide components of walls. We investigate the origins and evolution of the HRGP superfamily, which is commonly divided into three major multigene families: the arabinogalactan proteins (AGPs), extensins (EXTs), and proline-rich proteins. Using motif and amino acid bias, a newly developed bioinformatics pipeline, we identified HRGPs in sequences from the 1000 Plants transcriptome project (www.onekp.com). Our analyses provide new insights into the evolution of HRGPs across major evolutionary milestones, including the transition to land and the early radiation of angiosperms. Significantly, data mining reveals the origin of glycosylphosphatidylinositol (GPI)-anchored AGPs in green algae and a 3- to 4-fold increase in GPI-AGPs in liverworts and mosses. The first detection of cross-linking (CL)-EXTs is observed in bryophytes, which suggests that CL-EXTs arose though the juxtaposition of preexisting SP n EXT glycomotifs with refined Y-based motifs. We also detected the loss of CL-EXT in a few lineages, including the grass family (Poaceae), that have a cell wall composition distinct from other monocots and eudicots. A key challenge in HRGP research is tracking individual HRGPs throughout evolution. Using the 1000 Plants output, we were able to find putative orthologs of Arabidopsis pollen-specific GPI-AGPs in basal eudicots. © 2017 American Society of Plant Biologists. All Rights Reserved.

  13. Insights into the Evolution of Hydroxyproline-Rich Glycoproteins from 1000 Plant Transcriptomes1[OPEN

    Science.gov (United States)

    Cassin, Andrew M.; Soltis, Douglas E.; Miles, Nicholas W.; Melkonian, Michael; Melkonian, Barbara; Wu, Shuangxiu; Edger, Patrick P.; Carpenter, Eric J.

    2017-01-01

    The carbohydrate-rich cell walls of land plants and algae have been the focus of much interest given the value of cell wall-based products to our current and future economies. Hydroxyproline-rich glycoproteins (HRGPs), a major group of wall glycoproteins, play important roles in plant growth and development, yet little is known about how they have evolved in parallel with the polysaccharide components of walls. We investigate the origins and evolution of the HRGP superfamily, which is commonly divided into three major multigene families: the arabinogalactan proteins (AGPs), extensins (EXTs), and proline-rich proteins. Using motif and amino acid bias, a newly developed bioinformatics pipeline, we identified HRGPs in sequences from the 1000 Plants transcriptome project (www.onekp.com). Our analyses provide new insights into the evolution of HRGPs across major evolutionary milestones, including the transition to land and the early radiation of angiosperms. Significantly, data mining reveals the origin of glycosylphosphatidylinositol (GPI)-anchored AGPs in green algae and a 3- to 4-fold increase in GPI-AGPs in liverworts and mosses. The first detection of cross-linking (CL)-EXTs is observed in bryophytes, which suggests that CL-EXTs arose though the juxtaposition of preexisting SPn EXT glycomotifs with refined Y-based motifs. We also detected the loss of CL-EXT in a few lineages, including the grass family (Poaceae), that have a cell wall composition distinct from other monocots and eudicots. A key challenge in HRGP research is tracking individual HRGPs throughout evolution. Using the 1000 Plants output, we were able to find putative orthologs of Arabidopsis pollen-specific GPI-AGPs in basal eudicots. PMID:28446636

  14. Outcomes of the modified Brostrom procedure using suture anchors for chronic lateral ankle instability--a prospective, randomized comparison between single and double suture anchors.

    Science.gov (United States)

    Cho, Byung-Ki; Kim, Yong-Min; Kim, Dong-Soo; Choi, Eui-Sung; Shon, Hyun-Chul; Park, Kyoung-Jin

    2013-01-01

    The present prospective, randomized study was conducted to compare the clinical outcomes of the modified Brostrom procedure using single and double suture anchors for chronic lateral ankle instability. A total of 50 patients were followed up for more than 2 years after undergoing the modified Brostrom procedure. Of the 50 procedures, 25 each were performed using single and double suture anchors by 1 surgeon. The Karlsson scale had improved significantly to 89.8 points and 90.6 points in the single and double anchor groups, respectively. Using the Sefton grading system, 23 cases (92%) in the single anchor group and 22 (88%) in the double anchor group achieved satisfactory results. The talar tilt angle and anterior talar translation on stress radiographs using the Telos device had improved significantly to an average of 5.7° and 4.6 mm in the single anchor group and 4.5° and 4.3 mm in the double anchor group, respectively. The double anchor technique was superior with respect to the postoperative talar tilt. The single and double suture anchor techniques produced similar clinical and functional outcomes, with the exception of talar tilt as a reference of mechanical stability. The modified Brostrom procedure using both single and double suture anchors appears to be an effective treatment method for chronic lateral ankle instability. Copyright © 2013 American College of Foot and Ankle Surgeons. Published by Elsevier Inc. All rights reserved.

  15. Expression and phylogenetic analyses of the Gel/Gas proteins of Tuber melanosporum provide insights into the function and evolution of glucan remodeling enzymes in fungi.

    Science.gov (United States)

    Sillo, Fabiano; Gissi, Carmela; Chignoli, Daniele; Ragni, Enrico; Popolo, Laura; Balestrini, Raffaella

    2013-04-01

    The β(1,3)-glucanosyltransferases of the GH72 family are redundant enzymes that are essential for the formation and dynamic remodeling of the fungal wall during different stages of the life cycle. Four putative genes encoding glycosylphosphatidylinositol (GPI)-anchored β(1,3)-glucanosyltransferases, designated TmelGEL1, TmelGEL2, TmelGEL4 and TmelGAS4, have been annotated in the genome of Tuber melanosporum, an ectomycorrhizal fungus that also produces a hypogeous fruiting body (FB) of great commercial value (black truffle). This work focuses on the characterization and expression of this multigene family by taking advantage of a laser microdissection (LMD) technology that has been used to separate two distinct compartments in the FB, the hyphae and the asci containing the ascospores. Of the four genes, TmelGEL1 was the most up-regulated in the FB compared to the free-living mycelium. Inside the FB, the expression of TmelGEL1 was restricted to the hyphal compartment. A phylogenetic analysis of the Gel/Gas protein family of T. melanosporum was also carried out. A total of 237 GH72 proteins from 51 Ascomycotina and 3 Basidiomycota (outgroup) species were analyzed. The resulting tree provides insight into the evolution of the T. melanosporum proteins and identifies new GH72 paralogs/subfamilies. Moreover, it represents a starting point to formulate new hypotheses on the significance of the striking GH72 gene redundancy in fungal biology. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Formation of the food vacuole in Plasmodium falciparum: a potential role for the 19 kDa fragment of merozoite surface protein 1 (MSP1(19.

    Directory of Open Access Journals (Sweden)

    Anton R Dluzewski

    2008-08-01

    Full Text Available Plasmodium falciparum Merozoite Surface Protein 1 (MSP1 is synthesized during schizogony as a 195-kDa precursor that is processed into four fragments on the parasite surface. Following a second proteolytic cleavage during merozoite invasion of the red blood cell, most of the protein is shed from the surface except for the C-terminal 19-kDa fragment (MSP1(19, which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP1(19 during the parasite's subsequent intracellular development using immunochemical analysis of metabolically labeled MSP1(19, fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP1(19 remains intact and persists to the end of the intracellular cycle. This protein is the first marker for the biogenesis of the food vacuole; it is rapidly endocytosed into small vacuoles in the ring stage, which coalesce to form the single food vacuole containing hemozoin, and persists into the discarded residual body. The food vacuole is marked by the presence of both MSP1(19 and the chloroquine resistance transporter (CRT as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP1(19 does not simply follow a classical lysosome-like clearance pathway, instead, it may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase.

  17. Structural basis of sterol recognition and nonvesicular transport by lipid transfer proteins anchored at membrane contact sites.

    Science.gov (United States)

    Tong, Junsen; Manik, Mohammad Kawsar; Im, Young Jun

    2018-01-30

    Membrane contact sites (MCSs) in eukaryotic cells are hotspots for lipid exchange, which is essential for many biological functions, including regulation of membrane properties and protein trafficking. Lipid transfer proteins anchored at membrane contact sites (LAMs) contain sterol-specific lipid transfer domains [StARkin domain (SD)] and multiple targeting modules to specific membrane organelles. Elucidating the structural mechanisms of targeting and ligand recognition by LAMs is important for understanding the interorganelle communication and exchange at MCSs. Here, we determined the crystal structures of the yeast Lam6 pleckstrin homology (PH)-like domain and the SDs of Lam2 and Lam4 in the apo form and in complex with ergosterol. The Lam6 PH-like domain displays a unique PH domain fold with a conserved N-terminal α-helix. The Lam6 PH-like domain lacks the basic surface for phosphoinositide binding, but contains hydrophobic patches on its surface, which are critical for targeting to endoplasmic reticulum (ER)-mitochondrial contacts. Structures of the LAM SDs display a helix-grip fold with a hydrophobic cavity and a flexible Ω1-loop as a lid. Ergosterol is bound to the pocket in a head-down orientation, with its hydrophobic acyl group located in the tunnel entrance. The Ω1-loop in an open conformation is essential for ergosterol binding by direct hydrophobic interaction. Structural comparison suggested that the sterol binding mode of the Lam2 SD2 is likely conserved among the sterol transfer proteins of the StARkin superfamily. Structural models of full-length Lam2 correlated with the sterol transport function at the membrane contact sites.

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

    Science.gov (United States)

    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.

  19. Career anchors and learning plan (part one

    Directory of Open Access Journals (Sweden)

    Daniela Brečko

    2006-12-01

    Full Text Available The article is divided into three parts. The first part concentrates on how important career is for an individual, organization and society. The author establishes that understanding of career has changed dramatically and does not only refer to climbing up the career ladder, but also moving off or even down the career ladder. The notion of career, as a lifelong and professional path, encompasses all aspects of human personality and their roles acquired through one's life. On basis of vast and longitudinal research, where the author has studied career anchors of individuals, it is the objective of the author to find out on basis of what grounds do the individuals decide to take certain directions in their careers and how learning contributes to such decisions. As a source the author has used Shein's theory of career anchors. Part one describes in greater detail 8 different career anchors and introduces their main features with the findings of the research, which refer to the analysis of professions (work positions and established career anchors. The author thus verifies the hypothesis that career anchors do exist in our area.

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

    Science.gov (United States)

    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.

  1. Career anchors and values from different career management perspectives

    Directory of Open Access Journals (Sweden)

    Rodrigo Cunha da Silva

    2016-06-01

    Full Text Available Purpose – To analyze the relationships between career anchors and young Generation Y professionals’ values, from the career concept perspective. Design/methodology/approach – Research concerning the proposed objective was carried out through quantitative research involving 189 Business Administration majors from a Catholic university in São Paulo, Brazil. We used two instruments to identify the career anchors and values of respondents: Schein (1990 and Schwartz (1994, respectively. We used statistical techniques to explore the relationships between career anchors and values. Findings – Among the results, mention should be made to the statistical relationships found between analyzed career anchors and values. It is also important to stress that, although the Lifestyle career anchor was predominantly present in the conglomerate division, this anchor was the predominant characteristic in the differentiation of the smaller group of respondents, the new career group. The General Management Career Anchor, which presents a lower incidence, is the predominant characteristic of the larger group, referring to organizational careers. As well as the Lifestyle career anchor, the Hedonism value was predominant among respondents. Originality/value – The need to consider the following was found: Generation Y presents generational characteristics that drive people management to propose work structures that offer activities to generate learning, pleasure, self-fulfillment and conciliation between work and personal life.

  2. Test Score Equating Using Discrete Anchor Items versus Passage-Based Anchor Items: A Case Study Using "SAT"® Data. Research Report. ETS RR-14-14

    Science.gov (United States)

    Liu, Jinghua; Zu, Jiyun; Curley, Edward; Carey, Jill

    2014-01-01

    The purpose of this study is to investigate the impact of discrete anchor items versus passage-based anchor items on observed score equating using empirical data.This study compares an "SAT"® critical reading anchor that contains more discrete items proportionally, compared to the total tests to be equated, to another anchor that…

  3. On Moderator Detection in Anchoring Research: Implications of Ignoring Estimate Direction

    Directory of Open Access Journals (Sweden)

    Nathan N. Cheek

    2018-05-01

    Full Text Available Anchoring, whereby judgments assimilate to previously considered standards, is one of the most reliable effects in psychology. In the last decade, researchers have become increasingly interested in identifying moderators of anchoring effects. We argue that a drawback of traditional moderator analyses in the standard anchoring paradigm is that they ignore estimate direction—whether participants’ estimates are higher or lower than the anchor value. We suggest that failing to consider estimate direction can sometimes obscure moderation in anchoring tasks, and discuss three potential analytic solutions that take estimate direction into account. Understanding moderators of anchoring effects is essential for a basic understanding of anchoring and for applied research on reducing the influence of anchoring in real-world judgments. Considering estimate direction reduces the risk of failing to detect moderation.

  4. Proteins involved in invasion of human red blood cells by malaria parasites

    Directory of Open Access Journals (Sweden)

    Ewa Jaśkiewicz

    2010-11-01

    Full Text Available Malaria is a disease caused by parasites of Plasmodium species. It is responsible for around 1-2 million deaths annually, mainly children under the age of 5. It occurs mainly in tropical and subtropical areas.Malaria is caused by five Plasmodium species:[i] P. falciparum, P. malariae, P. vivax, P. knowlesi[/i] and [i]P. ovale[/i]. Mosquitoes spread the disease by biting humans. The malaria parasite has two stages of development: the human stage and the mosquito stage. The first stage occurs in the human body and is divided into two phases: the liver phase and the blood phase.The invasion of erythrocytes by [i]Plasmodium[/i] merozoites is a multistep process of specific protein interactions between the parasite and red blood cell. The first step is the reversible merozoite attachment to the erythrocyte followed by its apical reorientation, then formation of an irreversible “tight” junction and finally entry into the red cell in a parasitophorous vacuole.The blood phase is supported by a number of proteins produced by the parasite. The merozoite surface GPI-anchored proteins (MSP-1, 2, 4, 5, 8 and 10 assist in the process of recognition of susceptible erythrocytes, apical membrane antigen (AMA-1 may be directly responsible for apical reorientation of the merozoite and apical proteins which function in tight junction formation. These ligands are members of two families: Duffy binding-like (DBL and reticulocyte binding-like (RBL proteins. In [i]Plasmodium[/i] [i]falciparum[/i] the DBL family includes: EBA-175, EBA-140 (BAEBL, EBA-181 (JESEBL, EBA-165 (PEBL and EBL-1 ligands.To date, no effective antimalarial vaccine has been developed, but there are several studies for this purpose. Therefore, it is crucial to understand the molecular basis of host cells invasion by parasites. Major efforts are focused on developing a multiantigenic and multiepitope vaccine preventing all steps of [i]Plasmodium[/i] invasion.

  5. The late endosomal HOPS complex anchors active G-protein signaling essential for pathogenesis in magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Ravikrishna Ramanujam

    Full Text Available In Magnaporthe oryzae, the causal ascomycete of the devastating rice blast disease, the conidial germ tube tip must sense and respond to a wide array of requisite cues from the host in order to switch from polarized to isotropic growth, ultimately forming the dome-shaped infection cell known as the appressorium. Although the role for G-protein mediated Cyclic AMP signaling in appressorium formation was first identified almost two decades ago, little is known about the spatio-temporal dynamics of the cascade and how the signal is transmitted through the intracellular network during cell growth and morphogenesis. In this study, we demonstrate that the late endosomal compartments, comprising of a PI3P-rich (Phosphatidylinositol 3-phosphate highly dynamic tubulo-vesicular network, scaffold active MagA/GαS, Rgs1 (a GAP for MagA, Adenylate cyclase and Pth11 (a non-canonical GPCR in the likely absence of AKAP-like anchors during early pathogenic development in M. oryzae. Loss of HOPS component Vps39 and consequently the late endosomal function caused a disruption of adenylate cyclase localization, cAMP signaling and appressorium formation. Remarkably, exogenous cAMP rescued the appressorium formation defects associated with VPS39 deletion in M. oryzae. We propose that sequestration of key G-protein signaling components on dynamic late endosomes and/or endolysosomes, provides an effective molecular means to compartmentalize and control the spatio-temporal activation and rapid downregulation (likely via vacuolar degradation of cAMP signaling amidst changing cellular geometry during pathogenic development in M. oryzae.

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

    Science.gov (United States)

    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.

  7. Biased calculations: Numeric anchors influence answers to math equations

    Directory of Open Access Journals (Sweden)

    Andrew R. Smith

    2011-02-01

    Full Text Available People must often perform calculations in order to produce a numeric estimate (e.g., a grocery-store shopper estimating the total price of his or her shopping cart contents. The current studies were designed to test whether estimates based on calculations are influenced by comparisons with irrelevant anchors. Previous research has demonstrated that estimates across a wide range of contexts assimilate toward anchors, but none has examined estimates based on calculations. In two studies, we had participants compare the answers to math problems with anchors. In both studies, participants' estimates assimilated toward the anchor values. This effect was moderated by time limit such that the anchoring effects were larger when the participants' ability to engage in calculations was limited by a restrictive time limit.

  8. β-1,6-glucan synthesis-associated genes are required for proper spore wall formation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Pan, Hua-Ping; Wang, Ning; Tachikawa, Hiroyuki; Nakanishi, Hideki; Gao, Xiao-Dong

    2017-11-01

    The yeast spore wall is an excellent model to study the assembly of an extracellular macromolecule structure. In the present study, mutants defective in β-1,6-glucan synthesis, including kre1∆, kre6∆, kre9∆ and big1∆, were sporulated to analyse the effect of β-1,6-glucan defects on the spore wall. Except for kre6∆, these mutant spores were sensitive to treatment with ether, suggesting that the mutations perturb the integrity of the spore wall. Morphologically, the mutant spores were indistinguishable from wild-type spores. They lacked significant sporulation defects partly because the chitosan layer, which covers the glucan layer, compensated for the damage. The proof for this model was obtained from the effect of the additional deletion of CHS3 that resulted in the absence of the chitosan layer. Among the double mutants, the most severe spore wall deficiency was observed in big1∆ spores. The majority of the big1∆chs3∆ mutants failed to form visible spores at a higher temperature. Given that the big1∆ mutation caused a failure to attach a GPI-anchored reporter, Cwp2-GFP, to the spore wall, β-1,6-glucan is involved in tethering of GPI-anchored proteins in the spore wall as well as in the vegetative cell wall. Thus, β-1,6-glucan is required for proper organization of the spore wall. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  9. Different methods of membrane domains isolation result in similar 2-D distribution patterns of membrane domain proteins

    Czech Academy of Sciences Publication Activity Database

    Matoušek, Petr; Hodný, Zdeněk; Švandová, I.; Svoboda, Petr

    2003-01-01

    Roč. 81, č. 6 (2003), s. 365-372 ISSN 0829-8211 R&D Projects: GA MŠk LN00A026 Grant - others:Wellcome Trust(GB) xx Institutional research plan: CEZ:AV0Z5011922; CEZ:MSM 113100003; CEZ:AV0Z5039906 Keywords : membrane domain * G protein * two-dimensional electrophoresis * GPI-ancored proteins Subject RIV: CE - Biochemistry Impact factor: 2.456, year: 2003

  10. Midgut malrotation presenting with left-sided acute appendicitis and CT inversion sign

    Science.gov (United States)

    Çağlar, Emrah; Arıbaş, Bilgin; Tiken, Ramazan; Keskin, Suat

    2014-01-01

    In patients presenting with abdominal pain, appendicitis is the most common surgical disorder. Appendicitis causing pain in the left lower quadrant is extremely rare and can occur with congenital abnormalities that include true left-sided appendix or as an atypical presentation of right-sided long appendix, which projects into the left lower quadrant. We report a case of a 69-year-old man showing midgut malrotation with acute appendicitis presenting as left lower quadrant abdominal pain. PMID:24682135

  11. Arabidopsis thaliana FLA4 functions as a glycan-stabilized soluble factor via its carboxy-proximal Fasciclin 1 domain.

    Science.gov (United States)

    Xue, Hui; Veit, Christiane; Abas, Lindy; Tryfona, Theodora; Maresch, Daniel; Ricardi, Martiniano M; Estevez, José Manuel; Strasser, Richard; Seifert, Georg J

    2017-08-01

    Fasciclin-like arabinogalactan proteins (FLAs) are involved in numerous important functions in plants but the relevance of their complex structure to physiological function and cellular fate is unresolved. Using a fully functional fluorescent version of Arabidopsis thaliana FLA4 we show that this protein is localized at the plasma membrane as well as in endosomes and soluble in the apoplast. FLA4 is likely to be GPI-anchored, is highly N-glycosylated and carries two O-glycan epitopes previously associated with arabinogalactan proteins. The activity of FLA4 was resistant against deletion of the amino-proximal fasciclin 1 domain and was unaffected by removal of the GPI-modification signal, a highly conserved N-glycan or the deletion of predicted O-glycosylation sites. Nonetheless these structural changes dramatically decreased endoplasmic reticulum (ER)-exit and plasma membrane localization of FLA4, with N-glycosylation acting at the level of ER-exit and O-glycosylation influencing post-secretory fate. We show that FLA4 acts predominantly by molecular interactions involving its carboxy-proximal fasciclin 1 domain and that its amino-proximal fasciclin 1 domain is required for stabilization of plasma membrane localization. FLA4 functions as a soluble glycoprotein via its carboxy-proximal Fas1 domain and its normal cellular trafficking depends on N- and O-glycosylation. © 2017 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

  12. Decay accelerating factor (DAF) is anchored to membranes by a C-terminal glycolipid

    International Nuclear Information System (INIS)

    Medof, M.E.; Haas, R.; Walter, E.I.; Rosenberry, T.L.

    1986-01-01

    Purified 70 kDa membrane (m) DAF incorporates into cells when added in vitro. A 2 kDa smaller DAF form which functions extrinsically like C4bp but is unable to incorporate can be isolated from urine (u). Because of common deficits of mDAF and acetylcholinesterase (AChE) in erythrocytes (E) of patients with paroxysmal nocturnal hemoglobinuria (PNH), mDAF was analyzed for a O-terminal glycolipid membrane anchor similar to that in E AChE. Incubation of E with phosphatidylinositol-specific phospholipase C, an enzyme which cleaves a similar glycolipid anchor in trypanosome variant surface glycoproteins (mfVSGs), released 20% of the DAF antigen. The released DAF species resembled uDAF in size, extrinsic model of C4b2a decay, and lack of hydrophobicity. Reductive radiomethylation of mDAF with [ 14 C]HCHO and NaCNBH 3 revealed ethanolamine and glucosamine in proportions similar to those in the E AChE glycolipid anchor. Papain cleavage of radiomethylated mDAF released the labeled ethanolamine and glucosamine in small O-terminal fragments from the residual DAF that retained N-terminal Asp. Following labeling of the anchors of mDAF and E AChE with the lipophilic photoreagent 3-trifluoromethyl-3-(m-[ 125 I]iodophenyl)diazirine, cleavage at the glucosamine residue by deamination quantitatively released the label from both proteins. Biosynthetic labeling of Hela cells with [ 3 H]ethanolamine resulted in rapid 3 H incorporation into both 48 kDa proDAF and 70 kDa mDAF. These data indicate that mDAF is anchored by a glycolipid similar to that in E AChE, mfVSGs and Thy-1 antigen and raise the possibility that a defect in the assembly or attachment of this structure could account for the deficits of mDAF and E AChE in PNH

  13. Convenient method for resolving degeneracies due to symmetry of the magnetic susceptibility tensor and its application to pseudo contact shift-based protein-protein complex structure determination

    Energy Technology Data Exchange (ETDEWEB)

    Kobashigawa, Yoshihiro; Saio, Tomohide [Hokkaido University, Department of Structural Biology, Faculty of Advanced Life Science (Japan); Ushio, Masahiro [Hokkaido University, Graduate School of Life Science (Japan); Sekiguchi, Mitsuhiro [Astellas Pharma Inc., Analysis and Pharmacokinetics Research Labs, Department of Drug Discovery (Japan); Yokochi, Masashi; Ogura, Kenji; Inagaki, Fuyuhiko, E-mail: finagaki@pharm.hokudai.ac.jp [Hokkaido University, Department of Structural Biology, Faculty of Advanced Life Science (Japan)

    2012-05-15

    Pseudo contact shifts (PCSs) induced by paramagnetic lanthanide ions fixed in a protein frame provide long-range distance and angular information, and are valuable for the structure determination of protein-protein and protein-ligand complexes. We have been developing a lanthanide-binding peptide tag (hereafter LBT) anchored at two points via a peptide bond and a disulfide bond to the target proteins. However, the magnetic susceptibility tensor displays symmetry, which can cause multiple degenerated solutions in a structure calculation based solely on PCSs. Here we show a convenient method for resolving this degeneracy by changing the spacer length between the LBT and target protein. We applied this approach to PCS-based rigid body docking between the FKBP12-rapamycin complex and the mTOR FRB domain, and demonstrated that degeneracy could be resolved using the PCS restraints obtained from two-point anchored LBT with two different spacer lengths. The present strategy will markedly increase the usefulness of two-point anchored LBT for protein complex structure determination.

  14. 76 FR 30301 - Commercial Acquisition; Anchor Tenancy

    Science.gov (United States)

    2011-05-25

    ... NATIONAL AERONAUTICS AND SPACE ADMINISTRATION 48 CFR Part 1812 RIN 2700-AD64 Commercial... consistent with NASA's authority under Section 401 of the Commercial Space Competitiveness Act (CSCA) of 1992. NASA may enter into multi-year anchor tenancy contracts for commercial space goods or services. Anchor...

  15. A comparison of lateral ankle ligament suture anchor strength.

    Science.gov (United States)

    Barber, F Alan; Herbert, Morley A; Crates, John M

    2013-06-01

    Lateral ankle ligament repairs increasingly use suture anchors instead of bone tunnels. Our purpose was to compare the biomechanical properties of a knotted and knotless suture anchor appropriate for a lateral ankle ligament reconstruction. In porcine distal fibulae, 10 samples of 2 different PEEK anchors were inserted. The attached sutures were cyclically loaded between 10N and 60N for 200 cycles. A destructive pull was performed and failure loads, cyclic displacement, stiffness, and failure mode recorded. PushLock 2.5 anchors failed before 200 cycles. PushLock 100 cycle displacement was less than Morphix 2.5 displacement (panchors completing 200 cycles was 86.5N (PushLock) and 252.1N (Morphix) (panchor breaking and suture breakage. The knotted Morphix demonstrated more displacement and greater failure strength than the knotless PushLock. The PushLock failed consistently with suture breaking. The Morphix anchor failed both by anchor breaking and by suture breaking. Copyright © 2012 European Foot and Ankle Society. Published by Elsevier Ltd. All rights reserved.

  16. The effect of starvation and re-feeding on mitochondrial potential in the midgut of Neocaridina davidi (Crustacea, Malacostraca.

    Directory of Open Access Journals (Sweden)

    Agnieszka Włodarczyk

    Full Text Available The midgut in the freshwater shrimp Neocaridina davidi (previously named N. heteropoda (Crustacea, Malacostraca is composed of a tube-shaped intestine and a large hepatopancreas that is formed by numerous blind-ended tubules. The precise structure and ultrastructure of these regions were presented in our previous papers, while here we focused on the ultrastructural changes that occurred in the midgut epithelial cells (D-cells in the intestine, B- and F- cells in the hepatopancreas after long-term starvation and re-feeding. We used transmission electron microscopy, light and confocal microscopes and flow cytometry to describe all of the changes that occurred due to the stressor with special emphasis on mitochondrial alterations. A quantitative assessment of cells with depolarized mitochondria helped us to establish whether there is a relationship between starvation, re-feeding and the inactivation/activation of mitochondria. The results of our studies showed that in the freshwater shrimp N. davidi that were analyzed, long-term starvation activates the degeneration of epithelial cells at the ultrastructural level and causes an increase of cells with depolarized (non-active mitochondria. The process of re-feeding leads to the gradual regeneration of the cytoplasm of the midgut epithelial cells; however, these changes were observed at the ultrastructural level. Additionally, re-feeding causes the regeneration of mitochondrial ultrastructure. Therefore, we can state that the increase in the number of cells with polarized mitochondria occurs slowly and does not depend on ultrastructural alterations.

  17. The effect of starvation and re-feeding on mitochondrial potential in the midgut of Neocaridina davidi (Crustacea, Malacostraca).

    Science.gov (United States)

    Włodarczyk, Agnieszka; Sonakowska, Lidia; Kamińska, Karolina; Marchewka, Angelika; Wilczek, Grażyna; Wilczek, Piotr; Student, Sebastian; Rost-Roszkowska, Magdalena

    2017-01-01

    The midgut in the freshwater shrimp Neocaridina davidi (previously named N. heteropoda) (Crustacea, Malacostraca) is composed of a tube-shaped intestine and a large hepatopancreas that is formed by numerous blind-ended tubules. The precise structure and ultrastructure of these regions were presented in our previous papers, while here we focused on the ultrastructural changes that occurred in the midgut epithelial cells (D-cells in the intestine, B- and F- cells in the hepatopancreas) after long-term starvation and re-feeding. We used transmission electron microscopy, light and confocal microscopes and flow cytometry to describe all of the changes that occurred due to the stressor with special emphasis on mitochondrial alterations. A quantitative assessment of cells with depolarized mitochondria helped us to establish whether there is a relationship between starvation, re-feeding and the inactivation/activation of mitochondria. The results of our studies showed that in the freshwater shrimp N. davidi that were analyzed, long-term starvation activates the degeneration of epithelial cells at the ultrastructural level and causes an increase of cells with depolarized (non-active) mitochondria. The process of re-feeding leads to the gradual regeneration of the cytoplasm of the midgut epithelial cells; however, these changes were observed at the ultrastructural level. Additionally, re-feeding causes the regeneration of mitochondrial ultrastructure. Therefore, we can state that the increase in the number of cells with polarized mitochondria occurs slowly and does not depend on ultrastructural alterations.

  18. Post-installed concrete anchors in nuclear power plants: Performance and qualification

    International Nuclear Information System (INIS)

    Mahrenholtz, Philipp; Eligehausen, Rolf

    2015-01-01

    Graphical abstract: - Highlights: • Review of qualification and design regulations for anchors in nuclear power plants. • First complete set of nuclear anchor load–displacement data and its evaluation ever. • Demonstration of robust test behavior of a qualified post-installed anchor product. - Abstract: In nuclear power plants (NPPs), post-installed anchors are widely used for structural and non-structural connections to concrete. In many countries, anchor products employed for safety relevant applications have to be approved by the authorities. For the high safety standards in force for NPPs, special requirements have to be met to allow for extreme design situations. This paper presents an experimental test program conducted to evaluate the performance of anchors according to the German Guideline for Anchorages in Nuclear Power Plants and Nuclear Technology Installations (DIBt KKW Leitfaden, 2010). After a brief introduction to anchor behavior and the regulative context, the results of tension and shear tests carried out on undercut anchors are discussed. Robust load capacities and relatively small displacements determined for demanding load and crack cycling tests demonstrated the suitability of anchors qualified according to a state-of-the-art qualification guideline

  19. Anchorage Behaviors of Frictional Tieback Anchors in Silty Sand

    Science.gov (United States)

    Hsu, Shih-Tsung; Hsiao, Wen-Ta; Chen, Ke-Ting; Hu, Wen-Chi; Wu, Ssu-Yi

    2017-06-01

    Soil anchors are extensively used in geotechnical applications, most commonly serve as tieback walls in deep excavations. To investigate the anchorage mechanisms of this tieback anchor, a constitutive model that considers both strain hardening and softening and volume dilatancy entitled SHASOVOD model, and FLAC3D software are used to perform 3-D numerical analyses. The results from field anchor tests are compared with those calculated by numerical analyses to enhance the applicability of the numerical method. After the calibration, this research carried out the parameter studies by numerical analyses. The numerical results reveal that whether the yield of soil around an anchor develops to ground surface and/or touches the diaphragm wall depending on the overburden depth H and the embedded depth Z of an anchor, this study suggests the minimum overburden and embedded depths to avoid the yield of soils develop to ground surface and/or touch the diaphragm wall. When the embedded depth, overburden depth or fixed length of an anchor increases, the anchorage capacity also increases. Increasing fixed length should be the optimum method to increase the anchorage capacity for fixed length less than 20m. However, when the fixed length of an anchor exceeds 30 m, the increasing rate of anchorage capacity per fixed length decreases, and progressive yield occurs obviously between the fixed length and surrounding soil.

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

    Science.gov (United States)

    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.

  1. Molecular characterization of a peritrophic membrane protein from the silkworm, Bombyx mori.

    Science.gov (United States)

    Hu, Xiaolong; Chen, Lin; Yang, Rui; Xiang, Xingwei; Wu, Xiaofeng

    2013-02-01

    The peritrophic membrane lines the gut of most insects at one or more stages of their life cycles. It facilitates the digestive processes in the guts and protects from invasion by pathogens or food particles. In the current study, a novel PM protein, designated as BmMtch, was identified from the silkworm, Bombyx mori. The open reading frame of BmMtch is 888 bp in length, encoding 295 amino acid residues consisting of two domains (Mito_carr domains) and three transmembrane regions. They are localized on the 11th chromosome as single copy with one exon only. Quantitative real time PCR analysis (qRT-PCR) revealed that BmMtch was mainly expressed in larval fat bodies, Malpighian tubules, testis and ovaries, and could be detected through all stages of the life cycle of silkworm. Immuno-fluorescence analysis indicated that BmMtch was localized within the goblet cell of larval midgut. Western blotting analysis showed that BmMtch were detected in total proteins of PM and larval midgut. The characteristics of BmMtch indicated that BmMtch represents a novel member of insect PM proteins, without chitin-binding domains.

  2. Antigenicity analysis of human parvovirus B19-VP1u protein in the induction of anti-phospholipid syndrome.

    Science.gov (United States)

    Lin, Chun-Yu; Chiu, Chun-Ching; Cheng, Ju; Lin, Chia-Yun; Shi, Ya-Fang; Tsai, Chun-Chou; Tzang, Bor-Show; Hsu, Tsai-Ching

    2018-01-01

    Mounting evidence suggests a connection between human parvovirus B19 (B19) and autoimmune diseases, and especially an association between the B19-VP1 unique region (VP1u) and anti-phospholipid syndrome (APS). However, little is known about the antigenicity of B19-VP1u in the induction of APS-like syndrome. To elucidate the antigenicity of B19-VP1u in the induction of APS, N-terminal truncated B19-VP1u (tVP1u) proteins were prepared to immunize Balb/c mice to generate antibodies against B19-tVP1u proteins. The secreted phospholipase A2 (sPLA2) activities and binding specificity of mice anti-B19-tVP1u antibodies with cardiolipin (CL) and beta-2-glycoprotein I (β2GPI) were evaluated by performing immunoblot, ELISA and absorption experiments. A mice model of passively induced APS was adopted. Although sPLA2 activities were identified in all B19-tVP1u proteins, only amino acid residues 61-227 B19-tVP1u exhibited a higher sPLA2 activity. Autoantibodies against CL and β2GPI exhibited binding activities with all B19-tVP1u proteins. IgG that was purified from mice that had been immunized with amino acid residues 21-227 to 121-227 B19-tVP1u proteins exhibited significantly higher binding activity with CL. IgG that was purified from mice that had been immunized with amino acid residues 21-227, 31-227, 82-227 and 91-227 B19-tVP1u proteins exhibited significantly higher binding activity with β2GPI. Accordingly, significantly higher binding inhibition of CL was detected in the presence of amino acid residues 61-227 and 101-227 B19-tVP1u. Significantly higher binding inhibition of β2GPI was detected in the presence of amino acid residues 21-227, 31-227, 82-227 and 91-227 B19-tVP1u. The mice that received amino acid residues 31-227 or 61-227 anti-tB19-VP1u IgG revealed significant thrombocytopenia and those that received amino acid residues 21-227, 31-227, 61-227, 71-227, 82-227, 91-227, 101-227 or 114-227 anti-tB19-VP1u IgG exhibited significantly prolonged aPTT. These

  3. Link Anchors in Images: Is there Truth?

    NARCIS (Netherlands)

    Aly, Robin; McGuinness, Kevin; Kleppe, Martijn; Ordelman, Roeland J.F.; O'Connor, Noel; de Jong, Franciska M.G.

    2012-01-01

    While automatic linking in text collections is well understood, little is known about links in images. In this work, we investigate two aspects of anchors, the origin of a link, in images: 1) the requirements of users for such anchors, e.g. the things users would like more information on, and 2)

  4. A comparative study on oxidative stress response in the hepatopancreas and midgut of the white shrimp Litopenaeus vannamei under gradual changes to low or high pH environment.

    Science.gov (United States)

    Han, Si-Yin; Wang, Meng-Qiang; Wang, Bao-Jie; Liu, Mei; Jiang, Ke-Yong; Wang, Lei

    2018-05-01

    White shrimp Litopenaeus vannamei were reared under conditions of gradual changes to a low pH (gradual-low pH, 6.65-8.20) or a high pH (gradual-high pH, 8.20-9.81) versus a normal pH environment (8.14-8.31) during a 28-day period. Survival of shrimp, and ROS production, antioxidant responses and oxidative damage in the hepatopancreas and midgut were investigated. Consequently, shrimp enhanced MnSOD, GPx, and Hsp70 transcripts as early defense mechanism in the hepatopancreas and midgut to scavenge excessive ROS during short-term (≤ 7 days) gradual-low and high pH stress. Meanwhile, the hepatopancreas was more sensitive to ROS than midgut because of earlier ROS production increase, antioxidant response and oxidative damage. Then, suppressed antioxidant response in the hepatopancreas and midgut of shrimp suggested a loss of antioxidant regulatory capacity caused by aggravated oxidative damage after long-term (≥ 14 days) gradual-high pH stress, leading to continuous death. However, enhanced GPx, GST, and Hsp70 transcripts in the hepatopancreas and midgut might be long-term(≥ 14 days) antioxidant adaptation mechanism of shrimp to gradual-low pH stress, which could prevent further ROS perturbation and weaken oxidative damage to achieve a new immune homeostasis, contributing to stable survival rate. Therefore, we have a few insights that it is necessary to protect hepatopancreas for controlling shrimp death under gradual-high pH stress. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. Novel tumor necrosis factor-responsive mammalian neutral sphingomyelinase-3 is a C-tail-anchored protein.

    Science.gov (United States)

    Krut, Oleg; Wiegmann, Katja; Kashkar, Hamid; Yazdanpanah, Benjamin; Krönke, Martin

    2006-05-12

    Two genes encoding neutral sphingomyelinases-1 and -2 (sphingomyelin phosphodiesterases-2 and -3) have been recently identified that hydrolyze sphingomyelin to phosphorylcholine and ceramide. Data bank searches using a peptide sequence derived from a previously purified bovine neutral sphingomyelinase (nSMase) allowed us to identify a cDNA encoding a novel human sphingomyelinase, nSMase3, that shows only a little homology to nSMase1 and -2. nSMase3 was biochemically characterized by overexpression in a yeast strain, JK9-3ddeltaIsc1p, lacking endogenous SMase activity. Similar to nSMase2, nSMase3 is Mg2+-dependent and shows optimal activity at pH 7, which is enhanced in the presence of phosphatidylserine and inhibited by scyphostatin. nSMase3 is ubiquitously expressed as a 4.6-kb mRNA species. nSMase3 lacks an N-terminal signal peptide, yet contains a 23-amino-acid transmembrane domain close to the C terminus, which is indicative for the family of C-tail-anchored integral membrane proteins. Cellular localization studies with hemagglutinin-tagged nSMase3 demonstrated colocalization with markers of the endoplasmic reticulum as well as with Golgi markers. Tumor necrosis factor stimulates rapid activation of nSMase3 in MCF7 cells with peak activity at 1.5 min, which was impaired by expression of dominant negative FAN.

  6. The Effect of Mini and Midi Anchor Tests on Test Equating

    Science.gov (United States)

    Arikan, Çigdem Akin

    2018-01-01

    The main purpose of this study is to compare the test forms to the midi anchor test and the mini anchor test performance based on item response theory. The research was conducted with using simulated data which were generated based on Rasch model. In order to equate two test forms the anchor item nonequivalent groups (internal anchor test) was…

  7. G protein-coupled receptor 30 (GPR30) forms a plasma membrane complex with membrane-associated guanylate kinases (MAGUKs) and protein kinase A-anchoring protein 5 (AKAP5) that constitutively inhibits cAMP production.

    Science.gov (United States)

    Broselid, Stefan; Berg, Kelly A; Chavera, Teresa A; Kahn, Robin; Clarke, William P; Olde, Björn; Leeb-Lundberg, L M Fredrik

    2014-08-08

    GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. The Polerovirus Minor Capsid Protein Determines Vector Specificity and Intestinal Tropism in the Aphid

    Science.gov (United States)

    Brault, Véronique; Périgon, Sophie; Reinbold, Catherine; Erdinger, Monique; Scheidecker, Danièle; Herrbach, Etienne; Richards, Ken; Ziegler-Graff, Véronique

    2005-01-01

    Aphid transmission of poleroviruses is highly specific, but the viral determinants governing this specificity are unknown. We used a gene exchange strategy between two poleroviruses with different vectors, Beet western yellows virus (BWYV) and Cucurbit aphid-borne yellows virus (CABYV), to analyze the role of the major and minor capsid proteins in vector specificity. Virus recombinants obtained by exchanging the sequence of the readthrough domain (RTD) between the two viruses replicated in plant protoplasts and in whole plants. The hybrid readthrough protein of chimeric viruses was incorporated into virions. Aphid transmission experiments using infected plants or purified virions revealed that vector specificity is driven by the nature of the RTD. BWYV and CABYV have specific intestinal sites in the vectors for endocytosis: the midgut for BWYV and both midgut and hindgut for CABYV. Localization of hybrid virions in aphids by transmission electron microscopy revealed that gut tropism is also determined by the viral origin of the RTD. PMID:16014930

  9. Use of the ROC anchor in foot and ankle surgery. A retrospective study.

    Science.gov (United States)

    Kuwada, G T

    1999-05-01

    A retrospective study was conducted on the use of the ROC (Radial Osteo Compression) soft-tissue anchor in foot and ankle surgery. This article describes how the anchor is deployed, problematic aspects of using the anchor, and complications and success rates associated with the anchor in ankle stabilizations, posterior tibial tendon reconstruction, peroneus brevis tendon reconstruction after fracture of the base of the fifth metatarsal, and detachment and reattachment of the Achilles tendon. The ROC anchor consists of the anchor with nonabsorbable suture attached to the shaft, the deployment handle, and drill bits. The anchor and shaft are snapped into the deployment handle and inserted into the drill hole. Compression of the trigger deploys the anchor into the hole. The ROC anchor was found to be reliable, useful, and relatively easy to deploy, with outcomes similar to those of other soft-tissue anchors.

  10. Bacterial diversity analysis of larvae and adult midgut microflora using culture-dependent and culture-independent methods in lab-reared and field-collected Anopheles stephensi-an Asian malarial vector

    Directory of Open Access Journals (Sweden)

    Adak Tridibesh

    2009-05-01

    Full Text Available Abstract Background Mosquitoes are intermediate hosts for numerous disease causing organisms. Vector control is one of the most investigated strategy for the suppression of mosquito-borne diseases. Anopheles stephensi is one of the vectors of malaria parasite Plasmodium vivax. The parasite undergoes major developmental and maturation steps within the mosquito midgut and little is known about Anopheles-associated midgut microbiota. Identification and characterization of the mosquito midgut flora is likely to contribute towards better understanding of mosquito biology including longevity, reproduction and mosquito-pathogen interactions that are important to evolve strategies for vector control mechanisms. Results Lab-reared and field-collected A. stephensi male, female and larvae were screened by "culture-dependent and culture-independent" methods. Five 16S rRNA gene library were constructed form lab and field-caught A. stephensi mosquitoes and a total of 115 culturable isolates from both samples were analyzed further. Altogether, 68 genera were identified from midgut of adult and larval A. stephensi, 53 from field-caught and 15 from lab-reared mosquitoes. A total of 171 and 44 distinct phylotypes having 85 to 99% similarity with the closest database matches were detected among field and lab-reared A. stephensi midgut, respectively. These OTUs had a Shannon diversity index value of 1.74–2.14 for lab-reared and in the range of 2.75–3.49 for field-caught A. stephensi mosquitoes. The high species evenness values of 0.93 to 0.99 in field-collected adult and larvae midgut flora indicated the vastness of microbial diversity retrieved by these approaches. The dominant bacteria in field-caught adult male A. stephensi were uncultured Paenibacillaceae while in female and in larvae it was Serratia marcescens, on the other hand in lab-reared mosquitoes, Serratia marcescens and Cryseobacterium meninqosepticum bacteria were found to be abundant. Conclusion

  11. Acute appendicitis in a young adult with midgut malrotation: a case report

    International Nuclear Information System (INIS)

    Bider, K.; Kaim, A.; Wiesner, W.; Bongartz, G.

    2001-01-01

    Midgut malrotation is defined as a developmental anomaly that may cause atypical clinical symptoms in relatively common intestinal disorders due to altered anatomy. A 27-year-old woman presented with acute left-sided abdominal pain. Underlying type Ia malrotation prevented the correct clinical diagnosis of perforated, ulcerated appendicitis. Cross-sectional imaging demonstrated all the typical signs of this type of malrotation, i.e., right-sided duodenojejunal junction, left positioned cecum and ascending colon, inverted position of the superior mesenteric vessels, and hypoplasia of the uncinate process of pancreas, and surgical treatment was initiated. (orig.)

  12. Isolation and characterization of halotolerant bacteria associated with the midgut of Culex quinquefasciatus Say (Diptera: Culicidae).

    Science.gov (United States)

    Reegan, Appadurai Daniel; Paulraj, Michael Gabriel; Ignacimuthu, Savarimuthu

    2013-11-01

    We show for the first time that the midgut of Culex quinquefasciatus (Say) mosquito larvae harbors halotolerant bacteria. The midgut from field collected Cx. quinquefasciatus larvae were dissected under aseptic conditions, homogenized and plated on LB agar medium with 2% (w/v) NaCl. Two different colonies were successfully isolated and bacterial isolates were identified by 16S rRNA sequences. The halotolerant bacterial isolates were: Halobacillus litoralis (CxH1) and Staphylococcus cohnii (CxH2). The gene sequence of these isolates has been deposited in GenBank (JN016804 and JN183986). These halotolerant bacteria grew in the absence of salt (0%) as well as in the presence of relatively high salt concentrations in culture medium (20%), and grew best in the presence of 8-10% (w/v) NaCl. H. litoralis and S. cohnii showed growth up to 18 and 20% (w/v) NaCl, respectively. Optimum growth temperatures for both the bacteria were between 30-37 degrees C. H. litoralis was resistant to the antibiotics oxacillin, penicillin, polymixin and S. cohnii was resistant to the antibiotic oxacillin.

  13. On the Robustness of Anchoring Effects in WTP and WTA Experiments

    OpenAIRE

    Drew Fudenberg; David K Levine; Zacharias Maniadis

    2010-01-01

    We reexamine the effects of the anchoring manipulation of Ariely, Loewenstein, and Prelec (2003) on the evaluation of common market goods and find very weak anchoring effects. We perform the same manipulation on the evaluation of binary lotteries, and find no anchoring effects at all. This suggests limits on the robustness of anchoring effects. (JEL C91, D12, D44)

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

    Science.gov (United States)

    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…

  15. Stone anchors of India: Findings, classification and significance.

    Digital Repository Service at National Institute of Oceanography (India)

    Tripati, S.

    Various types of stone anchors have been observed during inshore and offshore explorations along the east and west coasts of India. The earliest stone anchors of India have been recorded from the Harappan sites (3rd millennium BC), but their shape...

  16. Investigation of suction anchor pullout capacity under undrained conditions

    OpenAIRE

    Jarand, Pollestad

    2015-01-01

    Master's thesis in Offshore technology Floating units are dependent on reliable mooring systems to ensure safety during marine operations. Suction anchors have proved to be a technologically viable and cost-effective concept. They are capable of precision installation, re-use, and provide large resistive capacity. This thesis investigates load capacity and failure modes of suction anchors subjected to vertical, horizontal (lateral), and incline loading. Suction anchor design co...

  17. Pyramidal anchor stone from Baga waters of Goa, west coast of India

    Digital Repository Service at National Institute of Oceanography (India)

    Tripati, S.

    . Pyramidal anchor stones have an apex hole which goes up to the round hole, however Goa anchor stone has no such perforation, but, instead has a rectangular cutting on the apex. The anchor stone is compared with Greek pyramidal anchor stones, and probably...

  18. Drosophila acetylcholinesterase: demonstration of a glycoinositol phospholipid anchor and an endogenous proteolytic cleavage

    International Nuclear Information System (INIS)

    Haas, R.; Marshall, T.L.; Rosenberry, T.L.

    1988-01-01

    The presence of a glycoinositol phospholipid anchor Drosophila acetylcholinesterase (AChE) was shown by several criteria. Chemical analysis of highly purified Drosophila AChE demonstrated approximately one residue of inositol per enzyme subunit. Selective cleavage by Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) was tested with Drosophila AChE radiolabeled by the photoactivatable affinity probe 3-(trifluoromethyl)-3-(m-[ 125 I]iodophenyl)diazirine ([ 125 I]TID), a reagent that specifically labels the lipid moiety of glycoinositol phospholipid-anchored proteins. Digestion with PI-PLC released 75% of this radiolabel from the protein. Gel electrophoresis of Drosophila AChE in sodium dodecyl sulfate indicated prominent 55- and 16-kDa bands and a faint 70-kDa band. The [ 125 ]I]TID label was localized on the 55-kDa fragment, suggesting that this fragment is the C-terminal portion of the protein. In support of this conclusion, a sensitive microsequencing procedure that involved manual Edman degradation combined with radiomethylation was used to determine residues 2-5 of the 16-kDa fragment. Comparison with the Drosophila AChE cDNA sequence confirmed that the 16-kDa fragment includes the N-terminus of AChE. Furthermore, the position of the N-terminal amino acid of the mature Drosophila AChE is closely homologous to that of Torpedo AChE. The presence of radiomethylatable ethanolamine in both 16- and 55-kDa fragments was also confirmed. Thus, Drosophila AChE may include a second posttranslational modification involving ethanolamine

  19. Stage-specific adhesion of Leishmania promastigotes to sand fly midguts assessed using an improved comparative binding assay.

    Directory of Open Access Journals (Sweden)

    Raymond Wilson

    2010-09-01

    Full Text Available The binding of Leishmania promastigotes to the midgut epithelium is regarded as an essential part of the life-cycle in the sand fly vector, enabling the parasites to persist beyond the initial blood meal phase and establish the infection. However, the precise nature of the promastigote stage(s that mediate binding is not fully understood.To address this issue we have developed an in vitro gut binding assay in which two promastigote populations are labelled with different fluorescent dyes and compete for binding to dissected sand fly midguts. Binding of procyclic, nectomonad, leptomonad and metacyclic promastigotes of Leishmania infantum and L. mexicana to the midguts of blood-fed, female Lutzomyia longipalpis was investigated. The results show that procyclic and metacyclic promastigotes do not bind to the midgut epithelium in significant numbers, whereas nectomonad and leptomonad promastigotes both bind strongly and in similar numbers. The assay was then used to compare the binding of a range of different parasite species (L. infantum, L. mexicana, L. braziliensis, L. major, L. tropica to guts dissected from various sand flies (Lu. longipalpis, Phlebotomus papatasi, P. sergenti. The results of these comparisons were in many cases in line with expectations, the natural parasite binding most effectively to its natural vector, and no examples were found where a parasite was unable to bind to its natural vector. However, there were interesting exceptions: L. major and L. tropica being able to bind to Lu. longipalpis better than L. infantum; L. braziliensis was able to bind to P. papatasi as well as L. major; and significant binding of L. major to P. sergenti and L. tropica to P. papatasi was observed.The results demonstrate that Leishmania gut binding is strictly stage-dependent, is a property of those forms found in the middle phase of development (nectomonad and leptomonad forms, but is absent in the early blood meal and final stages (procyclic

  20. Peptide-Mediated Liposome Fusion: The Effect of Anchor Positioning

    Directory of Open Access Journals (Sweden)

    Niek S. A. Crone

    2018-01-01

    Full Text Available A minimal model system for membrane fusion, comprising two complementary peptides dubbed “E” and “K” joined to a cholesterol anchor via a polyethyleneglycol spacer, has previously been developed in our group. This system promotes the fusion of large unilamellar vesicles and facilitates liposome-cell fusion both in vitro and in vivo. Whilst several aspects of the system have previously been investigated to provide an insight as to how fusion is facilitated, anchor positioning has not yet been considered. In this study, the effects of placing the anchor at either the N-terminus or in the center of the peptide are investigated using a combination of circular dichroism spectroscopy, dynamic light scattering, and fluorescence assays. It was discovered that anchoring the “K” peptide in the center of the sequence had no effect on its structure, its ability to interact with membranes, or its ability to promote fusion, whereas anchoring the ‘E’ peptide in the middle of the sequence dramatically decreases fusion efficiency. We postulate that anchoring the ‘E’ peptide in the middle of the sequence disrupts its ability to form homodimers with peptides on the same membrane, leading to aggregation and content leakage.