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Sample records for outer membrane-spanning proteins

  1. Secretion Trap Tagging of Secreted and Membrane-Spanning Proteins Using Arabidopsis Gene Traps

    Andrew T. Groover; Joseph R. Fontana; Juana M. Arroyo; Cristina Yordan; W. Richard McCombie; Robert A. Martienssen

    2003-01-01

    Secreted and membrane-spanning proteins play fundamental roles in plant development but pose challenges for genetic identification and characterization. We describe a "secretion trap" screen for gene trap insertions in genes encoding proteins routed through the secretory pathway. The gene trap transposon encodes a ß-glucuronidase reporter enzyme...

  2. cDNA for the human β2-adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor

    Kobilka, B.K.; Dixon, R.A.F.; Frielle, T.

    1987-01-01

    The authors have isolated and sequenced a cDNA encoding the human β 2 -adrenergic receptor. The deduced amino acid sequence (413 residues) is that of a protein containing seven clusters of hydrophobic amino acids suggestive of membrane-spanning domains. While the protein is 87% identical overall with the previously cloned hamster β 2 -adrenergic receptor, the most highly conserved regions are the putative transmembrane helices (95% identical) and cytoplasmic loops (93% identical), suggesting that these regions of the molecule harbor important functional domains. Several of the transmembrane helices also share lesser degrees of identity with comparable regions of select members of the opsin family of visual pigments. They have localized the gene for the β 2 -adrenergic receptor to q31-q32 on chromosome 5. This is the same position recently determined for the gene encoding the receptor for platelet-derived growth factor and is adjacent to that for the FMS protooncogene, which encodes the receptor for the macrophage colony-stimulating factor

  3. Membrane-spanning lipids for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer

    Schwarzmann, Günter; Breiden, Bernadette; Sandhoff, Konrad

    2015-01-01

    A Förster resonance energy transfer-based fusion and transfer assay was developed to study, in model membranes, protein-mediated membrane fusion and intermembrane lipid transfer of fluorescent sphingolipid analogs. For this assay, it became necessary to apply labeled reporter molecules that are resistant to spontaneous as well as protein-mediated intermembrane transfer. The novelty of this assay is the use of nonextractable fluorescent membrane-spanning bipolar lipids. Starting from the tetraether lipid caldarchaeol, we synthesized fluorescent analogs with fluorophores at both polar ends. In addition, we synthesized radioactive glycosylated caldarchaeols. These labeled lipids were shown to stretch through bilayer membranes rather than to loop within a single lipid layer of liposomes. More important, the membrane-spanning lipids (MSLs) in contrast to phosphoglycerides proved to be nonextractable by proteins. We could show that the GM2 activator protein (GM2AP) is promiscuous with respect to glycero- and sphingolipid transfer. Saposin (Sap) B also transferred sphingolipids albeit with kinetics different from GM2AP. In addition, we could unambiguously show that the recombinant activator protein Sap C x His6 induced membrane fusion rather than intermembrane lipid transfer. These findings showed that these novel MSLs, in contrast with fluorescent phosphoglycerolipids, are well suited for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer. PMID:26269359

  4. Membrane-spanning lipids for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer.

    Schwarzmann, Günter; Breiden, Bernadette; Sandhoff, Konrad

    2015-10-01

    A Förster resonance energy transfer-based fusion and transfer assay was developed to study, in model membranes, protein-mediated membrane fusion and intermembrane lipid transfer of fluorescent sphingolipid analogs. For this assay, it became necessary to apply labeled reporter molecules that are resistant to spontaneous as well as protein-mediated intermembrane transfer. The novelty of this assay is the use of nonextractable fluorescent membrane-spanning bipolar lipids. Starting from the tetraether lipid caldarchaeol, we synthesized fluorescent analogs with fluorophores at both polar ends. In addition, we synthesized radioactive glycosylated caldarchaeols. These labeled lipids were shown to stretch through bilayer membranes rather than to loop within a single lipid layer of liposomes. More important, the membrane-spanning lipids (MSLs) in contrast to phosphoglycerides proved to be nonextractable by proteins. We could show that the GM2 activator protein (GM2AP) is promiscuous with respect to glycero- and sphingolipid transfer. Saposin (Sap) B also transferred sphingolipids albeit with kinetics different from GM2AP. In addition, we could unambiguously show that the recombinant activator protein Sap C x His6 induced membrane fusion rather than intermembrane lipid transfer. These findings showed that these novel MSLs, in contrast with fluorescent phosphoglycerolipids, are well suited for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

  5. Structural Aspects of Bacterial Outer Membrane Protein Assembly.

    Calmettes, Charles; Judd, Andrew; Moraes, Trevor F

    2015-01-01

    The outer membrane of Gram-negative bacteria is predominantly populated by β-Barrel proteins and lipid anchored proteins that serve a variety of biological functions. The proper folding and assembly of these proteins is essential for bacterial viability and often plays a critical role in virulence and pathogenesis. The β-barrel assembly machinery (Bam) complex is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, whereas the localization of lipoproteins (Lol) system is required for proper targeting of lipoproteins to the outer membrane.

  6. Identification of outer membrane proteins of Yersinia pestis through biotinylation

    Smither, S.J.; Hill, J.; Baar, B.L.M. van; Hulst, A.G.; Jong, A.L. de; Titball, R.W.

    2007-01-01

    The outer membrane of Gram-negative bacteria contains proteins that might be good targets for vaccines, antimicrobials or detection systems. The identification of surface located proteins using traditional methods is often difficult. Yersinia pestis, the causative agent of plague, was labelled with

  7. The outer membrane protein assembly machinery of Neisseria meningitidis

    Volokhina, E.B.|info:eu-repo/dai/nl/304837202

    2009-01-01

    Gram-negative bacteria are characterized by a cell envelope consisting of an inner membrane (IM) and an outer membrane (OM), which are separated by the peptidoglycan-containing periplasm. While the integral IM proteins are alpha-helical, all but one known integral OM proteins (OMPs) are

  8. Periplasmic quality control in biogenesis of outer membrane proteins.

    Lyu, Zhi Xin; Zhao, Xin Sheng

    2015-04-01

    The β-barrel outer membrane proteins (OMPs) are integral membrane proteins that reside in the outer membrane of Gram-negative bacteria and perform a diverse range of biological functions. Synthesized in the cytoplasm, OMPs must be transported across the inner membrane and through the periplasmic space before they are assembled in the outer membrane. In Escherichia coli, Skp, SurA and DegP are the most prominent factors identified to guide OMPs across the periplasm and to play the role of quality control. Although extensive genetic and biochemical analyses have revealed many basic functions of these periplasmic proteins, the mechanism of their collaboration in assisting the folding and insertion of OMPs is much less understood. Recently, biophysical approaches have shed light on the identification of the intricate network. In the present review, we summarize recent advances in the characterization of these key factors, with a special emphasis on the multifunctional protein DegP. In addition, we present our proposed model on the periplasmic quality control in biogenesis of OMPs.

  9. Entry and exit of bacterial outer membrane proteins.

    Misra, Rajeev

    2015-08-01

    The sites of new outer membrane protein (OMP) deposition and the fate of pre-existing OMPs are still enigmatic despite numerous concerted efforts. Rassam et al. identified mid-cell regions as the primary entry points for new OMP insertion in clusters, driving the pre-existing OMP clusters towards cell poles for long-term storage. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli.

    Urfer, Matthias; Bogdanovic, Jasmina; Lo Monte, Fabio; Moehle, Kerstin; Zerbe, Katja; Omasits, Ulrich; Ahrens, Christian H; Pessi, Gabriella; Eberl, Leo; Robinson, John A

    2016-01-22

    Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Helicobacter pylori Outer Membrane Protein-Related Pathogenesis

    Yuichi Matsuo

    2017-03-01

    Full Text Available Helicobacter pylori colonizes the human stomach and induces inflammation, and in some cases persistent infection can result in gastric cancer. Attachment to the gastric mucosa is the first step in establishing bacterial colonization, and outer membrane proteins (OMPs play a pivotal role in binding to human cells. Some OMP interaction molecules are known in H. pylori, and their associated host cell responses have been gradually clarified. Many studies have demonstrated that OMPs are essential to CagA translocation into gastric cells via the Type IV secretion system of H. pylori. This review summarizes the mechanisms through which H. pylori utilizes OMPs to colonize the human stomach and how OMPs cooperate with the Type IV secretion system.

  12. Radioiodination of an outer membrane protein in intact Rickettsia prowazekii

    Smith, D.K.; Winkler, H.H.

    1980-01-01

    Intact Rickettsia prowazekii was radiolabeled with the glucose oxidase-lactoperoxidase method of iodination. Separation of the rickettsial extract into cytoplasmic, outer and inner membrane fractions demonstrated that the outer membrane was preferentially labeled. Analysis of the polypeptides of these fractions on high-resolution slab polyacrylamide gels showed that most of the 125 I was in polypeptide T49, an outer membrane constituent. Additional outer membrane polypeptides were iodinated in broken envelope preparations, demonstrating that T49 is uniquely accessible to the external environment and the asymmetric polypeptide organization of the outer membrane

  13. Nodulation outer proteins: double-edged swords of symbiotic rhizobia.

    Staehelin, Christian; Krishnan, Hari B

    2015-09-15

    Rhizobia are nitrogen-fixing bacteria that establish a nodule symbiosis with legumes. Nodule formation depends on signals and surface determinants produced by both symbiotic partners. Among them, rhizobial Nops (nodulation outer proteins) play a crucial symbiotic role in many strain-host combinations. Nops are defined as proteins secreted via a rhizobial T3SS (type III secretion system). Functional T3SSs have been characterized in many rhizobial strains. Nops have been identified using various genetic, biochemical, proteomic, genomic and experimental approaches. Certain Nops represent extracellular components of the T3SS, which are visible in electron micrographs as bacterial surface appendages called T3 (type III) pili. Other Nops are T3 effector proteins that can be translocated into plant cells. Rhizobial T3 effectors manipulate cellular processes in host cells to suppress plant defence responses against rhizobia and to promote symbiosis-related processes. Accordingly, mutant strains deficient in synthesis or secretion of T3 effectors show reduced symbiotic properties on certain host plants. On the other hand, direct or indirect recognition of T3 effectors by plant cells expressing specific R (resistance) proteins can result in effector triggered defence responses that negatively affect rhizobial infection. Hence Nops are double-edged swords that may promote establishment of symbiosis with one legume (symbiotic factors) and impair symbiotic processes when bacteria are inoculated on another legume species (asymbiotic factors). In the present review, we provide an overview of our current understanding of Nops. We summarize their symbiotic effects, their biochemical properties and their possible modes of action. Finally, we discuss future perspectives in the field of T3 effector research. © 2015 Authors; published by Portland Press Limited.

  14. Topological analysis of Chlamydia trachomatis L2 outer membrane protein 2

    Mygind, P; Christiansen, Gunna; Birkelund, Svend

    1998-01-01

    Using monospecific polyclonal antisera to different parts of Chlamydia trachomatis L2 outer membrane protein 2 (Omp2), we show that the protein is localized at the inner surface of the outer membrane. Omp2 becomes immunoaccessible when Chlamydia elementary bodies are treated with dithiothreitol...

  15. The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

    Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  16. Lateral release of proteins from the TOM complex into the outer membrane of mitochondria.

    Harner, Max; Neupert, Walter; Deponte, Marcel

    2011-07-15

    The TOM complex of the outer membrane of mitochondria is the entry gate for the vast majority of precursor proteins that are imported into the mitochondria. It is made up by receptors and a protein conducting channel. Although precursor proteins of all subcompartments of mitochondria use the TOM complex, it is not known whether its channel can only mediate passage across the outer membrane or also lateral release into the outer membrane. To study this, we have generated fusion proteins of GFP and Tim23 which are inserted into the inner membrane and, at the same time, are spanning either the TOM complex or are integrated into the outer membrane. Our results demonstrate that the TOM complex, depending on sequence determinants in the precursors, can act both as a protein conducting pore and as an insertase mediating lateral release into the outer membrane.

  17. A New Strain Collection for Improved Expression of Outer Membrane Proteins

    Ina Meuskens

    2017-11-01

    Full Text Available Almost all integral membrane proteins found in the outer membranes of Gram-negative bacteria belong to the transmembrane β-barrel family. These proteins are not only important for nutrient uptake and homeostasis, but are also involved in such processes as adhesion, protein secretion, biofilm formation, and virulence. As surface exposed molecules, outer membrane β-barrel proteins are also potential drug and vaccine targets. High production levels of heterologously expressed proteins are desirable for biochemical and especially structural studies, but over-expression and subsequent purification of membrane proteins, including outer membrane proteins, can be challenging. Here, we present a set of deletion mutants derived from E. coli BL21(DE3 designed for the over-expression of recombinant outer membrane proteins. These strains harbor deletions of four genes encoding abundant β-barrel proteins in the outer membrane (OmpA, OmpC, OmpF, and LamB, both single and in all combinations of double, triple, and quadruple knock-outs. The sequences encoding these outer membrane proteins were deleted completely, leaving only a minimal scar sequence, thus preventing the possibility of genetic reversion. Expression tests in the quadruple mutant strain with four test proteins, including a small outer membrane β-barrel protein and variants thereof as well as two virulence-related autotransporters, showed significantly improved expression and better quality of the produced proteins over the parent strain. Differences in growth behavior and aggregation in the presence of high salt were observed, but these phenomena did not negatively influence the expression in the quadruple mutant strain when handled as we recommend. The strains produced in this study can be used for outer membrane protein production and purification, but are also uniquely useful for labeling experiments for biophysical measurements in the native membrane environment.

  18. Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes

    Bunai Christine L

    2009-02-01

    Full Text Available Abstract Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr, an alkaline solution (180 mM Na2CO3, pH 11.3 and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14. Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries; (ii peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries; (iii cytoplasmic or ribosomal membrane-contaminating proteins (80 entries. Thirty-one proteins were experimentally associated with the outer membrane (OM. Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high Mr fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM proteins with two or more predicted transmembrane domains (TMDs were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six β-barrel OM protein families and 25 distinct IM transporter families.

  19. A Deg-protease family protein in marine Synechococcus is involved in outer membrane protein organization

    Rhona Kayra Stuart

    2014-06-01

    Full Text Available Deg-family proteases are a periplasm-associated group of proteins that are known to be involved in envelope stress responses and are found in most microorganisms. Orthologous genes SYNW2176 (in strain WH8102 and sync_2523 (strain CC9311 are predicted members of the Deg-protease family and are among the few genes induced by copper stress in both open ocean and coastal marine Synechococcus strains. In contrast to the lack of a phenotype in a similar knockout in Synechocystis PCC6803, a SYNW2176 knockout mutant in strain WH8102 was much more resistant to copper than the wild-type. The mutant also exhibited a significantly altered outer membrane protein composition which may contribute to copper resistance, longer lag phase after transfer, low-level consistent alkaline phosphatase activity, and an inability to induce high alkaline phosphatase activity in response to phosphate stress. This phenotype suggests a protein-quality-control role for SYNW2176, the absence of which leads to a constitutively activated stress response. Deg-protease family proteins in this ecologically important cyanobacterial group thus help to determine outer membrane responses to both nutrients and toxins.

  20. Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria

    Käser, Sandro; Oeljeklaus, Silke; Týč, Jiří; Vaughan, Sue; Warscheid, Bettina; Schneider, André

    2016-01-01

    Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance. PMID:27436903

  1. Comparative transcriptional and translational analysis of leptospiral outer membrane protein expression in response to temperature.

    Lo, Miranda; Cordwell, Stuart J; Bulach, Dieter M; Adler, Ben

    2009-12-08

    Leptospirosis is a global zoonosis affecting millions of people annually. Transcriptional changes in response to temperature were previously investigated using microarrays to identify genes potentially expressed upon host entry. Past studies found that various leptospiral outer membrane proteins are differentially expressed at different temperatures. However, our microarray studies highlighted a divergence between protein abundance and transcript levels for some proteins. Given the abundance of post-transcriptional expression control mechanisms, this finding highlighted the importance of global protein analysis systems. To complement our previous transcription study, we evaluated differences in the proteins of the leptospiral outer membrane fraction in response to temperature upshift. Outer membrane protein-enriched fractions from Leptospira interrogans grown at 30 degrees C or overnight upshift to 37 degrees C were isolated and the relative abundance of each protein was determined by iTRAQ analysis coupled with two-dimensional liquid chromatography and tandem mass spectrometry (2-DLC/MS-MS). We identified 1026 proteins with 99% confidence; 27 and 66 were present at elevated and reduced abundance respectively. Protein abundance changes were compared with transcriptional differences determined from the microarray studies. While there was some correlation between the microarray and iTRAQ data, a subset of genes that showed no differential expression by microarray was found to encode temperature-regulated proteins. This set of genes is of particular interest as it is likely that regulation of their expression occurs post-transcriptionally, providing an opportunity to develop hypotheses about the molecular dynamics of the outer membrane of Leptospira in response to changing environments. This is the first study to compare transcriptional and translational responses to temperature shift in L. interrogans. The results thus provide an insight into the mechanisms used by L

  2. Analysis of proteins in Chlamydia trachomatis L2 outer membrane complex, COMC

    Birkelund, Svend; Morgan-Fisher, Marie; Timmerman, Evy

    2009-01-01

    The protein composition and N-terminal sequences of proteins in the outer membrane of Chlamydia trachomatis L2 were analysed following isolation of N-terminal peptides using combined fractional diagonal chromatography and identification by liquid chromatography tandem MS. Acetylation of primary a...

  3. Leptospiral outer membrane protein microarray, a novel approach to identification of host ligand-binding proteins.

    Pinne, Marija; Matsunaga, James; Haake, David A

    2012-11-01

    Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens.

  4. The Xylella fastidiosa PD1063 protein is secreted in association with outer membrane vesicles.

    Pierce, Brittany K; Voegel, Tanja; Kirkpatrick, Bruce C

    2014-01-01

    Xylella fastidiosa is a gram-negative, xylem-limited plant pathogenic bacterium that causes disease in a variety of economically important agricultural crops including Pierce's disease of grapevines. Xylella fastidiosa biofilms formed in the xylem vessels of plants play a key role in early colonization and pathogenicity by providing a protected niche and enhanced cell survival. Here we investigate the role of Xylella fastidiosa PD1063, the predicted ortholog of Xanthomonas oryzae pv. oryzae PXO_03968, which encodes an outer membrane protein. To assess the function of the Xylella fastidiosa ortholog, we created Xylella fastidiosa mutants deleted for PD1063 and then assessed biofilm formation, cell-cell aggregation and cell growth in vitro. We also assessed disease severity and pathogen titers in grapevines mechanically inoculated with the Xylella fastidiosa PD1063 mutant. We found a significant decrease in cell-cell aggregation among PD1063 mutants but no differences in cell growth, biofilm formation, disease severity or titers in planta. Based on the demonstration that Xanthomonas oryzae pv. oryzae PXO_03968 encodes an outer membrane protein, secreted in association with outer membrane vesicles, we predicted that PD1063 would also be secreted in a similar manner. Using anti-PD1063 antibodies, we found PD1063 in the supernatant and secreted in association with outer membrane vesicles. PD1063 purified from the supernatant, outer membrane fractions and outer membrane vesicles was 19.2 kD, corresponding to the predicted size of the processed protein. Our findings suggest Xylella fastidiosa PD1063 is not essential for development of Pierce's disease in Vitis vinifera grapevines although further research is required to determine the function of the PD1063 outer membrane protein in Xylella fastidiosa.

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

    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

  6. UV-C Adaptation of Shigella: Morphological, Outer Membrane Proteins, Secreted Proteins, and Lipopolysaccharides Effects.

    Chourabi, Kalthoum; Campoy, Susana; Rodriguez, Jesus A; Kloula, Salma; Landoulsi, Ahmed; Chatti, Abdelwaheb

    2017-11-01

    Water UV disinfection remains extremely important, particularly in developing countries where drinking and reclaimed crop irrigation water may spread devastating infectious diseases. Enteric bacterial pathogens, among which Shigella, are possible contaminants of drinking and bathing water and foods. To study the effect of UV light on Shigella, four strains were exposed to different doses in a laboratory-made irradiation device, given that the ultraviolet radiation degree of inactivation is directly related to the UV dose applied to water. Our results showed that the UV-C rays are effective against all the tested Shigella strains. However, UV-C doses appeared as determinant factors for Shigella eradication. On the other hand, Shigella-survived strains changed their outer membrane protein profiles, secreted proteins, and lipopolysaccharides. Also, as shown by electron microscopy transmission, morphological alterations were manifested by an internal cytoplasm disorganized and membrane envelope breaks. Taken together, the focus of interest of our study is to know the adaptive mechanism of UV-C resistance of Shigella strains.

  7. Structure of Rotavirus Outer-Layer Protein VP7 Bound with a Neutralizing Fab

    Aoki, Scott T.; Settembre, Ethan C.; Trask, Shane D.; Greenberg, Harry B.; Harrison, Stephen C.; Dormitzer, Philip R.; (Stanford-MED); (CH-Boston)

    2009-06-17

    Rotavirus outer-layer protein VP7 is a principal target of protective antibodies. Removal of free calcium ions (Ca{sup 2+}) dissociates VP7 trimers into monomers, releasing VP7 from the virion, and initiates penetration-inducing conformational changes in the other outer-layer protein, VP4. We report the crystal structure at 3.4 angstrom resolution of VP7 bound with the Fab fragment of a neutralizing monoclonal antibody. The Fab binds across the outer surface of the intersubunit contact, which contains two Ca{sup 2+} sites. Mutations that escape neutralization by other antibodies suggest that the same region bears the epitopes of most neutralizing antibodies. The monovalent Fab is sufficient to neutralize infectivity. We propose that neutralizing antibodies against VP7 act by stabilizing the trimer, thereby inhibiting the uncoating trigger for VP4 rearrangement. A disulfide-linked trimer is a potential subunit immunogen.

  8. Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein.

    Deirdre R Ducken

    Full Text Available Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to

  9. Changes in outer membrane proteins of nontypable Haemophilus influenzae in patients with chronic obstructive pulmonary disease

    Groeneveld, K.; van Alphen, L.; Eijk, P. P.; Jansen, H. M.; Zanen, H. C.

    1988-01-01

    Five individual colonies of Haemophilus influenzae were isolated from each of one to three cultures of sputum collected from 18 patients with chronic obstructive pulmonary disease (COPD). The isolates were studied to investigate whether the major outer membrane proteins (MOMPs) changed during

  10. Analysis of the lipidated recombinant outer surface protein A from Borrelia burgdorferi by mass spectrometry

    Bouchon, B.; Klein, Michele; Bischoff, Rainer; Van Dorsselaer, A.; Roitsch, C.

    1997-01-01

    The outer surface protein A, OspA, from the spirochete Borrelia burgdorferi is a lipoprotein of 25 kDa. The recombinant OspA (rOspA) expressed in Escherichia coli has been purified and analyzed by electrospray mass spectrometry (ESMS). A heterogenous spectrum gave a measured mass of 28,462 +/- 9 Da

  11. Comparative transcriptional and translational analysis of leptospiral outer membrane protein expression in response to temperature.

    Miranda Lo

    Full Text Available BACKGROUND: Leptospirosis is a global zoonosis affecting millions of people annually. Transcriptional changes in response to temperature were previously investigated using microarrays to identify genes potentially expressed upon host entry. Past studies found that various leptospiral outer membrane proteins are differentially expressed at different temperatures. However, our microarray studies highlighted a divergence between protein abundance and transcript levels for some proteins. Given the abundance of post-transcriptional expression control mechanisms, this finding highlighted the importance of global protein analysis systems. METHODOLOGY/PRINCIPAL FINDINGS: To complement our previous transcription study, we evaluated differences in the proteins of the leptospiral outer membrane fraction in response to temperature upshift. Outer membrane protein-enriched fractions from Leptospira interrogans grown at 30 degrees C or overnight upshift to 37 degrees C were isolated and the relative abundance of each protein was determined by iTRAQ analysis coupled with two-dimensional liquid chromatography and tandem mass spectrometry (2-DLC/MS-MS. We identified 1026 proteins with 99% confidence; 27 and 66 were present at elevated and reduced abundance respectively. Protein abundance changes were compared with transcriptional differences determined from the microarray studies. While there was some correlation between the microarray and iTRAQ data, a subset of genes that showed no differential expression by microarray was found to encode temperature-regulated proteins. This set of genes is of particular interest as it is likely that regulation of their expression occurs post-transcriptionally, providing an opportunity to develop hypotheses about the molecular dynamics of the outer membrane of Leptospira in response to changing environments. CONCLUSIONS/SIGNIFICANCE: This is the first study to compare transcriptional and translational responses to temperature

  12. Structure Prediction of Outer Membrane Protease Protein of Salmonella typhimurium Using Computational Techniques

    Rozina Tabassum

    2016-03-01

    Full Text Available Salmonella typhimurium, a facultative gram-negative intracellular pathogen belonging to family Enterobacteriaceae, is the most frequent cause of human gastroenteritis worldwide. PgtE gene product, outer membrane protease emerges important in the intracellular phases of salmonellosis. The pgtE gene product of S. typhimurium was predicted to be capable of proteolyzing T7 RNA polymerase and localize in the outer membrane of these gram negative bacteria. PgtE product of S. enterica and OmpT of E. coli, having high sequence similarity have been revealed to degrade macrophages, causing salmonellosis and other diseases. The three-dimensional structure of the protein was not available through Protein Data Bank (PDB creating lack of structural information about E protein. In our study, by performing Comparative model building, the three dimensional structure of outer membrane protease protein was generated using the backbone of the crystal structure of Pla of Yersinia pestis, retrieved from PDB, with MODELLER (9v8. Quality of the model was assessed by validation tool PROCHECK, web servers like ERRAT and ProSA are used to certify the reliability of the predicted model. This information might offer clues for better understanding of E protein and consequently for developmet of better therapeutic treatment against pathogenic role of this protein in salmonellosis and other diseases.

  13. Exploring the interaction network of the Bacillus subtilis outer coat and crust proteins.

    Krajčíková, Daniela; Forgáč, Vladimír; Szabo, Adam; Barák, Imrich

    2017-11-01

    Bacillus subtilis spores, representatives of an exceptionally resistant dormant cell type, are encircled by a thick proteinaceous layer called the spore coat. More than 80 proteins assemble into four distinct coat layers: a basement layer, an inner coat, an outer coat and a crust. As the spore develops inside the mother cell, spore coat proteins synthesized in the cytoplasm are gradually deposited onto the prespore surface. A small set of morphogenetic proteins necessary for spore coat morphogenesis are thought to form a scaffold to which the rest of the coat proteins are attached. Extensive localization and proteomic studies using wild type and mutant spores have revealed the arrangement of individual proteins within the spore coat layers. In this study we examined the interactions between the proteins localized to the outer coat and crust using a bacterial two hybrid system. These two layers are composed of at least 25 components. Self-interactions were observed for most proteins and numerous novel interactions were identified. The most interesting contacts are those made with the morphogenetic proteins CotE, CotY and CotZ; these could serve as a basis for understanding the specific roles of particular proteins in spore coat morphogenesis. Copyright © 2017 Elsevier GmbH. All rights reserved.

  14. Pathogenicity of Vibrio anguillarum serogroup O1 strains compared to plasmids, outer membrane protein profiles and siderophore production

    Pedersen, K.; Gram, Lone; Austin, D.A.

    1997-01-01

    The virulence of 18 strains of Vibrio anguillarum serogroup 01 was compared to plasmid content, expression of siderophores and outer membrane proteins. All strains, irrespective of plasmid content, produced siderophores and inducible outer membrane proteins under iron-limited conditions. Only str...

  15. Comprehensive in silico prediction and analysis of chlamydial outer membrane proteins reflects evolution and life style of the Chlamydiae

    Myers Garry

    2009-12-01

    Full Text Available Abstract Background Chlamydiae are obligate intracellular bacteria comprising some of the most important bacterial pathogens of animals and humans. Although chlamydial outer membrane proteins play a key role for attachment to and entry into host cells, only few have been described so far. We developed a comprehensive, multiphasic in silico approach, including the calculation of clusters of orthologues, to predict outer membrane proteins using conservative criteria. We tested this approach using Escherichia coli (positive control and Bacillus subtilis (negative control, and applied it to five chlamydial species; Chlamydia trachomatis, Chlamydia muridarum, Chlamydia (a.k.a. Chlamydophila pneumoniae, Chlamydia (a.k.a. Chlamydophila caviae, and Protochlamydia amoebophila. Results In total, 312 chlamydial outer membrane proteins and lipoproteins in 88 orthologous clusters were identified, including 238 proteins not previously recognized to be located in the outer membrane. Analysis of their taxonomic distribution revealed an evolutionary conservation among Chlamydiae, Verrucomicrobia, Lentisphaerae and Planctomycetes as well as lifestyle-dependent conservation of the chlamydial outer membrane protein composition. Conclusion This analysis suggested a correlation between the outer membrane protein composition and the host range of chlamydiae and revealed a common set of outer membrane proteins shared by these intracellular bacteria. The collection of predicted chlamydial outer membrane proteins is available at the online database pCOMP http://www.microbial-ecology.net/pcomp and might provide future guidance in the quest for anti-chlamydial vaccines.

  16. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

    Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

    2016-05-01

    Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β. © FASEB.

  17. An evolutionarily conserved glycine-tyrosine motif forms a folding core in outer membrane proteins.

    Marcin Michalik

    Full Text Available An intimate interaction between a pair of amino acids, a tyrosine and glycine on neighboring β-strands, has been previously reported to be important for the structural stability of autotransporters. Here, we show that the conservation of this interacting pair extends to nearly all major families of outer membrane β-barrel proteins, which are thought to have originated through duplication events involving an ancestral ββ hairpin. We analyzed the function of this motif using the prototypical outer membrane protein OmpX. Stopped-flow fluorescence shows that two folding processes occur in the millisecond time regime, the rates of which are reduced in the tyrosine mutant. Folding assays further demonstrate a reduction in the yield of folded protein for the mutant compared to the wild-type, as well as a reduction in thermal stability. Taken together, our data support the idea of an evolutionarily conserved 'folding core' that affects the folding, membrane insertion, and thermal stability of outer membrane protein β-barrels.

  18. Adaptation of Salmonella enterica Hadar under static magnetic field: effects on outer membrane protein pattern

    Snoussi Sarra

    2012-02-01

    Full Text Available Abstract Background Salmonella enterica serovar Hadar (S. Hadar is a highly prevalent foodborne pathogen and therefore a major cause of human gastroenteritis worldwide. Outer membrane proteins whose production is often regulated by environmental conditions also play important roles in the adaptability of bacterial pathogens to various environments. Results The present study investigated the adaptation of S. Hadar under the effect of acute static magnetic field exposure (200 mT, 9 h and the impact on the outer membrane protein pattern. Via two-dimensional electrophoresis (2-DE and LC-MS/MS spectrometry, we compared the proteome of enriched-outer membrane fraction before and after exposure to a magnetic field. A total of 11 proteins, displaying more than a two-fold change, were differentially expressed in exposed cells, among which 7 were up-regulated and 4 down-regulated. These proteins were involved in the integrity of cell envelope (TolB, Pal, in the response to oxidative stress (OmpW, dihydrolipoamide dehydrogenase, UspF, in the oxidative stress status (bacterioferritin, in virulence (OmpX, Yfgl or in motility (FlgE and UspF. Complementary experiments associated the down-regulation of FlgE and UspF with an alteration of swarming, a flagella-driven motility, under SMF. Furthermore, the antibiotic disc diffusion method confirmed a decrease of gentamicin susceptibility in exposed cells. This decrease could be partly associated with the up-regulation of TolC, outer membrane component of an efflux pump. OmpA, a multifunctional protein, was up-regulated. Conclusions SMF (200 mT seems to maintain the cell envelope integrity and to submit the exposed cells to an oxidative stress. Some alterations suggest an increase of the ability of exposed cells to form biofilms.

  19. Monoclonal antibodies against the iron regulated outer membrane Proteins of Acinetobacter baumannii are bactericidal

    Goel Vikas

    2001-08-01

    Full Text Available Abstract Background Iron is an important nutrient required by all forms of life.In the case of human hosts,the free iron availability is 10-18M,which is far less than what is needed for the survival of the invading bacterial pathogen.To survive in such conditions, bacteria express new proteins in their outer membrane and also secrete iron chelators called siderophores. Results/ Discussion Acinetobacter baumannii ATCC 19606, a nosocomial pathogen which grows under iron restricted conditions, expresses four new outer membrane proteins,with molecular weight ranging from 77 kDa to 88 kDa, that are called Iron Regulated Outer Membrane Proteins (IROMPs. We studied the functional and immunological properties of IROMPs expressed by A.baumanii ATCC 19606.The bands corresponding to IROMPs were eluted from SDS-PAGE and were used to immunize BALB/c mice for the production of monoclonal antibodies. Hybridomas secreting specific antibodies against these IROMPs were selected after screening by ELISA and their reactivity was confirmed by Western Blot. The antibodies then generated belonged to IgM isotype and showed bactericidical and opsonising activities against A.baumanii in vitro.These antibodies also blocked siderophore mediated iron uptake via IROMPs in bacteria. Conclusion This proves that iron uptake via IROMPs,which is mediated through siderophores,may have an important role in the survival of A.baumanii inside the host,and helps establishing the infection.

  20. Reinforcement of Bacillus subtilis spores by cross-linking of outer coat proteins during maturation.

    Abhyankar, Wishwas; Pandey, Rachna; Ter Beek, Alexander; Brul, Stanley; de Koning, Leo J; de Koster, Chris G

    2015-02-01

    Resistance characteristics of bacterial endospores towards various environmental stresses such as chemicals and heat are in part attributed to their coat proteins. Heat resistance is developed in a late stage of sporulation and during maturation of released spores. Using our gel-free proteomic approach and LC-FT-ICR-MS/MS analysis we have monitored the efficiency of the tryptic digestion of proteins in the coat during spore maturation over a period of eight days, using metabolically (15)N labeled mature spores as reference. The results showed that during spore maturation the loss of digestion efficiency of outer coat and crust proteins synchronized with the increase in heat resistance. This implicates that spore maturation involves chemical cross-linking of outer coat and crust layer proteins leaving the inner coat layer proteins unmodified. It appears that digestion efficiencies of spore surface proteins can be linked to their location within the coat and crust layers. We also attempted to study a possible link between spore maturation and the observed heterogeneity in spore germination. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Effect of Leptospira interrogans outer membrane proteins LipL32 on HUVEC.

    Sun, Zhan; Bao, Lang; Li, DaoKun; Huang, Bi; Wu, Bingting

    2010-09-01

    Leptospira cause disease through a toxin-mediated process by inducing vascular injury, particularly a small-vessel vasculitis. Breakdown of vessel endothelial cell integrity may increase vessel permeability which is correlated with the changes of tight junction and/or apoptosis in vessel endothelial cells. The specific toxin responsible remains unidentified. In this study, we amplified outer membrane protein LipL32 from the genome of Leptospira interrogans serovar Lai, and it was subcloned in pET32a(+) vector to express thioredoxin(Trx)-LipL32 fusion protein in Escherichia coli BL21(DE3). The protein was expressed and purified, and Trx-LipL32 was administered to culture with human umbilical vein endothelial cells (HUVEC) to elucidate the role of leptospiral outer membrane proteins in vessel endothelial cell. The purified recombinant protein was capable to increase the permeability of HUVECs. And the protein was able to decrease the expression of ZO-1 and induce F-actin in HUVECs display thickening and clustering. Moreover, apoptosis of HUVEC was significantly accelerated. But the fusion partner had no effect in these regards. It is possible that LipL32 is involved in the vessel lesions. Copyright 2010 Elsevier Ltd. All rights reserved.

  2. Overexpression and surface localization of the Chlamydia trachomatis major outer membrane protein in Escherichia coli

    Koehler, JF; Birkelund, Svend; Stephens, RS

    1992-01-01

    The Chlamydia trachomatis major outer membrane protein (MOMP) is the quantitatively predominant surface protein which has important functional, structural and antigenic properties. We have cloned and overexpressed the MOMP in Escherichia coli. The MOMP is surface exposed in C. trachomatis....... The induction of MOMP expression had a rapidly lethal effect on the L2rMOMP E. coli clone. Although no genetic system exists for Chlamydia, development of a stable, inducible E. coli clone which overexpresses the chlamydial MOMP permits a study of the biological properties of the MOMP, including...

  3. The participation of outer membrane proteins in the bacterial sensitivity to nanosilver

    Anna Kędziora

    2016-06-01

    Full Text Available The presented study is to analyze the participation of outer membrane proteins of Gram- negative bacteria in sensitivity to silver nanomaterials. The mechanism of interaction of silver with the bacterial cell is best described in this group of microorganisms. There are several theories regarding the effectiveness of antimicrobial ions and nanosilver, and at the indicated differences in the way they work. Outer membrane proteins of Gram-negative bacteria are involved in the procurement of silver from the environment and contribute to the development mechanisms of resistance to nanometals. They are measurable parameter in the field of cell phenotypic response to the presence of Gram-negative bacteria in the environment silver nanoforms: its properties, chemical composition, content or times of action. Proteomic methods (including two dimensional electrophoresis and MALDI‑TOF MS are therefore relevant techniques for determining the susceptibility of bacteria to silver and the changes taking place in the outer membrane under the influence: uptime/exposure and physical and chemical parameters of silver nanomaterials. Many products containing nanosilver is still in the research phase in terms of physico‑chemical characteristics and biological activity, others have been already implemented in many industries. During the very fast nanotechnology developing and introduction to the market products based on the nanosilver the bacterial answer to nanosilver is needed.

  4. Analysis of the humoral immune response to Chlamydia outer membrane protein 2

    Mygind, P; Christiansen, Gunna; Persson, K

    1998-01-01

    The humoral immune response to Chlamydia outer membrane protein 2 (Omp2) was studied. Omp2 is a highly genus-conserved structural protein of all Chlamydia species, containing a variable N-terminal fragment. To analyze where the immunogenic parts were localized, seven highly purified truncated...... fusion proteins constituting different regions of the protein were produced (Chlamydia pneumoniae-Omp2aa23-aa93, Chlamydia psittaci-Omp2aa23-aa94, and Chlamydia trachomatis-Omp2aa23-aa84, aa87-aa547, aa23-aa182, aa167-aa434, aa420-aa547). By an enzyme-linked immunosorbent assay with serologically defined...... patient sera, Omp2 was found to be a major immunogen of both C. pneumoniae and C. trachomatis infections (P species-specific anti-Omp2 immunoglobulins were detected....

  5. Outer membrane proteins analysis of Shigella sonnei and evaluation of their antigenicity in Shigella infected individuals.

    Hemavathy Harikrishnan

    Full Text Available Bacillary dysentery caused by infection with Shigella spp. remains as serious and common health problem throughout the world. It is a highly multi drug resistant organism and rarely identified from the patient at the early stage of infection. S. sonnei is the most frequently isolated species causing shigellosis in industrialized countries. The antigenicity of outer membrane protein of this pathogen expressed during human infection has not been identified to date. We have studied the antigenic outer membrane proteins expressed by S. sonnei, with the aim of identifying presence of specific IgA and IgG in human serum against the candidate protein biomarkers. Three antigenic OMPs sized 33.3, 43.8 and 100.3 kDa were uniquely recognized by IgA and IgG from patients with S. sonnei infection, and did not cross-react with sera from patients with other types of infection. The antigenic proteome data generated in this study are a first for OMPs of S. sonnei, and they provide important insights of human immune responses. Furthermore, numerous prime candidate proteins were identified which will aid the development of new diagnostic tools for the detection of S. sonnei.

  6. Assembly and misassembly of cystic fibrosis transmembrane conductance regulator: folding defects caused by deletion of F508 occur before and after the calnexin-dependent association of membrane spanning domain (MSD) 1 and MSD2.

    Rosser, Meredith F N; Grove, Diane E; Chen, Liling; Cyr, Douglas M

    2008-11-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic membrane protein that functions as a Cl(-) channel and consists of two membrane spanning domains (MSDs), two cytosolic nucleotide binding domains (NBDs), and a cytosolic regulatory domain. Cytosolic 70-kDa heat shock protein (Hsp70), and endoplasmic reticulum-localized calnexin are chaperones that facilitate CFTR biogenesis. Hsp70 functions in both the cotranslational folding and posttranslational degradation of CFTR. Yet, the mechanism for calnexin action in folding and quality control of CFTR is not clear. Investigation of this question revealed that calnexin is not essential for CFTR or CFTRDeltaF508 degradation. We identified a dependence on calnexin for proper assembly of CFTR's membrane spanning domains. Interestingly, efficient folding of NBD2 was also found to be dependent upon calnexin binding to CFTR. Furthermore, we identified folding defects caused by deletion of F508 that occurred before and after the calnexin-dependent association of MSD1 and MSD2. Early folding defects are evident upon translation of the NBD1 and R-domain and are sensed by the RMA-1 ubiquitin ligase complex.

  7. Molecular biology of Neisseria meningitidis class 5 and H. 8 outer membrane proteins

    Kawula, T.H.

    1987-01-01

    One of the surface structures responsible for inter- and intrastrain antigenic variability in meningococci is the heat-modifiable class 5 (C.5) protein. Neisseria meningitidis strain FAM18 (a meningococcal disease isolate) expressed two different C.5 proteins (C.5a and C.5b) identifiable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. We generated two monoclonal antibodies (MAbs), each specific for one of the identified C.5 proteins. The MAbs, which were bactericidal for variants expressing the appropriate C.5 protein, were used to study C.5 expression changes in FAM18. The H.8 protein is an antigenically conserved outer membrane protein expressed almost exclusively by the pathogenic Neisseria. We have cloned and sequenced an H.8 gene from N. meningitidis FAM18. The predicted H.8 amino acid sequence indicated that the most probable signal peptide processing site matched the consensus prokaryotic lipoprotein processing/modification sequence. We then showed that the H.8 protein could be labeled with {sup 14}C-palmitic acid, confirming that H.8 was a lipoprotein. Processing of the H.8 protein was inhibited by globomycin in E. coli indicating that H.8 was modified by the described lipoprotein processing/modifying pathway described in both gram negative and gram positive genera.

  8. The VP7 Outer Capsid Protein of Rotavirus Induces Polyclonal B-Cell Activation

    Blutt, Sarah E.; Crawford, Sue E.; Warfield, Kelly L.; Lewis, Dorothy E.; Estes, Mary K.; Conner, Margaret E.

    2004-01-01

    The early response to a homologous rotavirus infection in mice includes a T-cell-independent increase in the number of activated B lymphocytes in the Peyer's patches. The mechanism of this activation has not been previously determined. Since rotavirus has a repetitively arranged triple-layered capsid and repetitively arranged antigens can induce activation of B cells, one or more of the capsid proteins could be responsible for the initial activation of B cells during infection. To address this question, we assessed the ability of rotavirus and virus-like particles to induce B-cell activation in vivo and in vitro. Using infectious rotavirus, inactivated rotavirus, noninfectious but replication-competent virus, and virus-like particles, we determined that neither infectivity nor RNA was necessary for B-cell activation but the presence of the rotavirus outer capsid protein, VP7, was sufficient for murine B-cell activation. Preincubation of the virus with neutralizing VP7 antibodies inhibited B-cell activation. Polymyxin B treatment and boiling of the virus preparation were performed, which ruled out possible lipopolysaccharide contamination as the source of activation and confirmed that the structural conformation of VP7 is important for B-cell activation. These findings indicate that the structure and conformation of the outer capsid protein, VP7, initiate intestinal B-cell activation during rotavirus infection. PMID:15194774

  9. Lack of Outer Membrane Protein A Enhances the Release of Outer Membrane Vesicles and Survival of Vibrio cholerae and Suppresses Viability of Acanthamoeba castellanii

    Soni Priya Valeru

    2014-01-01

    Full Text Available Vibrio cholerae, the causative agent of the diarrhoeal disease cholera, survives in aquatic environments. The bacterium has developed a survival strategy to grow and survive inside Acanthamoeba castellanii. It has been shown that V. cholerae expresses outer membrane proteins as virulence factors playing a role in the adherence to interacted host cells. This study examined the role of outer membrane protein A (OmpA and outer membrane vesicles (OMVs in survival of V. cholerae alone and during its interaction with A. castellanii. The results showed that an OmpA mutant of V. cholerae survived longer than wild-type V. cholerae when cultivated alone. Cocultivation with A. castellanii enhanced the survival of both bacterial strains and OmpA protein exhibited no effect on attachment, engulfment, and survival inside the amoebae. However, cocultivation of the OmpA mutant of V. cholerae decreased the viability of A. castellanii and this bacterial strain released more OMVs than wild-type V. cholerae. Surprisingly, treatment of amoeba cells with OMVs isolated from the OmpA mutant significantly decreased viable counts of the amoeba cells. In conclusion, the results might highlight a regulating rule for OmpA in survival of V. cholerae and OMVs as a potent virulence factor for this bacterium towards eukaryotes in the environment.

  10. Mapping of a Protective Epitope of the CopB Outer Membrane Protein of Moraxella catarrhalis

    Aebi, Christoph; Cope, Leslie D.; Latimer, Jo L.; Thomas, Sharon E.; Slaughter, Clive A.; McCracken, George H.; Hansen, Eric J.

    1998-01-01

    A monoclonal antibody (MAb) (MAb 10F3) directed against the CopB outer membrane protein of Moraxella catarrhalis previously was found to enhance pulmonary clearance of M. catarrhalis in an animal model (M. Helminen, I. Maciver, J. L. Latimer, L. D. Cope, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 61:2003–2010, 1993). In the present study, this same MAb was shown to exert complement-dependent bactericidal activity against this pathogen in vitro. Nucleotide sequence analysis of the ...

  11. Identification of immunogenic outer membrane proteins of Haemophilus influenzae type b in the infant rat model system

    Hansen, E.J.; Frisch, C.F.; McDade, R.L. Jr.; Johnston, K.H.

    1981-01-01

    Outer membrane proteins of Haemophilus influenzae type b which are immunogenic in infant rats were identified by a radioimmunoprecipitation method. Intact cells of H. influenzae type b were radioiodinated by a lactoperoxidase-catalyzed procedure, and an outer membrane-containing fraction was prepared from these cells. These radioiodinated outer membranes were mixed with sera obtained from rats convalescing from systemic H. influenzae type b disease induced at 6 days of age, and the resultant (antibody-outer membrane protein antigen) complexes were extracted from these membranes by treatment with nonionic detergent and ethylenediaminetetraacetic acid. These soluble antibody-antigen complexes were isolated by means of adsorption to protein A-bearing staphylococci, and the radioiodinated protein antigens were identified by gel electrophoresis followed by autoradiography. Infant rats were shown to mount a readily detectable antibody response to several different proteins present in the outer membrane of H. influenzae type b. Individual infant rats were found to vary both qualitatively and quantitatively in their immune response to these immunogenic outer membrane proteins

  12. Identification and Characterization of Outer Membrane Vesicle-Associated Proteins in Salmonella enterica Serovar Typhimurium

    Bai, Jaewoo; Kim, Seul I; Ryu, Sangryeol

    2014-01-01

    Salmonella enterica serovar Typhimurium is a primary cause of enteric diseases and has acquired a variety of virulence factors during its evolution into a pathogen. Secreted virulence factors interact with commensal flora and host cells and enable Salmonella to survive and thrive in hostile environments. Outer membrane vesicles (OMVs) released from many Gram-negative bacteria function as a mechanism for the secretion of complex mixtures, including virulence factors. We performed a proteomic analysis of OMVs that were isolated under standard laboratory and acidic minimal medium conditions and identified 14 OMV-associated proteins that were observed in the OMV fraction isolated only under the acidic minimal medium conditions, which reproduced the nutrient-deficient intracellular milieu. The inferred roles of these 14 proteins were diverse, including transporter, enzyme, and transcriptional regulator. The absence of these proteins influenced Salmonella survival inside murine macrophages. Eleven of these proteins were predicted to possess secretion signal sequences at their N termini, and three (HupA, GlnH, and PhoN) of the proteins were found to be translocated into the cytoplasm of host cells. The comparative proteomic profiling of OMVs performed in this study revealed different protein compositions in the OMVs isolated under the two different conditions, which indicates that the OMV cargo depends on the growth conditions and provides a deeper insight into how Salmonella utilizes OMVs to adapt to environmental changes. PMID:24935973

  13. Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation

    Overton, K. Wesley; Park, Dan M.; Yung, Mimi C.; Dohnalkova, Alice; Smit, John; Jiao, Yongqin

    2016-09-23

    ABSTRACT

    Surface layers, or S-layers, are two-dimensional protein arrays that form the outermost layer of many bacteria and archaea. They serve several functions, including physical protection of the cell from environmental threats. The high abundance of S-layer proteins necessitates a highly efficient export mechanism to transport the S-layer protein from the cytoplasm to the cell exterior.Caulobacter crescentusis unique in that it has two homologous, seemingly redundant outer membrane proteins, RsaFaand RsaFb, which together with other components form a type I protein translocation pathway for S-layer export. These proteins have homology toEscherichia coliTolC, the outer membrane channel of multidrug efflux pumps. Here we provide evidence that, unlike TolC, RsaFaand RsaFbare not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaFaand RsaFbare required to prevent intracellular accumulation and aggregation of the S-layer protein RsaA; deletion of RsaFaand RsaFbled to a general growth defect and lowered cellular fitness. Using Western blotting, transmission electron microscopy, and transcriptome sequencing (RNA-seq), we show that loss of both RsaFaand RsaFbled to accumulation of insoluble RsaA in the cytoplasm, which in turn caused upregulation of a number of genes involved in protein misfolding and degradation pathways. These findings provide new insight into the requirement for RsaFaand RsaFbin cellular fitness and tolerance to antimicrobial agents and further our understanding of the S-layer export mechanism on both the transcriptional and translational levels in

  14. Lack of protein-tyrosine sulfation disrupts photoreceptor outer segment morphogenesis, retinal function and retinal anatomy.

    Sherry, David M; Murray, Anne R; Kanan, Yogita; Arbogast, Kelsey L; Hamilton, Robert A; Fliesler, Steven J; Burns, Marie E; Moore, Kevin L; Al-Ubaidi, Muayyad R

    2010-11-01

    To investigate the role(s) of protein-tyrosine sulfation in the retina, we examined retinal function and structure in mice lacking tyrosylprotein sulfotransferases (TPST) 1 and 2. Tpst double knockout (DKO; Tpst1(-/-) /Tpst2 (-/-) ) retinas had drastically reduced electroretinographic responses, although their photoreceptors exhibited normal responses in single cell recordings. These retinas appeared normal histologically; however, the rod photoreceptors had ultrastructurally abnormal outer segments, with membrane evulsions into the extracellular space, irregular disc membrane spacing and expanded intradiscal space. Photoreceptor synaptic terminals were disorganized in Tpst DKO retinas, but established ultrastructurally normal synapses, as did bipolar and amacrine cells; however, the morphology and organization of neuronal processes in the inner retina were abnormal. These results indicate that protein-tyrosine sulfation is essential for proper outer segment morphogenesis and synaptic function, but is not critical for overall retinal structure or synapse formation, and may serve broader functions in neuronal development and maintenance. © 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  15. A conserved small RNA promotes silencing of the outer membrane protein YbfM

    Rasmussen, Anders Aamann; Johansen, Jesper; Nielsen, Jesper S

    2009-01-01

    important physiological role of regulatory RNA molecules in Gram-negative bacteria is to modulate the cell surface and/or to prevent accumulation of OMPs in the envelope. Here, we extend the OMP-sRNA network by showing that the expression of the outer membrane protein YbfM is silenced by a conserved sRNA......In the past few years an increasing number of small non-coding RNAs (sRNAs) in enterobacteria have been found to negatively regulate the expression of outer membrane proteins (OMPs) at the post-transcriptional level. These RNAs act under various growth and stress conditions, suggesting that one......, designated MicM (also known as RybC/SroB). The regulation is strictly dependent on the RNA chaperone Hfq, and mutational analysis indicates that MicM sequesters the ribosome binding site of ybfM mRNA by an antisense mechanism. Furthermore, we provide evidence that Hfq strongly enhances the on-rate of duplex...

  16. Comparative proteome analysis reveals pathogen specific outer membrane proteins of Leptospira.

    Dhandapani, Gunasekaran; Sikha, Thoduvayil; Rana, Aarti; Brahma, Rahul; Akhter, Yusuf; Gopalakrishnan Madanan, Madathiparambil

    2018-04-10

    Proteomes of pathogenic Leptospira interrogans and L. borgpetersenii and the saprophytic L. biflexa were filtered through computational tools to identify Outer Membrane Proteins (OMPs) that satisfy the required biophysical parameters for their presence on the outer membrane. A total of 133, 130, and 144 OMPs were identified in L. interrogans, L. borgpetersenii, and L. biflexa, respectively, which forms approximately 4% of proteomes. A holistic analysis of transporting and pathogenic characteristics of OMPs together with Clusters of Orthologous Groups (COGs) among the OMPs and their distribution across 3 species was made and put forward a set of 21 candidate OMPs specific to pathogenic leptospires. It is also found that proteins homologous to the candidate OMPs were also present in other pathogenic species of leptospires. Six OMPs from L. interrogans and 2 from L. borgpetersenii observed to have similar COGs while those were not found in any intermediate or saprophytic forms. These OMPs appears to have role in infection and pathogenesis and useful for anti-leptospiral strategies. © 2018 Wiley Periodicals, Inc.

  17. Brucella ovis PA mutants for outer membrane proteins Omp10, Omp19, SP41, and BepC are not altered in their virulence and outer membrane properties.

    Sidhu-Muñoz, Rebeca S; Sancho, Pilar; Vizcaíno, Nieves

    2016-04-15

    Mutants in several genes have been obtained on the genetic background of virulent rough (lacking O-polysaccharide) Brucella ovis PA. The target genes encode outer membrane proteins previously associated with the virulence of smooth (bearing O-polysaccharide chains in the lipopolysaccharide) Brucella strains. Multiple attempts to delete omp16, coding for a homologue to peptidoglycan-associated lipoproteins, were unsuccessful, which suggests that Omp16 is probably essential for in vitro survival of B. ovis PA. Single deletion of omp10 or omp19-that encode two other outer membrane lipoproteins--was achieved, but the simultaneous removal of both genes failed, suggesting an essential complementary function between both proteins. Two other deletion mutants, defective in the Tol-C-homologue BepC or in the SP41 adhesin, were also obtained. Surprisingly when compared to previous results obtained with smooth Brucella, none of the B. ovis mutants showed attenuation in the virulence, either in the mouse model or in cellular models of professional and non-professional phagocytes. Additionally, and in contrast to the observations reported with smooth Brucella strains, several properties related to the outer membrane remained almost unaltered. These results evidence new distinctive traits between naturally rough B. ovis and smooth brucellae. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Comparative Outer Membrane Protein Analysis of High and Low-Invasive Strains of Cronobacter malonaticus

    Maha A. Aldubyan

    2017-11-01

    Full Text Available Cronobacter are an important group of foodborne pathogens that has been linked to life-threatening infections in both infants and adults. The major infections associated with Cronobacter species are neonatal meningitis, necrotizing enterocolitis, and septicaemia. There are seven species in the Cronobacter genus, of which only three are of clinical importance; Cronobacter sakazakii, Cronobacter malonaticus, and Cronobacter turicensis. To date most studies have focussed on C. sakazakii as it is the major species associated with neonatal infections. However, recently C. malonaticus, in particular sequence type 7 (ST7, has been noted as being prevalent in adult infections and therefore warranting further investigation. In this study, eight strains of C. malonaticus ST7, that had been isolated from a wide range of sources and varied in their in vitro virulence, were chosen for proteomic analysis of their outer membrane proteins (OMPs. One-dimensional gel analysis revealed a ~29 kDa size band that was only present in the highly invasive strains. Subsequent mass spectrometric analysis identified several peptides that matched the flagellin protein. The presence of flagellin protein was confirmed in 2D gel spot. Mass spectrometry analysis of total OMPs revealed that the four highly invasive C. malonaticus strains expressed the main flagellum proteins that were absent from the four low invasive strains. These were the flagellar hook protein FlgE, flagellar hook-associated protein 1, flagellar hook-associated protein, flagellin, and flagellar hook-filament junction protein FlgL. This data indicates that C. malonaticus flagellar proteins may have an important role in the organism's invasion properties.

  19. Methylation and in vivo expression of the surface-exposed Leptospira interrogans outer-membrane protein OmpL32.

    Eshghi, Azad; Pinne, Marija; Haake, David A; Zuerner, Richard L; Frank, Ami; Cameron, Caroline E

    2012-03-01

    Recent studies have revealed that bacterial protein methylation is a widespread post-translational modification that is required for virulence in selected pathogenic bacteria. In particular, altered methylation of outer-membrane proteins has been shown to modulate the effectiveness of the host immune response. In this study, 2D gel electrophoresis combined with MALDI-TOF MS identified a Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 protein, corresponding to ORF LIC11848, which undergoes extensive and differential methylation of glutamic acid residues. Immunofluorescence microscopy implicated LIC11848 as a surface-exposed outer-membrane protein, prompting the designation OmpL32. Indirect immunofluorescence microscopy of golden Syrian hamster liver and kidney sections revealed expression of OmpL32 during colonization of these organs. Identification of methylated surface-exposed outer-membrane proteins, such as OmpL32, provides a foundation for delineating the role of this post-translational modification in leptospiral virulence.

  20. Eukaryote-wide sequence analysis of mitochondrial β-barrel outer membrane proteins

    Fujita Naoya

    2011-01-01

    Full Text Available Abstract Background The outer membranes of mitochondria are thought to be homologous to the outer membranes of Gram negative bacteria, which contain 100's of distinct families of β-barrel membrane proteins (BOMPs often forming channels for transport of nutrients or drugs. However, only four families of mitochondrial BOMPs (MBOMPs have been confirmed to date. Although estimates as high as 100 have been made in the past, the number of yet undiscovered MBOMPs is an open question. Fortunately, the recent discovery of a membrane integration signal (the β-signal for MBOMPs gave us an opportunity to look for undiscovered MBOMPs. Results We present the results of a comprehensive survey of eukaryotic protein sequences intended to identify new MBOMPs. Our search employs recent results on β-signals as well as structural information and a novel BOMP predictor trained on both bacterial and mitochondrial BOMPs. Our principal finding is circumstantial evidence suggesting that few MBOMPs remain to be discovered, if one assumes that, like known MBOMPs, novel MBOMPs will be monomeric and β-signal dependent. In addition to this, our analysis of MBOMP homologs reveals some exceptions to the current model of the β-signal, but confirms its consistent presence in the C-terminal region of MBOMP proteins. We also report a β-signal independent search for MBOMPs against the yeast and Arabidopsis proteomes. We find no good candidates MBOMPs in yeast but the Arabidopsis results are less conclusive. Conclusions Our results suggest there are no remaining MBOMPs left to discover in yeast; and if one assumes all MBOMPs are β-signal dependent, few MBOMP families remain undiscovered in any sequenced organism.

  1. Strain specific variation of outer membrane proteins of wild Yersinia pestis strains subjected to different growth temperatures

    Frederico Guilherme Coutinho Abath

    1990-03-01

    Full Text Available Three Yersinia pestis strains isolated from humans and one laboratory strain (EV76 were grown in rich media at 28§C and 37§C and their outer membrane protein composition compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-Page. Several proteins with molecular weights ranging from 34 kDa to 7 kDa were observed to change in relative abundance in samples grown at different temperatures. At least seven Y. pestis outer membrane proteins showed a temperature-dependent and strain-specific behaviour. Some differences between the outer membrane proteins of full-pathogenic wild isolates and the EV76 strain could aldso be detected and the relevance of this finding on the use of laboratory strains as a reference to the study of Y. pestis biological properties is discuted.

  2. Rapid outer-surface protein C DNA tattoo vaccination protects against Borrelia afzelii infection.

    Wagemakers, A; Mason, L M K; Oei, A; de Wever, B; van der Poll, T; Bins, A D; Hovius, J W R

    2014-12-01

    Borrelia afzelii is the predominant Borrelia species causing Lyme borreliosis in Europe. Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines against Borrelia burgdorferi sensu stricto. DNA tattooing is a novel vaccination method that can be applied in a rapid vaccination schedule. We vaccinated C3H/HeN mice with B. afzelii strain PKo OspC (outer-surface protein C) using a codon-optimized DNA vaccine tattoo and compared this with recombinant protein vaccination in a 0-2-4 week vaccination schedule. We also assessed protection by DNA tattoo in a 0-3-6 day schedule. DNA tattoo and recombinant OspC vaccination induced comparable total IgG responses, with a lower IgG1/IgG2a ratio after DNA tattoo. Two weeks after syringe-challenge with 5 × 10(5) B. afzelii spirochetes most vaccinated mice had negative B. afzelii tissue DNA loads and all were culture negative. Furthermore, DNA tattoo vaccination in a 0-3-6 day regimen also resulted in negative Borrelia loads and cultures after challenge. To conclude, DNA vaccination by tattoo was fully protective against B. afzelii challenge in mice in a rapid vaccination protocol, and induces a favorable humoral immunity compared to recombinant protein vaccination. Rapid DNA tattoo is a promising vaccination strategy against spirochetes.

  3. Methylation and in vivo expression of the surface-exposed Leptospira interrogans outer membrane protein OmpL32

    Recent studies have revealed that bacterial protein methylation is a widespread post-translational modification that is required for virulence in selected pathogenic bacteria. In particular, altered methylation of outer membrane proteins has been shown to modulate the effectiveness of the host immu...

  4. The major outer membrane proteins of enterobacteriaceae. Their immunological relatedness and their possible role in bacterial opsonization

    Hofstra, Harmen

    1981-01-01

    This thesis deals with immunological investigations of the major outer membrane proteins of the Enterobacteriaceae as a new group of enterobacterial common envelope antigens, and with some aspects of the possible role of antibodies, prepared against these proteins, in host defense mechanisms. ...

  5. Interaction between bacterial outer membrane proteins and periplasmic quality control factors: a kinetic partitioning mechanism.

    Wu, Si; Ge, Xi; Lv, Zhixin; Zhi, Zeyong; Chang, Zengyi; Zhao, Xin Sheng

    2011-09-15

    The OMPs (outer membrane proteins) of Gram-negative bacteria have to be translocated through the periplasmic space before reaching their final destination. The aqueous environment of the periplasmic space and high permeability of the outer membrane engender such a translocation process inevitably challenging. In Escherichia coli, although SurA, Skp and DegP have been identified to function in translocating OMPs across the periplasm, their precise roles and their relationship remain to be elucidated. In the present paper, by using fluorescence resonance energy transfer and single-molecule detection, we have studied the interaction between the OMP OmpC and these periplasmic quality control factors. The results of the present study reveal that the binding rate of OmpC to SurA or Skp is much faster than that to DegP, which may lead to sequential interaction between OMPs and different quality control factors. Such a kinetic partitioning mechanism for the chaperone-substrate interaction may be essential for the quality control of the biogenesis of OMPs.

  6. Residues in the membrane-spanning domain core modulate conformation and fusogenicity of the HIV-1 envelope glycoprotein

    Shang Liang; Hunter, Eric

    2010-01-01

    The membrane-spanning domain (MSD) of human immunodeficiency virus type I (HIV-1) envelope glycoprotein (Env) is critical for its biological activity. Initial studies have defined an almost invariant 'core' structure in the MSD and demonstrated that it is crucial for anchoring Env in the membrane and virus entry. We show here that amino acid substitutions in the MSD 'core' do not influence specific virus-cell attachment, nor CD4 receptor and CXCR4 coreceptor recognition by Env. However, substitutions within the MSD 'core' delayed the kinetics and reduced the efficiency of cell-cell fusion mediated by Env. Although we observed no evidence that membrane fusion mediated by the MSD core mutants was arrested at a hemifusion stage, impaired Env fusogenicity was correlated with minor conformational changes in the V2, C1, and C5 regions in gp120 and the immunodominant loop in gp41. These changes could delay initiation of the conformational changes required in the fusion process.

  7. Immunological characteristics of outer membrane protein omp31 of goat Brucella and its monoclonal antibody.

    Zheng, W Y; Wang, Y; Zhang, Z C; Yan, F

    2015-10-05

    We examined the immunological characteristics of outer membrane protein omp31 of goat Brucella and its monoclonal antibody. Genomic DNA from the M5 strain of goat Brucella was amplified by polymerase chain reaction and cloned into the prokaryotic expression vector pGEX-4T-1. The expression and immunological characteristics of the fusion protein GST-omp31 were subjected to preliminary western blot detection with goat Brucella rabbit immune serum. The Brucella immunized BALB/c mouse serum was detected using purified protein. The high-potency mouse splenocytes and myeloma Sp2/0 cells were fused. Positive clones were screened by enzyme-linked immunosorbent assay to establish a hybridoma cell line. Mice were inoculated intraperitoneally with hybridoma cells to prepare ascites. The mAb was purified using the n-caprylic acid-ammonium sulfate method. The characteristics of mAb were examined using western blotting and enzyme-linked immunosorbent assay. A 680-base pair band was observed after polymerase chain reaction. Enzyme digestion identification and sequencing showed that the pGEX-4T-1-omp31 prokaryotic expression vector was successfully established; a target band of approximately 57 kDa with an apparent molecular weight consistent with the size of the target fusion protein. At 25°C, the expression of soluble expression increased significantly; the fusion protein GST-omp31 was detected by western blotting. Anti-omp31 protein mAb was obtained from 2 strains of Brucella. The antibody showed strong specificity and sensitivity and did not cross-react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis, or Bacillus pyocyaneus. The pGEX-4T-1-omp31 prokaryotic expression vector was successfully established and showed good immunogenicity. The antibody also showed strong specificity and good sensitivity.

  8. Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium Xanthomonas campestris pv. campestris

    Niehaus Karsten

    2008-06-01

    Full Text Available Abstract Background Outer membrane vesicles (OMVs are released from the outer membrane of many Gram-negative bacteria. These extracellular compartments are known to transport compounds involved in cell-cell signalling as well as virulence associated proteins, e.g. the cytolysine from enterotoxic E. coli. Results We have demonstrated that Xanthomonas campestris pv. campestris (Xcc releases OMVs into the culture supernatant during growth. A proteome study identified 31 different proteins that associate with the OMV fraction of which half are virulence-associated. A comparison with the most abundant outer membrane (OM proteins revealed that some proteins are enriched in the OMV fraction. This may be connected to differences in the LPS composition between the OMVs and the OM. Furthermore, a comparison of the OMV proteomes from two different culture media indicated that the culture conditions have an impact on the protein composition. Interestingly, the proteins that are common to both culture conditions are mainly involved in virulence. Conclusion Outer membrane vesicles released from the OM of Xcc contain membrane- and virulence-associated proteins. Future experiments will prove whether these structures can serve as "vehicles" for the transport of virulence factors into the host membrane.

  9. Colistin resistance associated with outer membrane protein change in Klebsiella pneumoniae and Enterobacter asburiae.

    Kádár, Béla; Kocsis, Béla; Tóth, Ákos; Kristóf, Katalin; Felső, Péter; Kocsis, Béla; Böddi, Katalin; Szabó, Dóra

    2017-06-01

    In this study, outer membrane proteins (OMPs) of colistin-resistant Klebsiella pneumoniae and Enterobacter asburiae were analyzed. One colistin-susceptible and three colistin-resistant K. pneumoniae sequence type 258 strains as well as one colistin-susceptible E. asburiae and its colistin-heteroresistant counterpart strain were involved in the study. OMP analysis of each strain was performed by microchip method. Matrix-assisted laser desorption ionization time of flight/mass spectrometry (MALDI-TOF/MS) investigation was carried out after separation of OMPs by two-dimensional gel electrophoresis and in-gel digestion. The MALDI-TOF/MS analysis of OMPs in the colistin-susceptible K. pneumoniae found 16 kDa proteins belonging to the LysM domain/BON superfamily, as well as DNA starvation proteins, whereas OmpX and OmpW were detected in the colistin-resistant counterpart strains. OmpC and OmpW were detected in the colistin-susceptible E. asburiae, whereas OmpA and OmpX were identified in the colistin-resistant counterpart. This study demonstrated that OMP differences were between colistin-susceptible and -resistant counterpart strains. The altered Gram-negative cell wall may contribute to acquired colistin resistance in Enterobacteriaceae.

  10. Differences in outer membrane proteins of the lymphogranuloma venereum and trachoma biovars of Chlamydia trachomatis

    Batteiger, B.E.; Jones, R.B.

    1985-01-01

    The lymphogranuloma venereum (LGV) and trachoma biovars of Chlamydia trachomatis exhibit differences in biological properties both in vivo and in vitro. To identify analogous biochemical differences, the authors studied the molecular charges of chlamydial outer membrane proteins (OMPs) by means of isoelectric focusing and nonequilibrium pH gradient electrophoresis. Analysis of proteins of whole elementary bodies biosynthetically labeled with L-[35S]cysteine revealed that most chlamydial proteins were neutral or acidic. The major OMPs (MOMPs) of all strains tested were acidic and had apparent isoelectric points (pIs) that varied within narrow limits despite differences in molecular mass of up to 3,000 daltons (Da). However, a low-molecular-mass cysteine-rich OMP analogous to that previously described for Chlamydia psittaci varied consistently in molecular mass (12,500 versus 12,000 Da) and pI (5.4 versus 6.9) between LGV strains and trachoma strains, respectively. OMPs with a molecular mass of 60,000 Da in the trachoma biovar strains had pIs in the 7.3 to 7.7 range. However, analogous OMPs in the LGV strains existed as a doublet with a molecular mass of about 60,000 Da. These data indicate substantial differences in biochemical characteristics of analogous OMPs in the LGV and trachoma biovars. Such differences are the first structural differences described between LGV and trachoma strains which support their distinction into separate biovars and may be related to some of their biological differences

  11. Accelerated microevolution in an outer membrane protein (OMP of the intracellular bacteria Wolbachia

    Russell Jacob A

    2010-02-01

    Full Text Available Abstract Background Outer membrane proteins (OMPs of Gram-negative bacteria are key players in the biology of bacterial-host interactions. However, while considerable attention has been given to OMPs of vertebrate pathogens, relatively little is known about the role of these proteins in bacteria that primarily infect invertebrates. One such OMP is found in the intracellular bacteria Wolbachia, which are widespread symbionts of arthropods and filarial nematodes. Recent experimental studies have shown that the Wolbachia surface protein (WSP can trigger host immune responses and control cell death programming in humans, suggesting a key role of WSP for establishment and persistence of the symbiosis in arthropods. Results Here we performed an analysis of 515 unique alleles found in 831 Wolbachia isolates, to investigate WSP structure, microevolution and population genetics. WSP shows an eight-strand transmembrane β-barrel structure with four extracellular loops containing hypervariable regions (HVRs. A clustering approach based upon patterns of HVR haplotype diversity was used to group similar WSP sequences and to estimate the relative contribution of mutation and recombination during early stages of protein divergence. Results indicate that although point mutations generate most of the new protein haplotypes, recombination is a predominant force triggering diversity since the very first steps of protein evolution, causing at least 50% of the total amino acid variation observed in recently diverged proteins. Analysis of synonymous variants indicates that individual WSP protein types are subject to a very rapid turnover and that HVRs can accommodate a virtually unlimited repertoire of peptides. Overall distribution of WSP across hosts supports a non-random association of WSP with the host genus, although extensive horizontal transfer has occurred also in recent times. Conclusions In OMPs of vertebrate pathogens, large recombination impact, positive

  12. Outer Membrane Protein 25 of Brucella Activates Mitogen-Activated Protein Kinase Signal Pathway in Human Trophoblast Cells

    Jing Zhang

    2017-12-01

    Full Text Available Outer membrane protein 25 (OMP25, a virulence factor from Brucella, plays an important role in maintaining the structural stability of Brucella. Mitogen-activated protein kinase (MAPK signal pathway widely exists in eukaryotic cells. In this study, human trophoblast cell line HPT-8 and BALB/c mice were infected with Brucella abortus 2308 strain (S2308 and 2308ΔOmp25 mutant strain. The expression of cytokines and activation of MAPK signal pathway were detected. We found that the expressions of tumor necrosis factor-α, interleukin-1, and interleukin-10 (IL-10 were increased in HPT-8 cells infected with S2308 and 2308ΔOmp25 mutant. S2308 also activated p38 phosphorylation protein, extracellular-regulated protein kinases (ERK, and Jun-N-terminal kinase (JNK from MAPK signal pathway. 2308ΔOmp25 could not activate p38, ERK, and JNK branches. Immunohistochemistry experiments showed that S2308 was able to activate phosphorylation of p38 and ERK in BABL/c mice. However, 2308ΔOmp25 could weakly activate phosphorylation of p38 and ERK. These results suggest that Omp25 played an important role in the process of Brucella activation of the MAPK signal pathway.

  13. In silico local structure approach: a case study on outer membrane proteins.

    Martin, Juliette; de Brevern, Alexandre G; Camproux, Anne-Claude

    2008-04-01

    The detection of Outer Membrane Proteins (OMP) in whole genomes is an actual question, their sequence characteristics have thus been intensively studied. This class of protein displays a common beta-barrel architecture, formed by adjacent antiparallel strands. However, due to the lack of available structures, few structural studies have been made on this class of proteins. Here we propose a novel OMP local structure investigation, based on a structural alphabet approach, i.e., the decomposition of 3D structures using a library of four-residue protein fragments. The optimal decomposition of structures using hidden Markov model results in a specific structural alphabet of 20 fragments, six of them dedicated to the decomposition of beta-strands. This optimal alphabet, called SA20-OMP, is analyzed in details, in terms of local structures and transitions between fragments. It highlights a particular and strong organization of beta-strands as series of regular canonical structural fragments. The comparison with alphabets learned on globular structures indicates that the internal organization of OMP structures is more constrained than in globular structures. The analysis of OMP structures using SA20-OMP reveals some recurrent structural patterns. The preferred location of fragments in the distinct regions of the membrane is investigated. The study of pairwise specificity of fragments reveals that some contacts between structural fragments in beta-sheets are clearly favored whereas others are avoided. This contact specificity is stronger in OMP than in globular structures. Moreover, SA20-OMP also captured sequential information. This can be integrated in a scoring function for structural model ranking with very promising results. (c) 2007 Wiley-Liss, Inc.

  14. Immunochemical and biological characterization of outer membrane proteins of Porphyromonas endodontalis.

    Ogawa, T; Kuribayashi, S; Shimauchi, H; Toda, T; Hamada, S

    1992-01-01

    Outer membrane proteins (OMP) of Porphyromonas endodontalis HG 370 (ATCC 35406) were prepared from the cell envelope fraction of the organisms. The cell envelope that had been obtained by sonication of the whole cells was extracted in 2% lithium dodecyl sulfate and then successively chromatographed with Sephacryl S-200 HR and DEAE-Sepharose Fast Flow. Two OMP fractions, OMP-I and OMP-II, were obtained, and their immunochemical properties and induction of specific antibodies were examined. The OMP-I preparation consisted of a major protein with an apparent molecular mass of 31 kDa and other moderate to minor proteins of 40.3, 51.4, 67, and 71.6 kDa, while the OMP-II preparation contained 14-, 15.5-, 27-, and 44-kDa proteins as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. OMP-I was found to form hydrophilic diffusion pores by incorporation into artificial liposomes composed of egg yolk phosphatidylcholine and dicetylphosphate, indicating that OMP-I exhibited significant porin activity. However, the liposomes containing heat-denatured OMP-I were scarcely active. Spontaneous and antigen-specific immunoglobulin M (IgM)-, IgG-, and IgA-secreting spot-forming cells (SFC) enzymatically dissociated into single-cell suspensions from chronically inflamed periapical tissues and were enumerated by enzyme-linked immunospot assay. In patients with radicular cysts or dental granulomas, the major isotype of spontaneous SFC was IgG. In radicular cysts, the OMP-II-specific IgG SFC represented 0.13% of the total IgG SFC, while the antigen-specific IgA or IgM SFC was not observed. It was also found that none of these mononuclear cells produced antibodies specific for OMP-I or lipopolysaccharide of P. endodontalis. Images PMID:1328059

  15. A synthetic prestin reveals protein domains and molecular operation of outer hair cell piezoelectricity.

    Schaechinger, Thorsten J; Gorbunov, Dmitry; Halaszovich, Christian R; Moser, Tobias; Kügler, Sebastian; Fakler, Bernd; Oliver, Dominik

    2011-06-24

    Prestin, a transporter-like protein of the SLC26A family, acts as a piezoelectric transducer that mediates the fast electromotility of outer hair cells required for cochlear amplification and auditory acuity in mammals. Non-mammalian prestin orthologues are anion transporters without piezoelectric activity. Here, we generated synthetic prestin (SynPres), a chimera of mammalian and non-mammalian prestin exhibiting both, piezoelectric properties and anion transport. SynPres delineates two distinct domains in the protein's transmembrane core that are necessary and sufficient for generating electromotility and associated non-linear charge movement (NLC). Functional analysis of SynPres showed that the amplitude of NLC and hence electromotility are determined by the transport of monovalent anions. Thus, prestin-mediated electromotility is a dual-step process: transport of anions by an alternate access cycle, followed by an anion-dependent transition generating electromotility. The findings define structural and functional determinants of prestin's piezoelectric activity and indicate that the electromechanical process evolved from the ancestral transport mechanism.

  16. Peripheral-type benzodiazepine receptor: a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands

    Snyder, S.H.; Verma, A.; Trifiletti, R.R.

    1987-01-01

    The peripheral-type benzodiazepine receptor is a site identified by its nanomolar affinity for [ 3 H]diazepam, similar to the affinity of diazepam for the central-type benzodiazepine receptor in the brain. The peripheral type benzodiazepine receptor occurs in many peripheral tissues but has discrete localizations as indicated by autoradiographic studies showing uniquely high densities of the receptors in the adrenal cortex and in Leydig cells of the testes. Subcellular localization studies reveal a selective association of the receptors with the outer membrane of mitochondria. Photoaffinity labeling of the mitochondrial receptor with [ 3 H]flunitrazepam reveals two discrete labeled protein bands of 30 and 35 kDa, respectively. The 35-kDa band appears to be identical with the voltage-dependent anion channel protein porin. Fractionation of numerous peripheral tissues reveals a single principal endogenous ligand for the receptor, consisting of porphyrins, which display nanomolar affinity. Interactions of porphyrins with the mitochondrial receptor may clarify its physiological role and account for many pharmacological actions of benzodiazepines

  17. Evaluation of outer dense fiber-1 and -2 protein expression in asthenozoospermic infertile men

    Silvia W. Lestari

    2015-07-01

    Full Text Available Background: Most of male infertility are caused by defect in sperm motility (asthenozoospermia. The molecular mechanism of low sperm motility in asthenozoospermic patients has not been fully understood. Sperm motility is strongly related to the axoneme structure which is composed of microtubules and supported by outer dense fiber (ODF and fibrous sheath (FS protein. The objective of this study was to characterize the ODF (ODF1 and ODF2 expression in asthenozoospermic infertile male and control normozoospermic fertile male.Methods: Asthenozoospermic samples (n=18 were collected from infertile patients at Andrology Lab, Cipto Mangunkusumo Hospital Jakarta and control were taken from normozoospermic fertile donor (n=18. After motility analyses by computer-assisted sperm analysis (CASA, semen were divided into two parts, for Western blot and for immunocytochemistry analysis. Antibody against ODF1 and ODF2 protein were used in both analyses.Results: Analysis of ODF1 protein expression showed bands with molecular weight of ~30 kDa and ODF2 ~85 kDa. The mean band intensity of ODF1 and ODF2 protein were lower in the asthenozoospermic group (AG compared to normozoospermic group (NG. Moreover, both ODF proteins were less intense and less localized in the AG than NG. Sperm motility was lower in AG, compared to control NG, i.e. average path velocity (VAP = 32.07 ± 7.03 vs 37.58 ± 8.73 µm/s, p = 0.455; straight line velocity (VSL = 24.17 ± 6.90 vs 27.61 ± 4.50 µm/s, p = 0.317 and curvilinear velocity (VCL = 45.68 ± 7.91 vs 55.55 ± 16.40 µm/s, p = 0.099.Conclusion: There is down-regulation of ODF1 and ODF2 protein expression and less-compact localization in AG sperm compared to the NG. These changes might have caused disturbances in the sperm motility as observed in this study.

  18. Ferric-Pyoverdine Recognition by Fpv Outer Membrane Proteins of Pseudomonas protegens Pf-5

    Hartney, Sierra L.; Mazurier, Sylvie; Girard, Maëva K.; Mehnaz, Samina; Davis, Edward W.; Gross, Harald; Lemanceau, Philippe

    2013-01-01

    The soil bacterium Pseudomonas protegens Pf-5 (previously called P. fluorescens Pf-5) produces two siderophores, enantio-pyochelin and a compound in the large and diverse pyoverdine family. Using high-resolution mass spectroscopy, we determined the structure of the pyoverdine produced by Pf-5. In addition to producing its own siderophores, Pf-5 also utilizes ferric complexes of some pyoverdines produced by other strains of Pseudomonas spp. as sources of iron. Previously, phylogenetic analysis of the 45 TonB-dependent outer membrane proteins in Pf-5 indicated that six are in a well-supported clade with ferric-pyoverdine receptors (Fpvs) from other Pseudomonas spp. We used a combination of phylogenetics, bioinformatics, mutagenesis, pyoverdine structural determinations, and cross-feeding bioassays to assign specific ferric-pyoverdine substrates to each of the six Fpvs of Pf-5. We identified at least one ferric-pyoverdine that was taken up by each of the six Fpvs of Pf-5. Functional redundancy of the Pf-5 Fpvs was also apparent, with some ferric-pyoverdines taken up by all mutants with a single Fpv deletion but not by a mutant having deletions in two of the Fpv-encoding genes. Finally, we demonstrated that phylogenetically related Fpvs take up ferric complexes of structurally related pyoverdines, thereby establishing structure-function relationships that can be employed in the future to predict the pyoverdine substrates of Fpvs in other Pseudomonas spp. PMID:23222724

  19. [Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism].

    Zheng, Linli; Ge, Yumei; Hu, Weilin; Yan, Jie

    2013-03-01

    To determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism. OmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays. The bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P Expression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

  20. Outer surface protein B is critical for Borrelia burgdorferi adherence and survival within Ixodes ticks.

    Girish Neelakanta

    2007-03-01

    Full Text Available Survival of Borrelia burgdorferi in ticks and mammals is facilitated, at least in part, by the selective expression of lipoproteins. Outer surface protein (Osp A participates in spirochete adherence to the tick gut. As ospB is expressed on a bicistronic operon with ospA, we have now investigated the role of OspB by generating an OspB-deficient B. burgdorferi and examining its phenotype throughout the spirochete life cycle. Similar to wild-type isolates, the OspB-deficient B. burgdorferi were able to readily infect and persist in mice. OspB-deficient B. burgdorferi were capable of migrating to the feeding ticks but had an impaired ability to adhere to the tick gut and survive within the vector. Furthermore, the OspB-deficient B. burgdorferi bound poorly to tick gut extracts. The complementation of the OspB-deficient spirochete in trans, with a wild-type copy of ospB gene, restored its ability to bind tick gut. Taken together, these data suggest that OspB has an important role within Ixodes scapularis and that B. burgdorferi relies upon multiple genes to efficiently persist in ticks.

  1. The Role of Helicobacter pylori Outer Membrane Proteins in Adherence and Pathogenesis

    Oleastro, Mónica; Ménard, Armelle

    2013-01-01

    Helicobacter pylori is one of the most successful human pathogens, which colonizes the mucus layer of the gastric epithelium of more than 50% of the world’s population. This curved, microaerophilic, Gram-negative bacterium induces a chronic active gastritis, often asymptomatic, in all infected individuals. In some cases, this gastritis evolves to more severe diseases such as peptic ulcer disease, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. H. pylori has developed a unique set of factors, actively supporting its successful survival and persistence in its natural hostile ecological niche, the human stomach, throughout the individual’s life, unless treated. In the human stomach, the vast majority of H. pylori cells are motile in the mucus layer lining, but a small percentage adheres to the epithelial cell surfaces. Adherence to the gastric epithelium is important for the ability of H. pylori to cause disease because this intimate attachment facilitates: (1) colonization and persistence, by preventing the bacteria from being eliminated from the stomach, by mucus turnover and gastric peristalsis; (2) evasion from the human immune system and (3) efficient delivery of proteins into the gastric cell, such as the CagA oncoprotein. Therefore, bacteria with better adherence properties colonize the host at higher densities. H. pylori is one of the most genetically diverse bacterial species known and is equipped with an extraordinarily large set of outer membrane proteins, whose role in the infection and persistence process will be discussed in this review, as well as the different receptor structures that have been so far described for mucosal adherence. PMID:24833057

  2. The Role of Helicobacter pylori Outer Membrane Proteins in Adherence and Pathogenesis

    Armelle Ménard

    2013-08-01

    Full Text Available Helicobacter pylori is one of the most successful human pathogens, which colonizes the mucus layer of the gastric epithelium of more than 50% of the world’s population. This curved, microaerophilic, Gram-negative bacterium induces a chronic active gastritis, often asymptomatic, in all infected individuals. In some cases, this gastritis evolves to more severe diseases such as peptic ulcer disease, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. H. pylori has developed a unique set of factors, actively supporting its successful survival and persistence in its natural hostile ecological niche, the human stomach, throughout the individual’s life, unless treated. In the human stomach, the vast majority of H. pylori cells are motile in the mucus layer lining, but a small percentage adheres to the epithelial cell surfaces. Adherence to the gastric epithelium is important for the ability of H. pylori to cause disease because this intimate attachment facilitates: (1 colonization and persistence, by preventing the bacteria from being eliminated from the stomach, by mucus turnover and gastric peristalsis; (2 evasion from the human immune system and (3 efficient delivery of proteins into the gastric cell, such as the CagA oncoprotein. Therefore, bacteria with better adherence properties colonize the host at higher densities. H. pylori is one of the most genetically diverse bacterial species known and is equipped with an extraordinarily large set of outer membrane proteins, whose role in the infection and persistence process will be discussed in this review, as well as the different receptor structures that have been so far described for mucosal adherence.

  3. Elucidation of the outer membrane proteome of Salmonella enterica serovar Typhimurium utilising a lipid-based protein immobilization technique

    Appleton Hazel

    2010-02-01

    Full Text Available Abstract Background Salmonella enterica serovar Typhimurium (S. Typhimurium is a major cause of human gastroenteritis worldwide. The outer membrane proteins expressed by S. Typhimurium mediate the process of adhesion and internalisation within the intestinal epithelium of the host thus influencing the progression of disease. Since the outer membrane proteins are surface-exposed, they provide attractive targets for the development of improved antimicrobial agents and vaccines. Various techniques have been developed for their characterisation, but issues such as carryover of cytosolic proteins still remain a problem. In this study we attempted to characterise the surface proteome of S. Typhimurium using Lipid-based Protein Immobilisation technology in the form of LPI™ FlowCells. No detergents are required and no sample clean up is needed prior to downstream analysis. The immobilised proteins can be digested with proteases in multiple steps to increase sequence coverage, and the peptides eluted can be characterised directly by liquid chromatography - tandem mass spectrometry (LC-MS/MS and identified from mass spectral database searches. Results In this study, 54 outer membrane proteins, were identified with two or more peptide hits using a multi-step digest approach. Out of these 28 were lipoproteins, nine were involved in transport and three with enzyme activity These included the transporters BtuB which is responsible for the uptake of vitamin B12, LamB which is involved in the uptake of maltose and maltodextrins and LolB which is involved in the incorporation of lipoproteins in the outer membrane. Other proteins identified included the enzymes MltC which may play a role in cell elongation and division and NlpD which is involved in catabolic processes in cell wall formation as well as proteins involved in virulence such as Lpp1, Lpp2 and OmpX. Conclusion Using a multi-step digest approach the LPI™ technique enables the incorporation of a

  4. Overexpression of an Outer Membrane Protein Associated with Decreased Susceptibility to Carbapenems in Proteus mirabilis

    Tsai, Yi-Lin; Wang, Min-Cheng; Hsueh, Po-Ren; Liu, Ming-Che; Hu, Rouh-Mei; Wu, Yue-Jin; Liaw, Shwu-Jen

    2015-01-01

    Proteus mirabilis isolates commonly have decreased susceptibility to imipenem. Previously, we found P. mirabilis hfq mutant was more resistant to imipenem and an outer membrane protein (OMP) could be involved. Therefore, we investigated the role of this OMP in carbapenem susceptibility. By SDS-PAGE we found this OMP (named ImpR) was increased in hfq mutant and LC-MS/MS revealed it to be the homologue of Salmonella YbfM, which is a porin for chitobiose and subject to MicM (a small RNA) regulation. We demonstrated that ImpR overexpression resulted in increased carbapenem MICs in the laboratory strain and clinical isolates. Chitobiose induced expression of chb (a chitobiose utilization operon). Real-time RT-PCR and SDS-PAGE were performed to elucidate the relationship of hfq, impR, chb and MicM in P. mirabilis. We found MicM RNA was decreased in hfq mutant and chbBC-intergenic region (chbBC-IGR) overexpression strain (chbIGRov), while impR mRNA was increased in hfq mutant, micM mutant and chbIGRov strain. In addition, mutation of hfq or micM and overexpression of chbBC-IGR increased ImpR protein level. Accordingly, chitobiose made wild-type have higher levels of ImpR protein and are more resistant to carbapenems. Hfq- and MicM-complemented strains restored wild-type MICs. Mutation of both impR and hfq eliminated the increase in carbapenem MICs observed in hfq mutant and ImpR-complementation of hfq/impR double mutant resulted in MICs as hfq mutant, indicating that the ImpR-dependent decreased carbapenem susceptibility of hfq mutant. These indicate MicM was antisense to impR mRNA and was negatively-regulated by chbBC-IGR. Together, overexpression of ImpR contributed to the decreased carbapenem susceptibility in P. mirabilis. PMID:25756370

  5. Identification of two novel genes encoding 97- to 99-kilodalton outer membrane proteins of Chlamydia pneumoniae.Infect Immun. 1999 Jan;67(1):375-83

    Knudsen, K; Madsen, AS; Mygind, P

    1999-01-01

    Two genes encoding 97- to 99-kDa Chlamydia pneumoniae VR1310 outer membrane proteins (Omp4 and Omp5) with mutual similarity were cloned and sequenced. The proteins were shown to be constituents of the C. pneumoniae outer membrane complex, and the deduced amino acid sequences were similar to those...

  6. Clonality, outer-membrane proteins profile and efflux pump in KPC- producing Enterobacter sp. in Brazil.

    Rosa, Juliana Ferraz; Rizek, Camila; Marchi, Ana Paula; Guimaraes, Thais; Miranda, Lourdes; Carrilho, Claudia; Levin, Anna S; Costa, Silvia F

    2017-03-17

    Carbapenems resistance in Enterobacter spp. has increased in the last decade, few studies, however, described the mechanisms of resistance in this bacterium. This study evaluated clonality and mechanisms of carbapenems resistance in clinical isolates of Enterobacter spp. identified in three hospitals in Brazil (Hospital A, B and C) over 7-year. Antibiotics sensitivity, pulsed-field gel electrophoresis (PFGE), PCR for carbapenemase and efflux pump genes were performed for all carbapenems-resistant isolates. Outer-membrane protein (OMP) was evaluated based on PFGE profile. A total of 130 isolates of Enterobacter spp were analyzed, 44/105 (41, 9%) E. aerogenes and 8/25 (32,0%) E. cloacae were resistant to carbapenems. All isolates were susceptible to fosfomycin, polymyxin B and tigecycline. KPC was present in 88.6% of E. aerogenes and in all E. cloacae resistant to carbapenems. The carbapenems-resistant E. aerogenes identified in hospital A belonged to six clones, however, a predominant clone was identified in this hospital over the study period. There is a predominant clone in Hospital B and Hospital C as well. The mechanisms of resistance to carbapenems differ among subtypes. Most of the isolates co-harbored blaKPC, blaTEM and /or blaCTX associated with decreased or lost of 35-36KDa and or 39 KDa OMP. The efflux pump AcrAB-TolC gene was only identified in carbapenems-resistant E. cloacae. There was a predominant clone in each hospital suggesting that cross-transmission of carbapenems-resistant Enterobacter spp. was frequent. The isolates presented multiple mechanisms of resistance to carbapenems including OMP alteration.

  7. Quantitative Proteomics Reveals Distinct Differences in the Protein Content of Outer Membrane Vesicle Vaccines

    Waterbeemd, van de B.; Mommen, G.P.M.; Pennings, J.L.A.; Eppink, M.H.M.; Wijffels, R.H.; Pol, van der L.A.; Jong, de A.P.J.M.

    2013-01-01

    At present, only vaccines containing outer membrane vesicles (OMV) have successfully stopped Neisseria meningitidis serogroup B epidemics. These vaccines however require detergent-extraction to remove endotoxin, which changes immunogenicity and causes production difficulties. To investigate this in

  8. Antigen sequence typing of outer membrane protein (fetA gene of Neisseria meningitidis serogroup A from Delhi & adjoining areas

    S Dwivedi

    2014-01-01

    Full Text Available Background & objectives: Meningitis caused by Neisseria meningitidis is a fatal disease. Meningococcal meningitis is an endemic disease in Delhi and irregular pattern of outbreaks has been reported in India. All these outbreaks were associated with serogroup A. Detailed molecular characterization of N. meningitidis is required for the management of this fatal disease. In this study, we characterized antigenic diversity of surface exposed outer membrane protein (OMP FetA antigen of N. meningitidis serogroup A isolates obtained from cases of invasive meningococcal meningitis in Delhi, India. Methods: Eight isolates of N. meningitidis were collected from cerebrospinal fluid during October 2008 to May 2011 from occasional cases of meningococcal meningitis. Seven isolates were from outbreaks of meningococcal meningitis in 2005-2006 in Delhi and its adjoining areas. These were subjected to molecular typing of fetA gene, an outer membrane protein gene. Results: All 15 N. meningitides isolates studied were serogroup A. This surface exposed porin is putatively under immune pressure. Hence as a part of molecular characterization, genotyping was carried out to find out the diversity in outer membrane protein (FetA gene among the circulating isolates of N. meningitidis. All 15 isolates proved to be of the same existing allele type of FetA variable region (VR when matched with global database. The allele found was F3-1 for all the isolates. Interpretation & conclusions: There was no diversity reported in the outer membrane protein FetA in the present study and hence this protein appeared to be a stable molecule. More studies on molecular characterization of FetA antigen are required from different serogroups circulating in different parts of the world.

  9. [Identification and analysis of the proteins interacted with Prestin in cochlear outer hair cells of guinea pig].

    Luo, X; Wang, J Y; Zhang, F L; Xia, Y

    2018-01-07

    Objective: To explore the regulation and mechanism of Prestin protein by identifying the proteins interacted with Prestin in cochlear outer hair cell(OHC) and analyzing their biological function. Methods: Co-immunoprecipitation combined mass spectrometry technology was used to isolate and identify the proteins interacted with Prestin protein of OHC, bioinformatics was used to construct Prestin protein interaction network. The proteins interacted with Prestin in OHC of guinea pig were determined by matching primary interaction mass spectrometry with protein interaction network, and annotated their functions. Results: The results of co-immunoprecipitation combined with mass spectrometry showed that 116 kinds of credible proteins could interact with Prestin. By constructing Prestin protein interaction network, matching the results of mass spectrometry and analyzing of sub-cellular localization, eight kinds of proteins were confirmed that they interacted with Prestin directly, namely EEF2, HSP90AB1, FN1, FLNA, EEF1A1, HSP90B1, ATP5A1, and ERH, respectively, which were mainly involved in the synthesis and transportation, transmembrane folding and localization, structural stability and signal transduction of Prestin protein. Conclusion: EEF2, HSP90AB1, FN1, FLNA, EEF1A1, HSP90B1, ATP5A1 and ERH provide molecular basis for sensory amplification function of OHCs by participating in biotransformation, transmembrane folding and localization, signal transduction and other biological processes of Prestin protein.

  10. Targeting and Assembly of Components of the TOC Protein Import Complex at the Chloroplast Outer Envelope Membrane

    Lynn G.L. Richardson

    2014-06-01

    Full Text Available The translocon at the outer envelope membrane of chloroplasts (TOC initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β–barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

  11. Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane.

    Richardson, Lynn G L; Paila, Yamuna D; Siman, Steven R; Chen, Yi; Smith, Matthew D; Schnell, Danny J

    2014-01-01

    The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β-barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

  12. Expression, crystallization and preliminary X-ray crystallographic studies of the outer membrane protein OmpW from Escherichia coli

    Albrecht, Reinhard; Zeth, Kornelius; Söding, Johannes; Lupas, Andrei; Linke, Dirk

    2006-01-01

    The outer membrane protein OmpW from E. coli was overexpressed in inclusion bodies and refolded with the help of detergent. The protein has been crystallized and the crystals diffract to 3.5 Å resolution. OmpW is an eight-stranded 21 kDa molecular-weight β-barrel protein from the outer membrane of Gram-negative bacteria. It is a major antigen in bacterial infections and has implications in antibiotic resistance and in the oxidative degradation of organic compounds. OmpW from Escherichia coli was cloned and the protein was expressed in inclusion bodies. A method for refolding and purification was developed which yields properly folded protein according to circular-dichroism measurements. The protein has been crystallized and crystals were obtained that diffracted to a resolution limit of 3.5 Å. The crystals belong to space group P422, with unit-cell parameters a = 122.5, c = 105.7 Å. A homology model of OmpW is presented based on known structures of eight-stranded β-barrels, intended for use in molecular-replacement trials

  13. The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery.

    Zufferey, Mónica; Montandon, Cyrille; Douet, Véronique; Demarsy, Emilie; Agne, Birgit; Baginsky, Sacha; Kessler, Felix

    2017-04-28

    The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo However, kinases that phosphorylate the TOC159 A-domain to enable protein import have remained elusive. Here, using co-purification with TOC159 from Arabidopsis , we discovered a novel component of the chloroplast import machinery, the regulatory kinase at the outer chloroplast membrane 1 (KOC1). We found that KOC1 is an integral membrane protein facing the cytosol and stably associates with TOC. Moreover, KOC1 phosphorylated the A-domain of TOC159 in vitro , and in mutant koc1 chloroplasts, preprotein import efficiency was diminished. koc1 Arabidopsis seedlings had reduced survival rates after transfer from the dark to the light in which protein import into plastids is required to rapidly complete chloroplast biogenesis. In summary, our data indicate that KOC1 is a functional component of the TOC machinery that phosphorylates import receptors, supports preprotein import, and contributes to efficient chloroplast biogenesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Iodo-gen-catalysed iodination for identification of surface-exposed outer membrane proteins of Escherichia coli K12

    Ferreira, L.C.S.; Almeida, D.F. de

    1987-01-01

    Surface proteins of Escherichia coli K12 were identified by radiolabelling using 1,3,4,6 - tatrachloro, 3-alpha, 6-alpha - diphenylgycoluryl (Iodo-Gen) and 131 I. Labelled proteins were localized in the outer membrane of the cells. Using this technique it has been possible to observe technique it has been possible to observe that the eletrophoretic pattern of surface proteins changes according to the growth phases in culture. Radiolabelling of E.coli cells inculbated at 42 0 C showed that the syntheses of two surface proteins were temperature-inducible. At least one such protein may be involved in the process of cell division in E.coli K12. (author) [pt

  15. Iodo-gen-catalysed iodination for identification of surface-exposed outer membrane proteins of Escherichia coli K12

    Ferreira, L C.S.; Almeida, D.F. de

    1987-12-01

    Surface proteins of Escherichia coli K12 were identified by radiolabelling using 1,3,4,6 - tatrachloro, 3-alpha, 6-alpha - diphenylgycoluryl (Iodo-Gen) and /sup 131/I. Labelled proteins were localized in the outer membrane of the cells. Using this technique it has been possible to observe technique it has been possible to observe that the eletrophoretic pattern of surface proteins changes according to the growth phases in culture. Radiolabelling of E.coli cells inculbated at 42/sup 0/C showed that the syntheses of two surface proteins were temperature-inducible. At least one such protein may be involved in the process of cell division in E.coli K12.

  16. High-resolution diffraction from crystals of a membrane-protein complex: bacterial outer membrane protein OmpC complexed with the antibacterial eukaryotic protein lactoferrin

    Sundara Baalaji, N.; Acharya, K. Ravi; Singh, T. P.; Krishnaswamy, S.

    2005-01-01

    Crystals of the complex formed between the bacterial membrane protein OmpC and the antibacterial protein lactoferrin suitable for high-resolution structure determination have been obtained. The crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å. Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å, α = β = 90, γ = 120°. This indicated a Matthews coefficient (V M ) of 3.3 Å 3 Da −1 , corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 Å resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin

  17. Formation of the outer layer of the Dictyostelium spore coat depends on the inner-layer protein SP85/PsB.

    Metcalf, Talibah; Kelley, Karen; Erdos, Gregory W; Kaplan, Lee; West, Christopher M

    2003-02-01

    The Dictyostelium spore is surrounded by a 220 microm thick trilaminar coat that consists of inner and outer electron-dense layers surrounding a central region of cellulose microfibrils. In previous studies, a mutant strain (TL56) lacking three proteins associated with the outer layer exhibited increased permeability to macromolecular tracers, suggesting that this layer contributes to the coat permeability barrier. Electron microscopy now shows that the outer layer is incomplete in the coats of this mutant and consists of a residual regular array of punctate electron densities. The outer layer is also incomplete in a mutant lacking a cellulose-binding protein associated with the inner layer, and these coats are deficient in an outer-layer protein and another coat protein. To examine the mechanism by which this inner-layer protein, SP85, contributes to outer-layer formation, various domain fragments were overexpressed in forming spores. Most of these exert dominant negative effects similar to the deletion of outer-layer proteins, but one construct, consisting of a fusion of the N-terminal and Cys-rich C1 domain, induces a dense mat of novel filaments at the surface of the outer layer. Biochemical studies show that the C1 domain binds cellulose, and a combination of site-directed mutations that inhibits its cellulose-binding activity suppresses outer-layer filament induction. The results suggest that, in addition to a previously described early role in regulating cellulose synthesis, SP85 subsequently contributes a cross-bridging function between cellulose and other coat proteins to organize previously unrecognized structural elements in the outer layer of the coat.

  18. The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells.

    Yang, Chih-Wei; Wu, Mai-Szu; Pan, Ming-Jeng; Hsieh, Wang-Ju; Vandewalle, Alain; Huang, Chiu-Ching

    2002-08-01

    Tubulointerstitial nephritis is a main renal manifestation caused by pathogenic leptospira that accumulate mostly in the proximal tubules, thereby inducing tubular injury and tubulointerstitial nephritis. To elucidate the role of leptospira outer membrane proteins in tubulointerstitial nephritis, outer membrane proteins from pathogenic Leptospira shermani and nonpathogenic Leptospira patoc extracted by Triton X-114 were administered to cultured mouse proximal tubule cells. A dose-dependent increase of monocyte chemoattractant protein-1 (MCP-1), RANTES, nitrite, and tumor necrosis factor-alpha (TNF-alpha) in the culture supernatant was observed 48 h after incubating Leptospira shermani outer membrane proteins with mouse proximal tubule cells. RT competitive-PCR experiments showed that Leptospira shermani outer membrane proteins (0.2 microg/ml) increased the expression of MCP-1, nitric oxide synthase (iNOS), RANTES, and TNF-alpha mRNA by 3.0-, 9.4-, 2.5-, and 2.5-fold, respectively, when compared with untreated cells. Outer membrane proteins extract from avirulent Leptospira patoc did not induce significant effects. The pathogenic outer membrane proteins extract contain a major component of a 32-kD lipoprotein (LipL32), which is absent in the nonpathogenic leptospira outer membrane. An antibody raised against LipL32 prevented the stimulatory effect of Leptospira shermani outer membrane proteins extract on MCP-1 and iNOS mRNA expression in cultured proximal tubule cells, whereas recombinant LipL32 significantly stimulated the expression of MCP-1 and iNOS mRNAs and augmented nuclear binding of nuclear factor-kappaB (NF-kappaB) and AP-1 transcription factors in proximal tubule cells. An antibody raised against LipL32 also blunted the effects induced by the recombinant LipL32. This study demonstrates that LipL32 is a major component of pathogenic leptospira outer membrane proteins involved in the pathogenesis of tubulointerstitial nephritis.

  19. Differential expression of in vivo and in vitro protein profile of outer membrane of Acidovorax avenae subsp. avenae.

    Muhammad Ibrahim

    Full Text Available Outer membrane (OM proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium.

  20. Differential expression of in vivo and in vitro protein profile of outer membrane of Acidovorax avenae subsp. avenae.

    Ibrahim, Muhammad; Shi, Yu; Qiu, Hui; Li, Bin; Jabeen, Amara; Li, Liping; Liu, He; Kube, Michael; Xie, Guanlin; Wang, Yanli; Blondel, Carlos; Santiviago, Carlos A; Contreras, Ines; Sun, Guochang

    2012-01-01

    Outer membrane (OM) proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium.

  1. Solid-state NMR of the Yersinia pestis outer membrane protein Ail in lipid bilayer nanodiscs sedimented by ultracentrifugation

    Ding, Yi; Fujimoto, L. Miya; Yao, Yong; Marassi, Francesca M.

    2015-01-01

    Solid-state NMR studies of sedimented soluble proteins has been developed recently as an attractive approach for overcoming the size limitations of solution NMR spectroscopy while bypassing the need for sample crystallization or precipitation (Bertini et al. Proc Natl Acad Sci USA 108(26):10396–10399, 2011). Inspired by the potential benefits of this method, we have investigated the ability to sediment lipid bilayer nanodiscs reconstituted with a membrane protein. In this study, we show that nanodiscs containing the outer membrane protein Ail from Yersinia pestis can be sedimented for solid-state NMR structural studies, without the need for precipitation or lyophilization. Optimized preparations of Ail in phospholipid nanodiscs support both the structure and the fibronectin binding activity of the protein. The same sample can be used for solution NMR, solid-state NMR and activity assays, facilitating structure–activity correlation experiments across a wide range of timescales

  2. Solid-state NMR of the Yersinia pestis outer membrane protein Ail in lipid bilayer nanodiscs sedimented by ultracentrifugation

    Ding, Yi; Fujimoto, L. Miya; Yao, Yong; Marassi, Francesca M., E-mail: fmarassi@sbmri.org [Sanford-Burnham Medical Research Institute (United States)

    2015-04-15

    Solid-state NMR studies of sedimented soluble proteins has been developed recently as an attractive approach for overcoming the size limitations of solution NMR spectroscopy while bypassing the need for sample crystallization or precipitation (Bertini et al. Proc Natl Acad Sci USA 108(26):10396–10399, 2011). Inspired by the potential benefits of this method, we have investigated the ability to sediment lipid bilayer nanodiscs reconstituted with a membrane protein. In this study, we show that nanodiscs containing the outer membrane protein Ail from Yersinia pestis can be sedimented for solid-state NMR structural studies, without the need for precipitation or lyophilization. Optimized preparations of Ail in phospholipid nanodiscs support both the structure and the fibronectin binding activity of the protein. The same sample can be used for solution NMR, solid-state NMR and activity assays, facilitating structure–activity correlation experiments across a wide range of timescales.

  3. Undressing of Waddlia chondrophila to enrich its outer membrane proteins to develop a new species-specific ELISA

    J. Lienard

    2014-01-01

    Full Text Available Waddlia chondrophila, an obligate intracellular bacterium of the Chlamydiales order, is considered as an agent of bovine abortion and a likely cause of miscarriage in humans. Its role in respiratory diseases was questioned after the detection of its DNA in clinical samples taken from patients suffering from pneumonia or bronchiolitis. To better define the role of Waddlia in both miscarriage and pneumonia, a tool allowing large-scale serological investigations of Waddlia seropositivity is needed. Therefore, enriched outer membrane proteins of W. chondrophila were used as antigens to develop a specific ELISA. After thorough analytical optimization, the ELISA was validated by comparison with micro-immunofluorescence and it showed a sensitivity above 85% with 100% specificity. The ELISA was subsequently applied to human sera to specify the role of W. chondrophila in pneumonia. Overall, 3.6% of children showed antibody reactivity against W. chondrophila but no significant difference was observed between children with and without pneumonia. Proteomic analyses were then performed using mass spectrometry, highlighting members of the outer membrane protein family as the dominant proteins. The major Waddlia putative immunogenic proteins were identified by immunoblot using positive and negative human sera. The new ELISA represents an efficient tool with high throughput applications. Although no association with pneumonia and Waddlia seropositivity was observed, this ELISA could be used to specify the role of W. chondrophila in miscarriage and in other diseases.

  4. Outer membrane targeting of Pseudomonas aeruginosa proteins shows variable dependence on the components of Bam and Lol machineries.

    Hoang, Hanh H; Nickerson, Nicholas N; Lee, Vincent T; Kazimirova, Anastasia; Chami, Mohamed; Pugsley, Anthony P; Lory, Stephen

    2011-01-01

    In Gram-negative bacteria, the Lol and Bam machineries direct the targeting of lipidated and nonlipidated proteins, respectively, to the outer membrane (OM). Using Pseudomonas aeruginosa strains with depleted levels of specific Bam and Lol proteins, we demonstrated a variable dependence of different OM proteins on these targeting pathways. Reduction in the level of BamA significantly affected the ability of the β-barrel membrane protein OprF to localize to the OM, while the targeting of three secretins that are functionally related OM proteins was less affected (PilQ and PscC) or not at all affected (XcpQ). Depletion of LolB affected all lipoproteins examined and had a variable effect on the nonlipidated proteins. While the levels of OprF, PilQ, and PscC were significantly reduced by LolB depletion, XcpQ was unaffected and was correctly localized to the OM. These results suggest that certain β-barrel proteins such as OprF primarily utilize the complete Bam machinery. The Lol machinery participates in the OM targeting of secretins to variable degrees, likely through its involvement in the assembly of lipidated Bam components. XcpQ, but not PilQ or PscC, was shown to assemble spontaneously into liposomes as multimers. This work raises the possibility that there is a gradient of utilization of Bam and Lol insertion and targeting machineries. Structural features of individual proteins, including their β-barrel content, may determine the propensity of these proteins for folding (or misfolding) during periplasmic transit and OM insertion, thereby influencing the extent of utilization of the Bam targeting machinery, respectively. Targeting of lipidated and nonlipidated proteins to the outer membrane (OM) compartment in Gram-negative bacteria involves the transfer across the periplasm utilizing the Lol and Bam machineries, respectively. We show that depletion of Bam and Lol components in Pseudomonas aeruginosa does not lead to a general OM protein translocation defect

  5. Application of zwitterionic detergent to the solubilization of Klebsiella pneumoniae outer membrane proteins for two-dimensional gel electrophoresis.

    Bednarz-Misa, I; Serek, P; Dudek, B; Pawlak, A; Bugla-Płoskońska, G; Gamian, A

    2014-12-01

    Klebsiella pneumoniae is a frequent cause of nosocomial respiratory, urinary and gastrointestinal tract infections and septicemia with the multidrug-resistant K. pneumoniae being a major public health concern. Outer membrane proteins (OMPs) are important virulence factors responsible for the appropriate adaptation to the host environment. They constitute of the antigens being the first in contact with infected organism. However, K. pneumoniae strains are heavily capsulated and it is important to establish the OMPs isolation procedure prior to proteomics extensive studies. In this study we used Zwittergent Z 3-14® as a detergent to isolate the OMPs from K. pneumoniae cells and resolve them using two-dimensional electrophoresis (2-DE). As a result we identified 134 protein spots. The OMPs identified in this study are possible candidates for the development of a protein-based vaccine against K. pneumoniae infections. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. The Drosophila Pericentrin-like-protein (PLP cooperates with Cnn to maintain the integrity of the outer PCM

    Jennifer H. Richens

    2015-08-01

    Full Text Available Centrosomes comprise a pair of centrioles surrounded by a matrix of pericentriolar material (PCM. In vertebrate cells, Pericentrin plays an important part in mitotic PCM assembly, but the Drosophila Pericentrin-like protein (PLP appears to have a more minor role in mitotic fly cells. Here we investigate the function of PLP during the rapid mitotic cycles of the early Drosophila embryo. Unexpectedly, we find that PLP is specifically enriched in the outer-most regions of the PCM, where it largely co-localizes with the PCM scaffold protein Cnn. In the absence of PLP the outer PCM appears to be structurally weakened, and it rapidly disperses along the centrosomal microtubules (MTs. As a result, centrosomal MTs are subtly disorganized in embryos lacking PLP, although mitosis is largely unperturbed and these embryos develop and hatch at near-normal rates. Y2H analysis reveals that PLP can potentially form multiple interactions with itself and with the PCM recruiting proteins Asl, Spd-2 and Cnn. A deletion analysis suggests that PLP participates in a complex network of interactions that ultimately help to strengthen the PCM.

  7. Surface expression, single-channel analysis and membrane topology of recombinant Chlamydia trachomatis Major Outer Membrane Protein

    McClafferty Heather

    2005-01-01

    Full Text Available Abstract Background Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes. Results C. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded β-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands. Conclusions C. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with β-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP

  8. Protective immunity induced by 67 K outer membrane protein of phase I Coxiella burnetii in mice and guinea pigs

    Zhang, Y.X.; Zhi, N.; Yu, S.R.; Li, Q.J.; Yu, G.Q.; Zhang, X.

    1994-01-01

    A 67 K outer membrane protein (OMP) isolated from phase I Coxiella burnetii QiYi strain was purified with monoclonal antibodies (MoAb) coupled to CNBr-Sepharose 4B. Chemical analyses of the 67 K protein showed that it contained seventeen kids of amino acids and no lipopolysaccharides. The immunogenicity and protectivity of the 67 K protein against C. burnetii was evaluated in mice and guinea pigs bi in vitro lymphocyte proliferation assay, delayed-type skin test, antibody conversion rate, and immunization and challenge tests. Intraperitoneal injection of the 67 K protein resulted in antibody production against phase I and II whole cell antigens. The anti-67 K antibody conversion rate was found to be 100% in mice and guinea pigs as well. Lymphocytes were responses in vitro to specific antigen. In addition, delayed-type hypersensitivity appeared two weeks after immunization with the 67 K protein. Moreover, 199% of mice and guinea pigs inoculated with the 67 K protein were protected against a challenge with 10 3 ID 50 virulent C. burnetii. In conclusion, these results demonstrate that the 67 K OMP elicits in vivo and in vitro both B cell-mediated and T cell-mediated immunity in mice and guinea pigs. Thus the 67 K protein is a candidate for an effective subunit vaccine against Q fever. (author)

  9. Exposure of outer membrane proteins on the surface of Pseudomonas aeruginosa PA01 revealed by labelling with [125I]lactoperoxidase

    Lambert, P.A.; Booth, B.R.

    1982-01-01

    The authors have investigated the exposure of the major outer membrane proteins on the cell surface by treating whole cells of P. aeruginosa with [ 125 I]lactoperoxidase. This reagent catalyses the iodination of tyrosine and histidine residues of proteins in the presence of hydrogen peroxide. It is too large to penetrate the outer membrane (Msub(r) 77500), therefore it is assumed to label only those proteins which have such residues exposed on the cell surface and has been applied to a number of Gram-negative organisms. It is found that F was the major labelled protein, D1 and/or D2 were less heavily labelled, and G was very faintly labelled. In addition, two proteins (Msub(r) 72500 and 38000) which did not appear to be major outer membrane proteins were labelled. (Auth.)

  10. Cloning of Bordetella pertussis putative outer protein D (BopD) and Leucin/Isoleucine/Valin binding protein (LivJ)

    Öztürk, Burcu Emine Tefon

    2017-04-01

    Whooping cough also known as pertussis is a contagious acute upper respiratory disease primarily caused by Bordetella pertussis. It is known that this disease may be fatal especially in infants and recently, the number of pertussis cases has been increased. Despite the fact that there are numbers of acellular vaccines on the market, the current acellular vaccine compositions are inadequate for providing sustainable immunity and avoiding subclinical disease cases. Hence, exploring novel proteins with high immune protective capacities is essential to enhance the clinical efficacy of current vaccines. In this study, genes of selected immunogenic proteins via -omics studies, namely Putative outer protein D (BopD) and Leucin/Isoleucine/Valin Binding Protein (LivJ) were first cloned into pGEM-T Easy vector and transformed to into E. coli DH5α cells and then cloned into the expression vector pET-28a(+) and transformed into E. coli BL21 (DE3) cells to express the proteins.

  11. Identification and characterization of a novel outer membrane protein receptor required for hemin utilization in Vibrio vulnificus

    Datta, Shreya

    2011-01-01

    Vibrio vulnificus, the cause of septicemia and serious wound infection in humans and fishes, require iron for its pathogenesis. Hemin uptake through the outer membrane receptor, HupA, is one of its many mechanisms by which it acquires iron. We report here the identification of an additional TonB-dependent hemin receptor HvtA, that is needed in conjunction with the HupA protein for optimal hemin utilization. The HvtA protein is significantly homologous to other outer membrane hemin receptors and its expression in trans restored the uptake of hemin and hemoglobin, the latter to a weaker extent, in a mutant strain that was defective in both receptors. Quantitative RT-PCR suggested that transcription of the hvtA gene was iron regulated. The operon containing the hvtA gene is homologous to the operon in V. cholerae containing the hemin receptor gene hutR suggesting a vertical transmission of the hvtA cluster from V. cholerae to V. vulnificus. PMID:22015545

  12. The Outer Membrane Protein OmpW Forms an Eight-Stranded beta-Barrel with a Hydrophobic Channel

    Hong, H.; Patel, D.; Tamm, L.; van den Berg, B.

    2006-01-01

    Escherichia coli OmpW belongs to a family of small outer membrane (OM) proteins that are widespread in Gram-negative bacteria. Their functions are unknown, but recent data suggest that they may be involved in the protection of bacteria against various forms of environmental stress. In order to gain insight into the function of these proteins we have determined the crystal structure of Escherichia coli OmpW to 2.7 Angstroms resolution. The structure shows that OmpW forms an eight-stranded beta-barrel with a long and narrow hydrophobic channel that contains a bound LDAO detergent molecule. Single channel conductance experiments show that OmpW functions as an ion channel in planar lipid bilayers. The channel activity can be blocked by the addition of LDAO. Taken together, the data suggest that members of the OmpW family could be involved in the transport of small hydrophobic molecules across the bacterial OM

  13. Label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine.

    Dick, Lawrence W; Mehl, John T; Loughney, John W; Mach, Anna; Rustandi, Richard R; Ha, Sha; Zhang, Lan; Przysiecki, Craig T; Dieter, Lance; Hoang, Van M

    2015-01-01

    The development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90-110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques.

  14. Isolation and Identification of Outer Membrane Proteins of Helicobacter Pylori of Iranian Patient by SDS-PAGE

    M. Doosty

    1998-04-01

    Full Text Available The function of Helicobacter pylori (H.pylori is confirmed as one of the factors which motivates gastric and duodenal ulcer and gastritis. Various methods are used to diagnose the infection. Serological tests are the easiest and most harmless for the patients. Probably, H.pylori strains in Iran are different from the strains in other countries. Hence, it seems neccessary to design a specific serological test to recognize and identify different strains of bacterial antigenic proteins of Iranian patients."nSince the most manifest and specific to these bacterial antigens are the "Outer Membrane Protein" (OMP, therefore, the first necessary step is to separate and purify H.pylori OMP and then to identify antigenic proteins."nIn this study, we received bacteria colony that belonged to 15 patients with gastric or duodenal ulcer, which had been growed in blood agar or brucella broth. After processing such as washing, freezing and defreezing, sonicating, centrifugation with high speed (10,000 g and treatment with sarcosyl, the sarcosyl insoluble fraction was extracted. Sodium Dodecyl Sulfate - Poly Acrylamide Gel Electrophoresis (SDS-PAGE was preformed. From all 15 OMP specimens, we isolated protein bands."nThe first two bands with higher MW, were major bands and the two lighter bands were the minor bands. Approximate MW of these 4 proteins are equal to 67000, 61000, 30000 and 17000 dalton

  15. Unusual Self-Assembly of the Recombinant Chlamydia trachomatis Major Outer Membrane Protein-Based Fusion Antigen CTH522 Into Protein Nanoparticles

    Rose, Fabrice; Karlsen, Kasper; Jensen, Pernille

    2018-01-01

    Sexually transmitted Chlamydia trachomatis (Ct) infects more than 100 million people annually, and untreated chlamydia infections can cause severe complications. Therefore, there is an urgent need for a chlamydia vaccine. The Ct major outer membrane protein (MOMP) is highly immunogenic but is a c......Sexually transmitted Chlamydia trachomatis (Ct) infects more than 100 million people annually, and untreated chlamydia infections can cause severe complications. Therefore, there is an urgent need for a chlamydia vaccine. The Ct major outer membrane protein (MOMP) is highly immunogenic...... but is a challenging vaccine candidate by being an integral membrane protein, and the immunogenicity depends on a correctly folded structure. We investigated the biophysical properties of the recombinant MOMP-based fusion antigen CTH522, which is tested in early human clinical trials. It consists of a truncated......-defined secondary structural elements, and no thermal transitions were measurable. Chemical unfolding resulted monomers that upon removal of the denaturant self-assembled into higher order structures, comparable to the structure of the native protein. The conformation of CTH522 in nanoparticles is thus not entirely...

  16. Crystallization and preliminary crystallographic studies of the C-terminal domain of outer membrane protein A from enterohaemorrhagic Escherichia coli

    Gu, Jiang; Ji, Xiaowei; Qi, Jianxun; Ma, Ying; Mao, Xuhu; Zou, Quanming

    2010-01-01

    In this study, recombinant OmpAC from EHEC was purified and crystallized and a diffraction data set was collected to 2.7 Å resolution. Outer membrane protein A (OmpA) of enterohaemorrhagic Escherichia coli (EHEC) plays multiple roles in bacterial physiology and pathogenesis, such as mediation of bacterial conjunction, maintenance of cell shape, induction of adhesion of EHEC to host cells etc. Better understanding of the functions of OmpA will help in the control of EHEC infections. OmpA is composed of two domains: the N-terminal domain and the C-terminal domain. The N-terminal domain is a β-barrel structure and embeds in the outer membrane of the bacterium. The structure and function of the C-terminal domain of OmpA (OmpAC) remain elusive. In this study, recombinant OmpAC from EHEC was purified and crystallized and a diffraction data set was collected to 2.7 Å resolution. The crystals belonged to space group I4 1 32, with unit-cell parameter a = 158.99 Å. The Matthews coefficient and solvent content were calculated to be 2.55 Å 3 Da −1 and 51.77%, respectively, for two molecules in the asymmetric unit

  17. Involvement and necessity of the Cpx regulon in the event of aberrant β-barrel outer membrane protein assembly

    Gerken, Henri; Leiser, Owen P.; Bennion, Drew; Misra, Rajeev

    2010-01-01

    Summary The Cpx and σE regulons help maintain outer membrane integrity; the Cpx pathway monitors the biogenesis of cell surface structures, such as pili, while the σE pathway monitors the biogenesis of β-barrel outer membrane proteins (OMPs). In this study we revealed the importance of the Cpx regulon in the event of β-barrel OMP mis-assembly, by utilizing mutants expressing either a defective β-barrel OMP assembly machinery (Bam) or assembly defective β-barrel OMPs. Analysis of specific mRNAs showed that ΔcpxR bam double mutants failed to induce degP expression beyond the wild type level, despite activation of the σE pathway. The synthetic conditional lethal phenotype of ΔcpxR in mutant Bam or β-barrel OMP backgrounds was reversed by wild type DegP expressed from a heterologous plasmid promoter. Consistent with the involvement of the Cpx regulon in the event of aberrant β-barrel OMP assembly, the expression of cpxP, the archetypal member of the cpx regulon, was upregulated in defective Bam backgrounds or in cells expressing a single assembly-defective β-barrel OMP species. Together, these results showed that both the Cpx and σE regulons are required to reduce envelope stress caused by aberrant β-barrel OMP assembly, with the Cpx regulon principally contributing by controlling degP expression. PMID:20487295

  18. A proteomic screen reveals the mitochondrial outer membrane protein Mdm34p as an essential target of the F-box protein Mdm30p.

    Ota, Kazuhisa; Kito, Keiji; Okada, Satoshi; Ito, Takashi

    2008-10-01

    Ubiquitination plays various critical roles in eukaryotic cellular regulation and is mediated by a cascade of enzymes including ubiquitin protein ligase (E3). The Skp1-Cullin-F-box protein complex comprises the largest E3 family, in each member of which a unique F-box protein binds its targets to define substrate specificity. Although genome sequencing uncovers a growing number of F-box proteins, most of them have remained as "orphans" because of the difficulties in identification of their substrates. To address this issue, we tested a quantitative proteomic approach by combining the stable isotope labeling by amino acids in cell culture (SILAC), parallel affinity purification (PAP) that we had developed for efficient enrichment of ubiquitinated proteins, and mass spectrometry (MS). We applied this SILAC-PAP-MS approach to compare ubiquitinated proteins between yeast cells with and without over-expressed Mdm30p, an F-box protein implicated in mitochondrial morphology. Consequently, we identified the mitochondrial outer membrane protein Mdm34p as a target of Mdm30p. Furthermore, we found that mitochondrial defects induced by deletion of MDM30 are not only recapitulated by a mutant Mdm34p defective in interaction with Mdm30p but alleviated by ubiquitination-mimicking forms of Mdm34p. These results indicate that Mdm34p is a physiologically important target of Mdm30p.

  19. Nitazoxanide Inhibits Pilus Biogenesis by Interfering with Folding of the Usher Protein in the Outer Membrane.

    Chahales, Peter; Hoffman, Paul S; Thanassi, David G

    2016-04-01

    Many bacterial pathogens assemble surface fibers termed pili or fimbriae that facilitate attachment to host cells and colonization of host tissues. The chaperone/usher (CU) pathway is a conserved secretion system that is responsible for the assembly of virulence-associated pili by many different Gram-negative bacteria. Pilus biogenesis by the CU pathway requires a dedicated periplasmic chaperone and an integral outer membrane (OM) assembly and secretion platform termed the usher. Nitazoxanide (NTZ), an antiparasitic drug, was previously shown to inhibit the function of aggregative adherence fimbriae and type 1 pili assembled by the CU pathway in enteroaggregativeEscherichia coli, an important causative agent of diarrhea. We show here that NTZ also inhibits the function of type 1 and P pili from uropathogenicE. coli(UPEC). UPEC is the primary causative agent of urinary tract infections, and type 1 and P pili mediate colonization of the bladder and kidneys, respectively. By analysis of the different stages of the CU pilus biogenesis pathway, we show that treatment of bacteria with NTZ causes a reduction in the number of usher molecules in the OM, resulting in a loss of pilus assembly on the bacterial surface. In addition, we determine that NTZ specifically prevents proper folding of the usher β-barrel domain in the OM. Our findings demonstrate that NTZ is a pilicide with a novel mechanism of action and activity against diverse CU pathways. This suggests that further development of the NTZ scaffold may lead to new antivirulence agents that target the usher to prevent pilus assembly. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  20. Rapid outer-surface protein C DNA tattoo vaccination protects against Borrelia afzelii infection

    Wagemakers, A.; Mason, L. M. K.; Oei, A.; de Wever, B.; van der Poll, T.; Bins, A. D.; Hovius, J. W. R.

    2014-01-01

    Borrelia afzelii is the predominant Borrelia species causing Lyme borreliosis in Europe. Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines against Borrelia burgdorferi sensu stricto. DNA tattooing is a novel vaccination method

  1. Clearing the outer mitochondrial membrane from harmful proteins via lipid droplets

    Bischof, J.; Salzmann, M.; Streubel, M.K.; Hašek, Jiří; Geltinger, F.; Duschl, J.; Bresgen, N.; Briza, P.; Hašková, Danuša; Lejsková, Renata; Sopjani, M.; Richter, K.; Rinnerthaler, M.

    2017-01-01

    Roč. 3, March 20 (2017), č. článku 17016. E-ISSN 2058-7716 R&D Projects: GA ČR(CZ) GA16-05497S; GA MŠk(CZ) 7AMB16AT006 Institutional support: RVO:61388971 Keywords : mitochondrial membrane * harmful protein s * lipid droplets Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology

  2. Proteolytic cleavage of the Chlamydia pneumoniae major outer membrane protein in the absence of Pmp10

    Juul, Nicolai Stefan; Timmerman, E; Gevaert, K

    2007-01-01

    The genome of the obligate intracellular bacteria Chlamydia pneumoniae contains 21 genes encoding polymorphic membrane proteins (Pmp). While no function has yet been attributed to the Pmps, they may be involved in an antigenic variation of the Chlamydia surface. It has previously been demonstrated...

  3. High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes

    Yao, Yong; Dutta, Samit Kumar; Park, Sang Ho; Rai, Ratan; Fujimoto, L. Miya; Bobkov, Andrey A.; Opella, Stanley J.; Marassi, Francesca M.

    2017-01-01

    The outer membrane protein Ail (Adhesion invasion locus) is one of the most abundant proteins on the cell surface of Yersinia pestis during human infection. Its functions are expressed through interactions with a variety of human host proteins, and are essential for microbial virulence. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but those samples contained detergents that interfere with functionality, thus, precluding analysis of the structural basis for Ail’s biological activity. Here, we demonstrate that high-resolution solid-state NMR spectra can be obtained from samples of Ail in detergent-free phospholipid liposomes, prepared with a lipid to protein molar ratio of 100. The spectra, obtained with 13 C or 1 H detection, have very narrow line widths (0.40–0.60 ppm for 13 C, 0.11–0.15 ppm for 1 H, and 0.46–0.64 ppm for 15 N) that are consistent with a high level of sample homogeneity. The spectra enable resonance assignments to be obtained for N, CO, CA and CB atomic sites from 75 out of 156 residues in the sequence of Ail, including 80% of the transmembrane region. The 1 H-detected solid-state NMR 1 H/ 15 N correlation spectra obtained for Ail in liposomes compare very favorably with the solution NMR 1 H/ 15 N TROSY spectra obtained for Ail in nanodiscs prepared with a similar lipid to protein molar ratio. These results set the stage for studies of the molecular basis of the functional interactions of Ail with its protein partners from human host cells, as well as the development of drugs targeting Ail.

  4. High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes

    Yao, Yong; Dutta, Samit Kumar [Sanford Burnham Prebys Medical Discovery Institute (United States); Park, Sang Ho; Rai, Ratan [University of California San Diego, Department of Chemistry and Biochemistry (United States); Fujimoto, L. Miya; Bobkov, Andrey A. [Sanford Burnham Prebys Medical Discovery Institute (United States); Opella, Stanley J. [University of California San Diego, Department of Chemistry and Biochemistry (United States); Marassi, Francesca M., E-mail: fmarassi@sbp.edu [Sanford Burnham Prebys Medical Discovery Institute (United States)

    2017-03-15

    The outer membrane protein Ail (Adhesion invasion locus) is one of the most abundant proteins on the cell surface of Yersinia pestis during human infection. Its functions are expressed through interactions with a variety of human host proteins, and are essential for microbial virulence. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but those samples contained detergents that interfere with functionality, thus, precluding analysis of the structural basis for Ail’s biological activity. Here, we demonstrate that high-resolution solid-state NMR spectra can be obtained from samples of Ail in detergent-free phospholipid liposomes, prepared with a lipid to protein molar ratio of 100. The spectra, obtained with {sup 13}C or {sup 1}H detection, have very narrow line widths (0.40–0.60 ppm for {sup 13}C, 0.11–0.15 ppm for {sup 1}H, and 0.46–0.64 ppm for {sup 15}N) that are consistent with a high level of sample homogeneity. The spectra enable resonance assignments to be obtained for N, CO, CA and CB atomic sites from 75 out of 156 residues in the sequence of Ail, including 80% of the transmembrane region. The {sup 1}H-detected solid-state NMR {sup 1}H/{sup 15}N correlation spectra obtained for Ail in liposomes compare very favorably with the solution NMR {sup 1}H/{sup 15}N TROSY spectra obtained for Ail in nanodiscs prepared with a similar lipid to protein molar ratio. These results set the stage for studies of the molecular basis of the functional interactions of Ail with its protein partners from human host cells, as well as the development of drugs targeting Ail.

  5. [Study on immunogenicity of group A and group C meningococcal conjugate vaccine with coupling group B meningococcal outer membrane protein].

    Ma, Fu-Bao; Tao, Hong; Wang, Hong-Jun

    2009-10-01

    To evaluate the Immunogenicity of Group A and Group C Meningococcal conjugate Vaccine with coupling Group B Meningococcal Outer Membrane Protein (Men B-OMP). 458 healthy children aged 3-5 months, 6-23 months, 2-6 years and 7-24 years were given the Groups A and C conjugate Vaccine with MenB-OMP or other vaccine as control group to measure the pre-and post-vaccination Men A and C and B by Serum Bactericidal Assay (SBA) in the double-blind randomized controlled trial. 97.65%-100% were 4 times or greater increase in SBA titer for the healthy children given the Groups A and C conjugate Vaccine with MenB-OMP, The geometric mean titer of SBA were 1:194-1:420, which significantly higber than controls. The Group A and C conjugate Vaccine with MenB-OMP was safe and well immunogenic.

  6. Expression, purification and preliminary X-ray analysis of the Neisseria meningitidis outer membrane protein PorB

    Tanabe, Mikio; Iverson, Tina M.; (Vanderbilt)

    2010-01-28

    The Neisseria meningitidis outer membrane protein PorB was expressed in Escherichia coli and purified from inclusion bodies by denaturation in urea followed by refolding in buffered LDAO on a size-exclusion column. PorB has been crystallized in three different crystal forms: C222, R32 and P6{sub 3}. The C222 crystal form may contain either one or two PorB monomers in the asymmetric unit, while both the R32 and P6{sub 3} crystal forms contained one PorB monomer in the asymmetric unit. Of the three, the P6{sub 3} crystal form had the best diffraction quality, yielding data extending to 2.3 {angstrom} resolution.

  7. Outer membrane protein A (OmpA: a new player in shigella flexneri protrusion formation and inter-cellular spreading.

    Cecilia Ambrosi

    Full Text Available Outer membrane protein A (OmpA is a multifaceted predominant outer membrane protein of Escherichia coli and other Enterobacteriaceae whose role in the pathogenesis of various bacterial infections has recently been recognized. Here, the role of OmpA on the virulence of Shigella flexneri has been investigated. An ompA mutant of wild-type S. flexneri 5a strain M90T was constructed (strain HND92 and it was shown to be severely impaired in cell-to-cell spreading since it failed to plaque on HeLa cell monolayers. The lack of OmpA significantly reduced the levels of IcsA while the levels of cell associated and released IcsP-cleaved 95 kDa amino-terminal portion of the mature protein were similar. Nevertheless, the ompA mutant displayed IcsA exposed across the entire bacterial surface. Surprisingly, the ompA mutant produced proper F-actin comet tails, indicating that the aberrant IcsA exposition at bacterial lateral surface did not affect proper activation of actin-nucleating proteins, suggesting that the absence of OmpA likely unmasks mature or cell associated IcsA at bacterial lateral surface. Moreover, the ompA mutant was able to invade and to multiply within HeLa cell monolayers, although internalized bacteria were found to be entrapped within the host cell cytoplasm. We found that the ompA mutant produced significantly less protrusions than the wild-type strain, indicating that this defect could be responsible of its inability to plaque. Although we could not definitely rule out that the ompA mutation might exert pleiotropic effects on other S. flexneri genes, complementation of the ompA mutation with a recombinant plasmid carrying the S. flexneri ompA gene clearly indicated that a functional OmpA protein is required and sufficient for proper IcsA exposition, plaque and protrusion formation. Moreover, an independent ompA mutant was generated. Since we found that both mutants displayed identical virulence profile, these results further supported the

  8. Is the C-terminal insertional signal in Gram-negative bacterial outer membrane proteins species-specific or not?

    Paramasivam Nagarajan

    2012-09-01

    Full Text Available Abstract Background In Gram-negative bacteria, the outer membrane is composed of an asymmetric lipid bilayer of phopspholipids and lipopolysaccharides, and the transmembrane proteins that reside in this membrane are almost exclusively β-barrel proteins. These proteins are inserted into the membrane by a highly conserved and essential machinery, the BAM complex. It recognizes its substrates, unfolded outer membrane proteins (OMPs, through a C-terminal motif that has been speculated to be species-specific, based on theoretical and experimental results from only two species, Escherichia coli and Neisseria meningitidis, where it was shown on the basis of individual sequences and motifs that OMPs from the one cannot easily be over expressed in the other, unless the C-terminal motif was adapted. In order to determine whether this species specificity is a general phenomenon, we undertook a large-scale bioinformatics study on all predicted OMPs from 437 fully sequenced proteobacterial strains. Results We were able to verify the incompatibility reported between Escherichia coli and Neisseria meningitidis, using clustering techniques based on the pairwise Hellinger distance between sequence spaces for the C-terminal motifs of individual organisms. We noticed that the amino acid position reported to be responsible for this incompatibility between Escherichia coli and Neisseria meningitidis does not play a major role for determining species specificity of OMP recognition by the BAM complex. Instead, we found that the signal is more diffuse, and that for most organism pairs, the difference between the signals is hard to detect. Notable exceptions are the Neisseriales, and Helicobacter spp. For both of these organism groups, we describe the specific sequence requirements that are at the basis of the observed difference. Conclusions Based on the finding that the differences between the recognition motifs of almost all organisms are small, we assume that

  9. Localization of the fourth membrane spanning domain as a ligand binding site in the human platelet α2-adrenergic receptor

    Matsui, Hiroaki; Lefkowitz, R.J.; Caron, M.G.; Regan, J.W.

    1989-01-01

    The human platelet α 2 -adrenergic receptor is an integral membrane protein which binds epinephrine. The gene for this receptor has been cloned, and the primary structure is thus known. A model of its secondary structure predicts that the receptor has seven transmembrane spanning domains. By covalent labeling and peptide mapping, the authors have identified a region of the receptor that is directly involved with ligand binding. Partially purified preparations of the receptor were covalently radiolabeled with either of two specific photoaffinity ligands: [ 3 H]SKF 102229 (an antagonist) or p-azido[ 3 H]clonidine (an agonist). The radiolabeled receptors were then digested with specific endopeptidases, and peptides containing the covalently bound radioligands were identified. Lysylendopeptidase treatment of [ 3 H]SKF 102229 labeled receptor yielded one peptide of M r 2400 as the product of a complete digest. Endopeptidase Arg-C gave a labeled peptide of M r 4000, which was further digested to the M r 2400 peptide by additional treatment with lysylendopeptidase. Using p-azido[ 3 H]clonidine-labeled receptor, a similar M r 2400 peptide was obtained by lysylendopeptidase cleavage. This M r 2400 peptide corresponds to the fourth transmembrane spanning domain of the receptor. These data suggest that this region forms part of the ligand binding domain of the human platelet α 2 -adrenergic receptor

  10. Protective Immunity and Reduced Renal Colonization Induced by Vaccines Containing Recombinant Leptospira interrogans Outer Membrane Proteins and Flagellin Adjuvant

    Monaris, D.; Sbrogio-Almeida, M. E.; Dib, C. C.; Canhamero, T. A.; Souza, G. O.; Vasconcellos, S. A.; Ferreira, L. C. S.

    2015-01-01

    Leptospirosis is a global zoonotic disease caused by different Leptospira species, such as Leptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinant Leptospira interrogans outer membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigAC) and six novel antigens, combined with aluminum hydroxide (alum) or Salmonella flagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigAC, either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigAC or LigAC coadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen. PMID:26108285

  11. Immunoprotective efficacy of Acinetobacter baumannii outer membrane protein, FilF, predicted in silico as a potential vaccine candidate

    Ravinder eSingh

    2016-02-01

    Full Text Available Acinetobacter baumannii is emerging as a serious nosocomial pathogen with multidrug resistance that has made it difficult to cure and development of efficacious treatment against this pathogen is direly needed. This has led to investigate vaccine approach to prevent and treat A. baumannii infections. In this work, an outer membrane putative pilus assembly protein, FilF, was predicted as vaccine candidate by in silico analysis of A. baumannii proteome and was found to be conserved among the A. baumannii strains. It was cloned and expressed in E. coli BL21(DE3 and purified by Ni-NTA chromatography. Immunization with FilF generated high antibody titer (>64000 and provided 50% protection against a standardized lethal dose (10*8 CFU of A. baumannii in murine pneumonia model. FilF immunization reduced the bacterial load in lungs by 2 and 4 log cycles, 12 and 24 h post infection as compared to adjuvant control; reduced the levels of pro-inflammatory cytokines TNF-α, IL-6, IL-33, IFN-γ and IL-1β significantly and histology of lung tissue supported the data by showing considerably reduced damage and infiltration of neutrophils in lungs. These results demonstrate the in vivo validation of immunoprotective efficacy of a protein predicted as a vaccine candidate by in silico proteomic analysis and open the possibilities for exploration of a large array of uncharacterized proteins.

  12. Putative outer membrane proteins of Leptospira interrogans stimulate human umbilical vein endothelial cells (HUVECS) and express during infection.

    Gómez, Ricardo M; Vieira, Monica L; Schattner, Mirta; Malaver, Elisa; Watanabe, Monica M; Barbosa, Angela S; Abreu, Patricia A E; de Morais, Zenaide M; Cifuente, Javier O; Atzingen, Marina V; Oliveira, Tatiane R; Vasconcellos, Silvio A; Nascimento, Ana L T O

    2008-01-01

    Cell adhesion molecules (CAMs) are surface receptors present in eukaryotic cells that mediate cell-cell or cell-extracellular matrix interactions. Vascular endothelium stimulation in vitro that lead to the upregulation of CAMs was reported for the pathogenic spirochaetes, including rLIC10365 of Leptospira interrogans. In this study, we report the cloning of LIC10507, LIC10508, LIC10509 genes of L. interrogans using Escherichia coli as a host system. The rational for selecting these sequences is due to their location in L. interrogans serovar Copenhageni genome that has a potential involvement in pathogenesis. The genes encode for predicted lipoproteins with no assigned functions. The purified recombinant proteins were capable to promote the upregulation of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on monolayers of human umbilical vein endothelial cells (HUVECS). In addition, the coding sequences are expressed in the renal tubules of animal during bacterial experimental infection. The proteins are probably located at the outer membrane of the bacteria since they are detected in detergent-phase of L. interrogans Triton X-114 extract. Altogether our data suggest a possible involvement of these proteins during bacterial infection and provide new insights into the role of this region in the pathogenesis of Leptospira.

  13. Shigella outer membrane protein PSSP-1 is broadly protective against Shigella infection.

    Kim, Jae-Ouk; Rho, Semi; Kim, Su Hee; Kim, Heejoo; Song, Hyo Jin; Kim, Eun Jin; Kim, Ryang Yeo; Kim, Eun Hye; Sinha, Anuradha; Dey, Ayan; Yang, Jae Seung; Song, Man Ki; Nandy, Ranjan Kumar; Czerkinsky, Cecil; Kim, Dong Wook

    2015-04-01

    In developing countries, Shigella is a primary cause of diarrhea in infants and young children. Although antibiotic therapy is an effective treatment for shigellosis, therapeutic options are narrowing due to the emergence of antibiotic resistance. Thus, preventive vaccination could become the most efficacious approach for controlling shigellosis. We have identified several conserved protein antigens that are shared by multiple Shigella serotypes and species. Among these, one antigen induced cross-protection against experimental shigellosis, and we have named it pan-Shigella surface protein 1 (PSSP-1). PSSP-1-induced protection requires a mucosal administration route and coadministration of an adjuvant. When PSSP-1 was administered intranasally, it induced cross-protection against Shigella flexneri serotypes 2a, 5a, and 6, Shigella boydii, Shigella sonnei, and Shigella dysenteriae serotype 1. Intradermally administered PSSP-1 induced strong serum antibody responses but failed to induce protection in the mouse lung pneumonia model. In contrast, intranasal administration elicited efficient local and systemic antibody responses and production of interleukin 17A and gamma interferon. Interestingly, blood samples from patients with recent-onset shigellosis showed variable but significant mucosal antibody responses to other conserved Shigella protein antigens but not to PSSP-1. We suggest that PSSP-1 is a promising antigen for a broadly protective vaccine against Shigella. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. A novel Geobacteraceae-specific outer membrane protein J (OmpJ is essential for electron transport to Fe (III and Mn (IV oxides in Geobacter sulfurreducens

    Schiffer Marianne

    2005-07-01

    Full Text Available Abstract Background Metal reduction is thought to take place at or near the bacterial outer membrane and, thus, outer membrane proteins in the model dissimilatory metal-reducing organism Geobacter sulfurreducens are of interest to understand the mechanisms of Fe(III reduction in the Geobacter species that are the predominant Fe(III reducers in many environments. Previous studies have implicated periplasmic and outer membrane cytochromes in electron transfer to metals. Here we show that the most abundant outer membrane protein of G. sulfurreducens, OmpJ, is not a cytochrome yet it is required for metal respiration. Results When outer membrane proteins of G. sulfurreducens were separated via SDS-PAGE, one protein, designated OmpJ (outer membrane protein J, was particularly abundant. The encoding gene, which was identified from mass spectrometry analysis of peptide fragments, is present in other Geobacteraceae, but not in organisms outside this family. The predicted localization and structure of the OmpJ protein suggested that it was a porin. Deletion of the ompJ gene in G. sulfurreducens produced a strain that grew as well as the wild-type strain with fumarate as the electron acceptor but could not grow with metals, such as soluble or insoluble Fe (III and insoluble Mn (IV oxide, as the electron acceptor. The heme c content in the mutant strain was ca. 50% of the wild-type and there was a widespread loss of multiple cytochromes from soluble and membrane fractions. Transmission electron microscopy analyses of mutant cells revealed an unusually enlarged periplasm, which is likely to trigger extracytoplasmic stress response mechanisms leading to the degradation of periplasmic and/or outer membrane proteins, such as cytochromes, required for metal reduction. Thus, the loss of the capacity for extracellular electron transport in the mutant could be due to the missing c-type cytochromes, or some more direct, but as yet unknown, role of OmpJ in metal

  15. Evasion of IFN-γ signaling by Francisella novicida is dependent upon Francisella outer membrane protein C.

    Kalyan C Nallaparaju

    2011-03-01

    Full Text Available Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of the lethal disease tularemia. An outer membrane protein (FTT0918 of F. tularensis subsp. tularensis has been identified as a virulence factor. We generated a F. novicida (F. tularensis subsp. novicida FTN_0444 (homolog of FTT0918 fopC mutant to study the virulence-associated mechanism(s of FTT0918.The ΔfopC strain phenotype was characterized using immunological and biochemical assays. Attenuated virulence via the pulmonary route in wildtype C57BL/6 and BALB/c mice, as well as in knockout (KO mice, including MHC I, MHC II, and µmT (B cell deficient, but not in IFN-γ or IFN-γR KO mice was observed. Primary bone marrow derived macrophages (BMDM prepared from C57BL/6 mice treated with rIFN-γ exhibited greater inhibition of intracellular ΔfopC than wildtype U112 strain replication; whereas, IFN-γR KO macrophages showed no IFN-γ-dependent inhibition of ΔfopC replication. Moreover, phosphorylation of STAT1 was downregulated by the wildtype strain, but not the fopC mutant, in rIFN-γ treated macrophages. Addition of NG-monomethyl-L-arginine, an NOS inhibitor, led to an increase of ΔfopC replication to that seen in the BMDM unstimulated with rIFN-γ. Enzymatic screening of ΔfopC revealed aberrant acid phosphatase activity and localization. Furthermore, a greater abundance of different proteins in the culture supernatants of ΔfopC than that in the wildtype U112 strain was observed.F. novicida FopC protein facilitates evasion of IFN-γ-mediated immune defense(s by down-regulation of STAT1 phosphorylation and nitric oxide production, thereby promoting virulence. Additionally, the FopC protein also may play a role in maintaining outer membrane stability (integrity facilitating the activity and localization of acid phosphatases and other F. novicida cell components.

  16. Engineering of an E. coli outer membrane protein FhuA with increased channel diameter

    Dworeck Tamara

    2011-08-01

    Full Text Available Abstract Background Channel proteins like FhuA can be an alternative to artificial chemically synthesized nanopores. To reach such goals, channel proteins must be flexible enough to be modified in their geometry, i.e. length and diameter. As continuation of a previous study in which we addressed the lengthening of the channel, here we report the increasing of the channel diameter by genetic engineering. Results The FhuA Δ1-159 diameter increase has been obtained by doubling the amino acid sequence of the first two N-terminal β-strands, resulting in variant FhuA Δ1-159 Exp. The total number of β-strands increased from 22 to 24 and the channel surface area is expected to increase by ~16%. The secondary structure analysis by circular dichroism (CD spectroscopy shows a high β-sheet content, suggesting the correct folding of FhuA Δ1-159 Exp. To further prove the FhuA Δ1-159 Exp channel functionality, kinetic measurement using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine were conducted. The results indicated a 17% faster diffusion kinetic for FhuA Δ1-159 Exp as compared to FhuA Δ1-159, well correlated to the expected channel surface area increase of ~16%. Conclusion In this study using a simple "semi rational" approach the FhuA Δ1-159 diameter was enlarged. By combining the actual results with the previous ones on the FhuA Δ1-159 lengthening a new set of synthetic nanochannels with desired lengths and diameters can be produced, broadening the FhuA Δ1-159 applications. As large scale protein production is possible our approach can give a contribution to nanochannel industrial applications.

  17. Yersinia outer protein YopE affects the actin cytoskeleton in Dictyostelium discoideum through targeting of multiple Rho family GTPases

    Vlahou, Georgia

    2009-07-14

    Abstract Background All human pathogenic Yersinia species share a virulence-associated type III secretion system that translocates Yersinia effector proteins into host cells to counteract infection-induced signaling responses and prevent phagocytosis. Dictyostelium discoideum has been recently used to study the effects of bacterial virulence factors produced by internalized pathogens. In this study we explored the potential of Dictyostelium as model organism for analyzing the effects of ectopically expressed Yersinia outer proteins (Yops). Results The Yersinia pseudotuberculosis virulence factors YopE, YopH, YopM and YopJ were expressed de novo within Dictyostelium and their effects on growth in axenic medium and on bacterial lawns were analyzed. No severe effect was observed for YopH, YopJ and YopM, but expression of YopE, which is a GTPase activating protein for Rho GTPases, was found to be highly detrimental. GFP-tagged YopE expressing cells had less conspicuous cortical actin accumulation and decreased amounts of F-actin. The actin polymerization response upon cAMP stimulation was impaired, although chemotaxis was unaffected. YopE also caused reduced uptake of yeast particles. These alterations are probably due to impaired Rac1 activation. We also found that YopE predominantly associates with intracellular membranes including the Golgi apparatus and inhibits the function of moderately overexpressed RacH. Conclusion The phenotype elicited by YopE in Dictyostelium can be explained, at least in part, by inactivation of one or more Rho family GTPases. It further demonstrates that the social amoeba Dictyostelium discoideum can be used as an efficient and easy-to-handle model organism in order to analyze the function of a translocated GAP protein of a human pathogen.

  18. Borrelia burgdorferi outer surface protein C (OspC) binds complement component C4b and confers bloodstream survival.

    Caine, Jennifer A; Lin, Yi-Pin; Kessler, Julie R; Sato, Hiromi; Leong, John M; Coburn, Jenifer

    2017-12-01

    Borrelia burgdorferi (Bb) is the causative agent of Lyme disease in the United States, a disease that can result in carditis, and chronic and debilitating arthritis and/or neurologic symptoms if left untreated. Bb survives in the midgut of the Ixodes scapularis tick, or within tissues of immunocompetent hosts. In the early stages of infection, the bacteria are present in the bloodstream where they must resist clearance by the innate immune system of the host. We have found a novel role for outer surface protein C (OspC) from B. burgdorferi and B. garinii in interactions with the complement component C4b and bloodstream survival in vivo. Our data show that OspC inhibits the classical and lectin complement pathways and competes with complement protein C2 for C4b binding. Resistance to complement is important for maintenance of the lifecycle of Bb, enabling survival of the pathogen within the host as well as in the midgut of a feeding tick when ospC expression is induced. © 2017 John Wiley & Sons Ltd.

  19. Outer capsid proteins induce the formation of pores in epithelial cells

    Ruiz, M; Abad M; Michelangely, F; Charpilienne, A; Cohen, J

    1995-01-01

    Two mechanisms of entrance in cell of the rotavirus, during the infection, were proposed: a direct entrance through the plasmatic membrane or by means of endocytosis. In the two cases, a permeabilization mechanism of the membrane (cellular or of the endocytic vesicle, respectively) should occur. It has been shown that the rotavirus induces permeabilization of liposomes and of membrane vesicles. In this work, are studied the changes of intact cells permeability, measuring the entrance of e tide bromides. Viral particles of double capsid of the RF stump produce an increase of the cells membrane MA104 permeability, while the simple capsid ones don't induce effect. This phenomenon requires the particles trypsinization, and occurs in a means where the concentration of free Ca is lower to 1 micromolar. The temporary course of the fluorescence increase is sigmoid. The latency, the speed and the width depend on the relationship of virus / cell, and it can be observed up to 100% of permeabilization in relation to the effect of digitonin. The pores induced in the membrane by the rotavirus are irreversible. The permeabilizer effect of the rotavirus on the membrane was observed in other cellular lines as Hela and HT29, but not in the L929 ones. These results suggest that one or more proteins of the external capsid are responsible s of the effect. These could be involved in the penetration process of the virus towards the cytoplasm and could be one of the restrictive factor of the cell infection by means of the virus [es

  20. Peptidoglycan-associated outer membrane protein Mep45 of rumen anaerobe Selenomonas ruminantium forms a non-specific diffusion pore via its C-terminal transmembrane domain.

    Kojima, Seiji; Hayashi, Kanako; Tochigi, Saeko; Kusano, Tomonobu; Kaneko, Jun; Kamio, Yoshiyuki

    2016-10-01

    The major outer membrane protein Mep45 of Selenomonas ruminantium, an anaerobic Gram-negative bacterium, comprises two distinct domains: the N-terminal S-layer homologous (SLH) domain that protrudes into the periplasm and binds to peptidoglycan, and the remaining C-terminal transmembrane domain, whose function has been unknown. Here, we solubilized and purified Mep45 and characterized its function using proteoliposomes reconstituted with Mep45. We found that Mep45 forms a nonspecific diffusion channel via its C-terminal region. The channel was permeable to solutes smaller than a molecular weight of roughly 600, and the estimated pore radius was 0.58 nm. Truncation of the SLH domain did not affect the channel property. On the basis of the fact that Mep45 is the most abundant outer membrane protein in S. ruminantium, we conclude that Mep45 serves as a main pathway through which small solutes diffuse across the outer membrane of this bacterium.

  1. Shewanella putrefaciens mtrB encodes an outer membrane protein required for Fe(III) and Mn(IV) reduction.

    Beliaev, A S; Saffarini, D A

    1998-12-01

    Iron and manganese oxides or oxyhydroxides are abundant transition metals, and in aquatic environments they serve as terminal electron acceptors for a large number of bacterial species. The molecular mechanisms of anaerobic metal reduction, however, are not understood. Shewanella putrefaciens is a facultative anaerobe that uses Fe(III) and Mn(IV) as terminal electron acceptors during anaerobic respiration. Transposon mutagenesis was used to generate mutants of S. putrefaciens, and one such mutant, SR-21, was analyzed in detail. Growth and enzyme assays indicated that the mutation in SR-21 resulted in loss of Fe(III) and Mn(IV) reduction but did not affect its ability to reduce other electron acceptors used by the wild type. This deficiency was due to Tn5 inactivation of an open reading frame (ORF) designated mtrB. mtrB encodes a protein of 679 amino acids and contains a signal sequence characteristic of secreted proteins. Analysis of membrane fractions of the mutant, SR-21, and wild-type cells indicated that MtrB is located on the outer membrane of S. putrefaciens. A 5.2-kb DNA fragment that contains mtrB was isolated and completely sequenced. A second ORF, designated mtrA, was found directly upstream of mtrB. The two ORFs appear to be arranged in an operon. mtrA encodes a putative 10-heme c-type cytochrome of 333 amino acids. The N-terminal sequence of MtrA contains a potential signal sequence for secretion across the cell membrane. The amino acid sequence of MtrA exhibited 34% identity to NrfB from Escherichia coli, which is involved in formate-dependent nitrite reduction. To our knowledge, this is the first report of genes encoding proteins involved in metal reduction.

  2. Outer membrane protein complex of Meningococcus enhances the antipolysaccharide antibody response to pneumococcal polysaccharide-CRM₁₉₇ conjugate vaccine.

    Lai, Zengzu; Schreiber, John R

    2011-05-01

    Bacterial polysaccharides (PS) are T cell-independent antigens that do not induce immunologic memory and are poor immunogens in infants. Conjugate vaccines in which the PS is covalently linked to a carrier protein have enhanced immunogenicity that resembles that of T cell-dependent antigens. The Haemophilus influenzae type b (Hib) conjugate vaccine, which uses the outer membrane protein complex (OMPC) from meningococcus as a carrier protein, elicits protective levels of anti-capsular PS antibody (Ab) after a single dose, in contrast to other conjugate vaccines, which require multiple doses. We have previously shown that OMPC robustly engages Toll-like receptor 2 (TLR2) and enhances the early anti-Hib PS Ab titer associated with an increase in TLR2-mediated induction of cytokines. We now show that the addition of OMPC to the 7-valent pneumococcal PS-CRM₁₉₇ conjugate vaccine during immunization significantly increases the anti-PS IgG and IgM responses to most serotypes of pneumococcus contained in the vaccine. The addition of OMPC also increased the likelihood of anti-PS IgG3 production against serotypes 4, 6B, 9V, 18C, 19F, and 23F. Splenocytes from mice who had received OMPC with the pneumococcal conjugate vaccine produced significantly more interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) than splenocytes from mice who received phosphate-buffered saline (PBS) plus the conjugate vaccine. We conclude that OMPC enhances the anti-PS Ab response to pneumococcal PS-CRM₁₉₇ conjugate vaccine, an effect associated with a distinct change in cytokine profile. It may be possible to reduce the number of conjugate vaccine doses required to achieve protective Ab levels by priming with adjuvants that are TLR2 ligands.

  3. Expression of the recombinant bacterial outer surface protein A in tobacco chloroplasts leads to thylakoid localization and loss of photosynthesis.

    Hennig, Anna; Bonfig, Katharina; Roitsch, Thomas; Warzecha, Heribert

    2007-11-01

    Bacterial lipoproteins play crucial roles in host-pathogen interactions and pathogenesis and are important targets for the immune system. A prominent example is the outer surface protein A (OspA) of Borrelia burgdorferi, which has been efficiently used as a vaccine for the prevention of Lyme disease. In a previous study, OspA could be produced in tobacco chloroplasts in a lipidated and immunogenic form. To further explore the potential of chloroplasts for the production of bacterial lipoproteins, the role of the N-terminal leader sequence was investigated. The amount of recombinant OspA could be increased up to ten-fold by the variation of the insertion site in the chloroplast genome. Analysis of OspA mutants revealed that replacement of the invariant cysteine residue as well as deletion of the leader sequence abolishes palmitolyation of OspA. Also, decoration of OspA with an N-terminal eukaryotic lipidation motif does not lead to palmitoylation in chloroplasts. Strikingly, the bacterial signal peptide of OspA efficiently targets the protein to thylakoids, and causes a mutant phenotype. Plants accumulating OspA at 10% total soluble protein could not grow without exogenously supplied sugars and rapidly died after transfer to soil under greenhouse conditions. The plants were found to be strongly affected in photosystem II, as revealed by the analyses of temporal and spatial dynamics of photosynthetic activity by chlorophyll fluorescence imaging. Thus, overexpression of OspA in chloroplasts is limited by its concentration-dependent interference with essential functions of chloroplastic membranes required for primary metabolism.

  4. Outer membrane protein changes during bacteroid development are independent of nitrogen fixation and differ between indeterminate and determinate nodulating host plants of Rhizobium leguminosarum

    Roest, H.P.; Goosen-de Roo, L.; Wijffelman, C.A.; Maagd, de R.A.; Lugtenberg, B.J.J.

    1995-01-01

    The outer membrane of bacteroids contains largely decreased levels of protein antigen groups II and III in comparison with that of free-living rhizobia (R. A. de Maagd, R. de Rijk, I. H. M. Mulders, and B. J, J. Lugtenberg, J.Bacteriol, 171:1136-1142, 1989). Since we intend to study the molecular

  5. Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

    Gentry-Weeks, C R; Hultsch, A L; Kelly, S M; Keith, J M; Curtiss, R

    1992-01-01

    Three gene libraries of Bordetella avium 197 DNA were prepared in Escherichia coli LE392 by using the cosmid vectors pCP13 and pYA2329, a derivative of pCP13 specifying spectinomycin resistance. The cosmid libraries were screened with convalescent-phase anti-B. avium turkey sera and polyclonal rabbit antisera against B. avium 197 outer membrane proteins. One E. coli recombinant clone produced a 56-kDa protein which reacted with convalescent-phase serum from a turkey infected with B. avium 197. In addition, five E. coli recombinant clones were identified which produced B. avium outer membrane proteins with molecular masses of 21, 38, 40, 43, and 48 kDa. At least one of these E. coli clones, which encoded the 21-kDa protein, reacted with both convalescent-phase turkey sera and antibody against B. avium 197 outer membrane proteins. The gene for the 21-kDa outer membrane protein was localized by Tn5seq1 mutagenesis, and the nucleotide sequence was determined by dideoxy sequencing. DNA sequence analysis of the 21-kDa protein revealed an open reading frame of 582 bases that resulted in a predicted protein of 194 amino acids. Comparison of the predicted amino acid sequence of the gene encoding the 21-kDa outer membrane protein with protein sequences in the National Biomedical Research Foundation protein sequence data base indicated significant homology to the OmpA proteins of Shigella dysenteriae, Enterobacter aerogenes, E. coli, and Salmonella typhimurium and to Neisseria gonorrhoeae outer membrane protein III, Haemophilus influenzae protein P6, and Pseudomonas aeruginosa porin protein F. The gene (ompA) encoding the B. avium 21-kDa protein hybridized with 4.1-kb DNA fragments from EcoRI-digested, chromosomal DNA of Bordetella pertussis and Bordetella bronchiseptica and with 6.0- and 3.2-kb DNA fragments from EcoRI-digested, chromosomal DNA of B. avium and B. avium-like DNA, respectively. A 6.75-kb DNA fragment encoding the B. avium 21-kDa protein was subcloned into the

  6. Outer Membrane Protein A Conservation among Orientia tsutsugamushi Isolates Suggests Its Potential as a Protective Antigen and Diagnostic Target

    Sean M. Evans

    2018-06-01

    Full Text Available Scrub typhus threatens one billion people in the Asia-Pacific area and cases have emerged outside this region. It is caused by infection with any of the multitude of strains of the bacterium Orientia tsutsugamushi. A vaccine that affords heterologous protection and a commercially-available molecular diagnostic assay are lacking. Herein, we determined that the nucleotide and translated amino acid sequences of outer membrane protein A (OmpA are highly conserved among 51 O. tsutsugamushi isolates. Molecular modeling revealed the predicted tertiary structure of O. tsutsugamushi OmpA to be very similar to that of the phylogenetically-related pathogen, Anaplasma phagocytophilum, including the location of a helix that contains residues functionally essential for A. phagocytophilum infection. PCR primers were developed that amplified ompA DNA from all O. tsutsugamushi strains, but not from negative control bacteria. Using these primers in quantitative PCR enabled sensitive detection and quantitation of O. tsutsugamushi ompA DNA from organs and blood of mice that had been experimentally infected with the Karp or Gilliam strains. The high degree of OmpA conservation among O. tsutsugamushi strains evidences its potential to serve as a molecular diagnostic target and justifies its consideration as a candidate for developing a broadly-protective scrub typhus vaccine.

  7. Role of Tim50 in the transfer of precursor proteins from the outer to the inner membrane of mitochondria.

    Mokranjac, Dejana; Sichting, Martin; Popov-Celeketić, Dusan; Mapa, Koyeli; Gevorkyan-Airapetov, Lada; Zohary, Keren; Hell, Kai; Azem, Abdussalam; Neupert, Walter

    2009-03-01

    Transport of essentially all matrix and a number of inner membrane proteins is governed, entirely or in part, by N-terminal presequences and requires a coordinated action of the translocases of outer and inner mitochondrial membranes (TOM and TIM23 complexes). Here, we have analyzed Tim50, a subunit of the TIM23 complex that is implicated in transfer of precursors from TOM to TIM23. Tim50 is recruited to the TIM23 complex via Tim23 in an interaction that is essentially independent of the rest of the translocase. We find Tim50 in close proximity to the intermembrane space side of the TOM complex where it recognizes both types of TIM23 substrates, those that are to be transported into the matrix and those destined to the inner membrane, suggesting that Tim50 recognizes presequences. This function of Tim50 depends on its association with TIM23. We conclude that the efficient transfer of precursors between TOM and TIM23 complexes requires the concerted action of Tim50 with Tim23.

  8. Relationship of Triamine-Biocide Tolerance of Salmonella enterica Serovar Senftenberg to Antimicrobial Susceptibility, Serum Resistance and Outer Membrane Proteins.

    Futoma-Kołoch, Bożena; Dudek, Bartłomiej; Kapczyńska, Katarzyna; Krzyżewska, Eva; Wańczyk, Martyna; Korzekwa, Kamila; Rybka, Jacek; Klausa, Elżbieta; Bugla-Płoskońska, Gabriela

    2017-07-11

    A new emerging phenomenon is the association between the incorrect use of biocides in the process of disinfection in farms and the emergence of cross-resistance in Salmonella populations. Adaptation of the microorganisms to the sub-inhibitory concentrations of the disinfectants is not clear, but may result in an increase of sensitivity or resistance to antibiotics, depending on the biocide used and the challenged Salmonella serovar. Exposure of five Salmonella enterica subsp. enterica serovar Senftenberg ( S. Senftenberg) strains to triamine-containing disinfectant did not result in variants with resistance to antibiotics, but has changed their susceptibility to normal human serum (NHS). Three biocide variants developed reduced sensitivity to NHS in comparison to the sensitive parental strains, while two isolates lost their resistance to serum. For S. Senftenberg, which exhibited the highest triamine tolerance (6 × MIC) and intrinsic sensitivity to 22.5% and 45% NHS, a downregulation of flagellin and enolase has been demonstrated, which might suggest a lower adhesion and virulence of the bacteria. This is the first report demonstrating the influence of biocide tolerance on NHS resistance. In conclusion, there was a potential in S. Senftenberg to adjust to the conditions, where the biocide containing triamine was present. However, the adaptation did not result in the increase of antibiotic resistance, but manifested in changes within outer membrane proteins' patterns. The strategy of bacterial membrane proteins' analysis provides an opportunity to adjust the ways of infection treatments, especially when it is connected to the life-threating bacteremia caused by Salmonella species.

  9. Inhibition of K+ transport through Na+, K+-ATPase by capsazepine: role of membrane span 10 of the α-subunit in the modulation of ion gating.

    Mahmmoud, Yasser A; Shattock, Michael; Cornelius, Flemming; Pavlovic, Davor

    2014-01-01

    Capsazepine (CPZ) inhibits Na+,K+-ATPase-mediated K+-dependent ATP hydrolysis with no effect on Na+-ATPase activity. In this study we have investigated the functional effects of CPZ on Na+,K+-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na+,K+-ATPase. In isolated rat cardiomyocytes, CPZ abolished Na+,K+-ATPase current in the presence of extracellular K+. In contrast, CPZ stimulated pump current in the absence of extracellular K+. Similar conclusions were attained using HEK293 cells loaded with the Na+ sensitive dye Asante NaTRIUM green. Proteolytic cleavage of pig kidney Na+,K+-ATPase indicated that CPZ stabilizes ion interaction with the K+ sites. The distal part of membrane span 10 (M10) of the α-subunit was exposed to trypsin cleavage in the presence of guanidinum ions, which function as Na+ congener at the Na+ specific site. This effect of guanidinium was amplified by treatment with CPZ. Fluorescence of the membrane potential sensitive dye, oxonol VI, was measured following addition of substrates to reconstituted inside-out Na+,K+-ATPase. CPZ increased oxonol VI fluorescence in the absence of K+, reflecting increased Na+ efflux through the pump. Surprisingly, CPZ induced an ATP-independent increase in fluorescence in the presence of high extravesicular K+, likely indicating opening of an intracellular pathway selective for K+. As revealed by the recent crystal structure of the E1.AlF4-.ADP.3Na+ form of the pig kidney Na+,K+-ATPase, movements of M5 of the α-subunit, which regulate ion selectivity, are controlled by the C-terminal tail that extends from M10. We propose that movements of M10 and its cytoplasmic extension is affected by CPZ, thereby regulating ion selectivity and transport through the K+ sites in Na+,K+-ATPase.

  10. Inhibition of K+ transport through Na+, K+-ATPase by capsazepine: role of membrane span 10 of the α-subunit in the modulation of ion gating.

    Yasser A Mahmmoud

    Full Text Available Capsazepine (CPZ inhibits Na+,K+-ATPase-mediated K+-dependent ATP hydrolysis with no effect on Na+-ATPase activity. In this study we have investigated the functional effects of CPZ on Na+,K+-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na+,K+-ATPase. In isolated rat cardiomyocytes, CPZ abolished Na+,K+-ATPase current in the presence of extracellular K+. In contrast, CPZ stimulated pump current in the absence of extracellular K+. Similar conclusions were attained using HEK293 cells loaded with the Na+ sensitive dye Asante NaTRIUM green. Proteolytic cleavage of pig kidney Na+,K+-ATPase indicated that CPZ stabilizes ion interaction with the K+ sites. The distal part of membrane span 10 (M10 of the α-subunit was exposed to trypsin cleavage in the presence of guanidinum ions, which function as Na+ congener at the Na+ specific site. This effect of guanidinium was amplified by treatment with CPZ. Fluorescence of the membrane potential sensitive dye, oxonol VI, was measured following addition of substrates to reconstituted inside-out Na+,K+-ATPase. CPZ increased oxonol VI fluorescence in the absence of K+, reflecting increased Na+ efflux through the pump. Surprisingly, CPZ induced an ATP-independent increase in fluorescence in the presence of high extravesicular K+, likely indicating opening of an intracellular pathway selective for K+. As revealed by the recent crystal structure of the E1.AlF4-.ADP.3Na+ form of the pig kidney Na+,K+-ATPase, movements of M5 of the α-subunit, which regulate ion selectivity, are controlled by the C-terminal tail that extends from M10. We propose that movements of M10 and its cytoplasmic extension is affected by CPZ, thereby regulating ion selectivity and transport through the K+ sites in Na+,K+-ATPase.

  11. Leptospiral outer membrane protein LipL41 is not essential for acute leptospirosis but requires a small chaperone protein, lep, for stable expression.

    King, Amy M; Bartpho, Thanatchaporn; Sermswan, Rasana W; Bulach, Dieter M; Eshghi, Azad; Picardeau, Mathieu; Adler, Ben; Murray, Gerald L

    2013-08-01

    Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira spp., but knowledge of leptospiral pathogenesis remains limited. However, the development of mutagenesis systems has allowed the investigation of putative virulence factors and their involvement in leptospirosis. LipL41 is the third most abundant lipoprotein found in the outer membranes of pathogenic leptospires and has been considered a putative virulence factor. LipL41 is encoded on the large chromosome 28 bp upstream of a small open reading frame encoding a hypothetical protein of unknown function. This gene was named lep, for LipL41 expression partner. In this study, lipL41 was found to be cotranscribed with lep. Two transposon mutants were characterized: a lipL41 mutant and a lep mutant. In the lep mutant, LipL41 protein levels were reduced by approximately 90%. Lep was shown through cross-linking and coexpression experiments to bind to LipL41. Lep is proposed to be a molecular chaperone essential for the stable expression of LipL41. The roles of LipL41 and Lep in the pathogenesis of Leptospira interrogans were investigated; surprisingly, neither of these two unique proteins was essential for acute leptospirosis.

  12. FmvB: A Francisella tularensis Magnesium-Responsive Outer Membrane Protein that Plays a Role in Virulence.

    Xiaojun Wu

    Full Text Available Francisella tularensis is the causative agent of the lethal disease tularemia. Despite decades of research, little is understood about why F. tularensis is so virulent. Bacterial outer membrane proteins (OMPs are involved in various virulence processes, including protein secretion, host cell attachment, and intracellular survival. Many pathogenic bacteria require metals for intracellular survival and OMPs often play important roles in metal uptake. Previous studies identified three F. tularensis OMPs that play roles in iron acquisition. In this study, we examined two previously uncharacterized proteins, FTT0267 (named fmvA, for Francisella metal and virulence and FTT0602c (fmvB, which are homologs of the previously studied F. tularensis iron acquisition genes and are predicted OMPs. To study the potential roles of FmvA and FmvB in metal acquisition and virulence, we first examined fmvA and fmvB expression following pulmonary infection of mice, finding that fmvB was upregulated up to 5-fold during F. tularensis infection of mice. Despite sequence homology to previously-characterized iron-acquisition genes, FmvA and FmvB do not appear to be involved iron uptake, as neither fmvA nor fmvB were upregulated in iron-limiting media and neither ΔfmvA nor ΔfmvB exhibited growth defects in iron limitation. However, when other metals were examined in this study, magnesium-limitation significantly induced fmvB expression, ΔfmvB was found to express significantly higher levels of lipopolysaccharide (LPS in magnesium-limiting medium, and increased numbers of surface protrusions were observed on ΔfmvB in magnesium-limiting medium, compared to wild-type F. tularensis grown in magnesium-limiting medium. RNA sequencing analysis of ΔfmvB revealed the potential mechanism for increased LPS expression, as LPS synthesis genes kdtA and wbtA were significantly upregulated in ΔfmvB, compared with wild-type F. tularensis. To provide further evidence for the potential

  13. The novel 2Fe–2S outer mitochondrial protein mitoNEET displays conformational flexibility in its N-terminal cytoplasmic tethering domain

    Conlan, Andrea R.; Paddock, Mark L.; Axelrod, Herbert L.; Cohen, Aina E.; Abresch, Edward C.; Wiley, Sandra; Roy, Melinda; Nechushtai, Rachel; Jennings, Patricia A.

    2009-01-01

    The crystal structure of the anti-diabetic drug target mitoNEET obtained from a GFP fusion construct (1.4 Å resolution, R factor = 20.2%) shows that the CDGSH 2Fe–2S binding domains are superimposable with previously determined non-fused constructs. However, there is considerable flexibility in the position of the outer mitochondrial tethering arms resulting in two different conformations in the crystal structure. A primary role for mitochondrial dysfunction is indicated in the pathogenesis of insulin resistance. A widely used drug for the treatment of type 2 diabetes is pioglitazone, a member of the thiazolidinedione class of molecules. MitoNEET, a 2Fe–2S outer mitochondrial membrane protein, binds pioglitazone [Colca et al. (2004 ▶), Am. J. Physiol. Endocrinol. Metab.286, E252–E260]. The soluble domain of the human mitoNEET protein has been expressed C-terminal to the superfolder green fluorescent protein and the mitoNEET protein has been isolated. Comparison of the crystal structure of mitoNEET isolated from cleavage of the fusion protein (1.4 Å resolution, R factor = 20.2%) with other solved structures shows that the CDGSH domains are superimposable, indicating proper assembly of mitoNEET. Furthermore, there is considerable flexibility in the position of the cytoplasmic tethering arms, resulting in two different conformations in the crystal structure. This flexibility affords multiple orientations on the outer mitochondrial membrane

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

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

    2014-01-01

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

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

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

    2014-04-11

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

  16. Helicobacter pylori Outer Membrane Protein 18 (Hp1125 Is Involved in Persistent Colonization by Evading Interferon-γ Signaling

    Yuqun Shan

    2015-01-01

    Full Text Available Outer membrane proteins (OMPs can induce an immune response. Omp18 (HP1125 of H. pylori is a powerful antigen that can induce significant interferon-γ (IFN-γ levels. Previous studies have suggested that IFN-γ plays an important role in H. pylori clearance. However, H. pylori has multiple mechanisms to avoid host immune surveillance for persistent colonization. We generated an omp18 mutant (H. pylori 26695 and H. pylori SS1 strain to examine whether Omp18 interacts with IFN-γ and is involved in H. pylori colonization. qRT-PCR revealed that IFN-γ induced Omp18 expression. qRT-PCR and western blot analysis revealed reduced expressions of virulence factors CagA and NapA in H. pylori 26695 with IFN-γ treatment, but they were induced in the Δomp18 strain. In C57BL/6 mice infected with H. pylori SS1 and the Δomp18 strain, the Δomp18 strain conferred defective colonization and activated a stronger inflammatory response. Signal transducer phosphorylation and transcription 1 (STAT1 activator was downregulated by the wild-type strain but not the Δomp18 strain in IFN-γ-treated macrophages. Furthermore, Δomp18 strain survival rates were poor in macrophages compared to the wild-type strain. We concluded that H. pylori Omp18 has an important function influencing IFN-γ-mediated immune response to participate in persistent colonization.

  17. Outer magnetosphere

    Schardt, A.W.; Behannon, K.W.; Lepping, R.P.; Carbary, J.F.; Eviatar, A.; Siscoe, G.L.

    1984-01-01

    Similarities between the Saturnian and terrestrial outer magnetosphere are examined. Saturn, like earth, has a fully developed magnetic tail, 80 to 100 RS in diameter. One major difference between the two outer magnetospheres is the hydrogen and nitrogen torus produced by Titan. This plasma is, in general, convected in the corotation direction at nearly the rigid corotation speed. Energies of magnetospheric particles extend to above 500 keV. In contrast, interplanetary protons and ions above 2 MeV have free access to the outer magnetosphere to distances well below the Stormer cutoff. This access presumably occurs through the magnetotail. In addition to the H+, H2+, and H3+ ions primarily of local origin, energetic He, C, N, and O ions are found with solar composition. Their flux can be substantially enhanced over that of interplanetary ions at energies of 0.2 to 0.4 MeV/nuc

  18. The pro-apoptotic BH3-only protein Bim interacts with components of the translocase of the outer mitochondrial membrane (TOM).

    Frank, Daniel O; Dengjel, Jörn; Wilfling, Florian; Kozjak-Pavlovic, Vera; Häcker, Georg; Weber, Arnim

    2015-01-01

    The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.

  19. The pro-apoptotic BH3-only protein Bim interacts with components of the translocase of the outer mitochondrial membrane (TOM.

    Daniel O Frank

    Full Text Available The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM. In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20 by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.

  20. Antibody and Cytokine Responses of Koalas (Phascolarctos cinereus) Vaccinated with Recombinant Chlamydial Major Outer Membrane Protein (MOMP) with Two Different Adjuvants

    Khan, Shahneaz Ali; Desclozeaux, Marion; Waugh, Courtney; Hanger, Jon; Loader, Jo; Gerdts, Volker; Potter, Andrew; Polkinghorne, Adam; Beagley, Kenneth; Timms, Peter

    2016-01-01

    Developing a vaccine against Chlamydia is key to combating widespread mortalities and morbidities associated with this infection in koalas (Phascolarctos cinereus). In previous studies, we have shown that two or three doses of a Recombinant Major Outer Membrane Protein (rMOMP) antigen-based vaccine, combined with immune stimulating complex (ISC) adjuvant, results in strong cellular and humoral immune responses in koalas. We have also separately evaluated a single dose vaccine, utilising a tri...

  1. Disease association with two Helicobacter pylori duplicate outer membrane protein genes, homB and homA.

    Oleastro, Monica; Cordeiro, Rita; Yamaoka, Yoshio; Queiroz, Dulciene; Mégraud, Francis; Monteiro, Lurdes; Ménard, Armelle

    2009-06-22

    homB encodes a Helicobacter pylori outer membrane protein. This gene was previously associated with peptic ulcer disease (PUD) and was shown to induce activation of interleukin-8 secretion in vitro, as well as contributing to bacterial adherence. Its 90%-similar gene, homA, was previously correlated with gastritis. The present study aimed to evaluate the gastric disease association with homB and homA, as well as with the H. pylori virulence factors cagA, babA and vacA, in 415 H. pylori strains isolated from patients from East Asian and Western countries. The correlation among these genotypes was also evaluated. Both homB and homA genes were heterogeneously distributed worldwide, with a marked difference between East Asian and Western strains. In Western strains (n = 234, 124 PUD and 110 non-ulcer dyspepsia (NUD), homB, cagA and vacA s1 were all significantly associated with PUD (p = 0.025, p = 0.014, p = 0.039, respectively), and homA was closely correlated with NUD (p = 0.072). In East Asian strains (n = 138, 73 PUD and 65 NUD), homB was found more frequently than homA, and none of these genes was associated with the clinical outcome. Overall, homB was associated with the presence of cagA (p = 0.043) and vacA s1 (p homA was found more frequently in cagA-negative (p = 0.062) and vacA s2 (p homA copy number were observed, with a clear geographical specificity, suggesting an involvement of these genes in host adaptation. A correlation between the homB two-copy genotype and PUD was also observed, emphasizing the role of homB in the virulence of the strain. The global results suggest that homB and homA contribute to the determination of clinical outcome.

  2. Anal lymphogranuloma venereum infection screening with IgA anti-Chlamydia trachomatis-specific major outer membrane protein serology.

    de Vries, Henry J C; Smelov, Vitaly; Ouburg, Sander; Pleijster, Jolein; Geskus, Ronald B; Speksnijder, Arjen G C L; Fennema, Johannes S A; Morré, Servaas A

    2010-12-01

    Anal lymphogranuloma venereum (LGV) infections, caused by Chlamydia trachomatis biovar L (Ct+/LGV+), are endemic among men who have sex with men (MSM). Anal non-LGV biovar Ct infections (Ct+/LGV-) can be eradicated with 1 week doxycycline, whereas Ct+/LGV+ infections require 3-week doxycycline. To differentiate Ct+/LGV+ from Ct+/LGV- infections, biovar-specific Nucleic Acid Amplification Test (NAAT) are standard, but also expensive and laborious. A chlamydia-specific serological assay could serve as an alternative test. MSM were screened for anal Ct+/LGV+ and Ct+/LGV- infections with a commercial nonspecific NAAT and an in house biovar L-specific NAAT. Serum samples were evaluated with chlamydia-specific anti-Major Outer Membrane Protein (MOMP) and antilipopolysaccharide assays of IgA and IgG classes. Asymptomatic patients were identified as: (1) no anal complaints or (2) no microscopic inflammation (i.e., <10 leucocytes per high power field in anal smears). The best differentiating assay was subsequently evaluated in 100 Ct+/LGV+ and 100 Ct+/LGV- MSM using different cut-off points. The anti-MOMP IgA assay was the most accurate to differentiate Ct+/LGV+ (n = 42) from Ct+/LGV- (n = 19) with 85.7% sensitivity (95% confidence interval [CI], 72.2-93.3) and 84.2% specificity (95% CI, 62.4-94.5), even among asymptomatic patients. In a population comprising 98 Ct+/LGV+ and 105 Ct+/LGV- patients, the anti-MOMP IgA assay scored most accurate when the cut-off point was set to 2.0 with 75.5% (95% CI, 65.8-83.6) sensitivity and 74.3% (95% CI, 64.8-82.3) specificity. The IgA anti-MOMP assay can identify a considerable proportion of the (asymptomatic) anal LGV infections correctly. Yet, biovar L-specific NAAT are still the preferred diagnostic tests in clinical settings.

  3. Sorting of an integral outer membrane protein via the lipoprotein-specific Lol pathway and a dedicated lipoprotein pilotin.

    Collin, Séverine; Guilvout, Ingrid; Nickerson, Nicholas N; Pugsley, Anthony P

    2011-05-01

    The lipoprotein PulS is a dedicated chaperone that is required to target the secretin PulD to the outer membrane in Klebsiella or Escherichia coli, and to protect it from proteolysis. Here, we present indirect evidence that PulD protomers do not assemble into the secretin dodecamer before they reach the outer membrane, and that PulS reaches the outer membrane in a soluble heterodimer with the general lipoprotein chaperone LolA. However, we could not find any direct evidence for PulD protomer association with the PulS-LolA heterodimer. Instead, in cells producing PulD and a permanently locked PulS-LolA dimer (in which LolA carries an R43L substitution that prevents lipoprotein transfer to LolB in the outer membrane), LolAR43L was found in the inner membrane, probably still associated with PulS bound to PulD that had been incorrectly targeted because of the LolAR43L substitution. It is speculated that PulD protomers normally cross the periplasm together with PulS bound to LolA but when the latter cannot be separated (due to the mutation in lolA), the PulD protomers form dodecamers that insert into the inner membrane. © 2011 Blackwell Publishing Ltd.

  4. A comparison of the endotoxin biosynthesis and protein oxidation pathways in the biogenesis of the outer membrane of Escherichia coli and Neisseria meningitidis

    Susannah ePiek

    2012-12-01

    Full Text Available The Gram-negative bacterial cell envelope consists of an inner membrane (IM that surrounds the cytoplasm, and an asymmetrical outer-membrane (OM that forms a protective barrier to the external environment. The OM consists of lipopolysaccahride (LPS, phospholipids, outer membrane proteins (OMPs and lipoproteins. Oxidative protein folding mediated by periplasmic oxidoreductases is required for the correct biogenesis of the protein components, mainly constituents of virulence determinants such as pili, flagella and toxins, of the Gram-negative OM. Recently, periplasmic oxidoreductases have been implicated in LPS biogenesis of Escherichia coli and Neisseria meningitidis. Differences in OM biogenesis, in particular the transport pathways for endotoxin to the OM, the composition and role of the protein oxidation and isomerisation pathways and the regulatory networks that control them have been found in these two Gram-negative species suggesting that although form and function of the OM is conserved, these conserved pathways have been modified to suit the lifestyle of each organism.

  5. Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bond-dependent calcium-binding capability

    Dai, Shuyan; Sun, Cancan; Tan, Kemin; Ye, Sheng; Zhang, Rongguang

    2017-09-01

    Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca2+) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca2+-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer in gram-negative bacteria. Here, we report the crystal structure of the periplasmic region of OmpA from Capnocytophaga gingivalis, which contains a linker region comprising five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well-ordered architecture organized around two tightly-coordinated Ca2+ and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca2+-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds, which help to fix a conserved glycine residue at its proper position for Ca2+ coordination. Additionally, despite lacking inter repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.

  6. Expression of the major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis in Escherichia coli using an arabinose-inducible plasmid vector.

    Hoelzle, L E; Hoelzle, K; Wittenbrink, M M

    2003-10-01

    The ompA genes encoding the 40 kDa major outer membrane protein (MOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were cloned into the arabinose-inducible plasmid vector pBADMycHis, and recombinant MOMPs (rMOMP) from the three chlamydial species were expressed at high levels in Escherichia (E.) coli. The proteins lacking the 22 aa N-terminal signal peptide were expressed as insoluble cytoplasmic inclusion bodies which were readily purified using immobilized metal-affinity chromatography. The rMOMPs including the N-terminal signal peptide were expressed and translocated as a surface-exposed immunoaccessible protein into the outer membrane of E. coli. Transformants expressing this full-length rMOMP were significantly reduced in viability. Purified native elementary bodies (EB) and rMOMPs of the three chlamydial species purified from the E. coli cytoplasm were used for immunization of rabbits. The resulting sera were analysed for their ability to recognize homologous and heterologous rMOMP and native EB. When testing rMOMP antisera against rMOMP and EB antigens, marked cross-reactivities were detected between the three species. Using EB antisera and rMOMPs as antigens, a significant species-specific reactivity was measured.

  7. A Western-blot assay for the detection of antibodies against pathogenic Leptospira serogroups with recombinant outer membrane protein LipL32

    Hong-yuan DUAN; Zhi-guo LIU; Shao-fu QIU; Bin HE; Hai ZHAO; Li-hua SONG; Hong ZHU; Qing DUAN

    2011-01-01

    Objective To provide a possible antigen for rapid serodiagnosis of leptospirosis,the present study focused on the activity of immune-reaction and cross-reaction between outer membrane protein LipL32 and multi-serogroup anti-pathogenic Leptospira antibodies.Methods Based on the given sequence of LipL32 gene of Leptospira icterohaemorrhagiae strain 56601,the primer pair was designed and the DNA fragment was amplified by PCR.The amplified product was inserted into vector pET-28a-(c) to construct...

  8. The Xanthomonas Ax21 protein is processed by the general secretory system and is secreted in association with outer membrane vesicles

    Ofir Bahar

    2014-01-01

    Full Text Available Pattern recognition receptors (PRRs play an important role in detecting invading pathogens and mounting a robust defense response to restrict infection. In rice, one of the best characterized PRRs is XA21, a leucine rich repeat receptor-like kinase that confers broad-spectrum resistance to multiple strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo. In 2009 we reported that an Xoo protein, called Ax21, is secreted by a type I-secretion system and that it serves to activate XA21-mediated immunity. This report has recently been retracted. Here we present data that corrects our previous model. We first show that Ax21 secretion does not depend on the predicted type I secretion system and that it is processed by the general secretion (Sec system. We further show that Ax21 is an outer membrane protein, secreted in association with outer membrane vesicles. Finally, we provide data showing that ax21 knockout strains do not overcome XA21-mediated immunity.

  9. Overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of the periplasmic domain of outer membrane protein A from Acinetobacter baumannii

    Park, Jeong Soon; Lee, Woo Cheol; Choi, Saehae; Yeo, Kwon Joo; Song, Jung Hyun; Han, Young-Hyun; Lee, Je Chul; Kim, Seung Il; Jeon, Young Ho; Cheong, Chaejoon; Kim, Hye-Yeon

    2011-01-01

    The crystallization of the OmpA periplasmic domain from A. baumannii is described. Outer membrane protein A from Acinetobacter baumannii (AbOmpA) is a major outer membrane protein and a key player in the bacterial pathogenesis that induces host cell death. AbOmpA is presumed to consist of an N-terminal β-barrel transmembrane domain and a C-terminal periplasmic OmpA-like domain. In this study, the recombinant C-terminal periplasmic domain of AbOmpA was overexpressed in Escherichia coli, purified and crystallized using the vapour-diffusion method. A native diffraction data set was collected to a resolution of 2.0 Å using synchrotron radiation. The space group of the crystal was P2 1 , with unit-cell parameters a = 58.24, b = 98.59, c = 97.96 Å, β = 105.92°. The native crystal contained seven or eight molecules per asymmetric unit and had a calculated Matthews coefficient of 2.93 or 2.56 Å 3 Da −1

  10. The Major Outer Membrane Protein MopB Is Required for Twitching Movement and Affects Biofilm Formation and Virulence in Two Xylella fastidiosa strains.

    Chen, Hongyu; Kandel, Prem P; Cruz, Luisa F; Cobine, Paul A; De La Fuente, Leonardo

    2017-11-01

    MopB is a major outer membrane protein (OMP) in Xylella fastidiosa, a bacterial plant pathogen that causes losses on many economically important crops. Based on in silico analysis, the uncharacterized MopB protein of X. fastidiosa contains a β-barrel structure with an OmpA-like domain and a predicted calcium-binding motif. Here, MopB function was studied by mutational analysis taking advantage of the natural competence of X. fastidiosa. Mutants of mopB were constructed in two different X. fastidiosa strains, the type strain Temecula and the more virulent WM1-1. Deletion of the mopB gene impaired cell-to-cell aggregation, surface attachment, and biofilm formation in both strains. Interestingly, mopB deletion completely abolished twitching motility. Electron microscopy of the bacterial cell surface revealed that mopB deletion eliminated type IV and type I pili formation, potentially caused by destabilization of the outer membrane. Both mopB mutants showed reduced virulence using tobacco (Nicotiana tabacum) as a host under greenhouse conditions. These results suggest that MopB has pleiotropic functions in biofilm formation and twitching motility and is important for virulence of X. fastidiosa.

  11. An overlapping region between the two terminal folding units of the outer surface protein A (OspA) controls its folding behavior.

    Makabe, Koki; Nakamura, Takashi; Dhar, Debanjan; Ikura, Teikichi; Koide, Shohei; Kuwajima, Kunihiro

    2018-04-27

    Although many naturally occurring proteins consist of multiple domains, most studies on protein folding to date deal with single-domain proteins or isolated domains of multi-domain proteins. Studies of multi-domain protein folding are required for further advancing our understanding of protein folding mechanisms. Borrelia outer surface protein A (OspA) is a β-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer β-sheet. Thus, OspA is particularly suited as a model system for studying the interplays of domains in protein folding. Here, we studied the equilibria and kinetics of the urea-induced folding-unfolding reactions of OspA probed with tryptophan fluorescence and ultraviolet circular dichroism. Global analysis of the experimental data revealed compelling lines of evidence for accumulation of an on-pathway intermediate during kinetic refolding and for the identity between the kinetic intermediate and a previously described equilibrium unfolding intermediate. The results suggest that the intermediate has the fully native structure in the N-terminal domain and the single layer β-sheet, with the C-terminal domain still unfolded. The observation of the productive on-pathway folding intermediate clearly indicates substantial interactions between the two domains mediated by the single-layer β-sheet. We propose that a rigid and stable intervening region between two domains creates an overlap between two folding units and can energetically couple their folding reactions. Copyright © 2018. Published by Elsevier Ltd.

  12. Cloning and Characterization of an Outer Membrane Protein of Vibrio vulnificus Required for Heme Utilization: Regulation of Expression and Determination of the Gene Sequence

    Litwin, Christine M.; Byrne, Burke L.

    1998-01-01

    Vibrio vulnificus is a halophilic, marine pathogen that has been associated with septicemia and serious wound infections in patients with iron overload and preexisting liver disease. For V. vulnificus, the ability to acquire iron from the host has been shown to correlate with virulence. V. vulnificus is able to use host iron sources such as hemoglobin and heme. We previously constructed a fur mutant of V. vulnificus which constitutively expresses at least two iron-regulated outer membrane proteins, of 72 and 77 kDa. The N-terminal amino acid sequence of the 77-kDa protein purified from the V. vulnificus fur mutant had 67% homology with the first 15 amino acids of the mature protein of the Vibrio cholerae heme receptor, HutA. In this report, we describe the cloning, DNA sequence, mutagenesis, and analysis of transcriptional regulation of the structural gene for HupA, the heme receptor of V. vulnificus. DNA sequencing of hupA demonstrated a single open reading frame of 712 amino acids that was 50% identical and 66% similar to the sequence of V. cholerae HutA and similar to those of other TonB-dependent outer membrane receptors. Primer extension analysis localized one promoter for the V. vulnificus hupA gene. Analysis of the promoter region of V. vulnificus hupA showed a sequence homologous to the consensus Fur box. Northern blot analysis showed that the transcript was strongly regulated by iron. An internal deletion in the V. vulnificus hupA gene, done by using marker exchange, resulted in the loss of expression of the 77-kDa protein and the loss of the ability to use hemin or hemoglobin as a source of iron. The hupA deletion mutant of V. vulnificus will be helpful in future studies of the role of heme iron in V. vulnificus pathogenesis. PMID:9632577

  13. Use of T7 RNA polymerase to direct expression of outer Surface Protein A (OspA) from the Lyme disease Spirochete, Borrelia burgdorferi

    Dunn, John J.; Lade, Barbara N.

    1991-01-01

    The OspA gene from a North American strain of the Lyme disease Spirochete, Borrelia burgdorferi, was cloned under the control of transciption and translation signals from bacteriophage T7. Full-length OspA protein, a 273 amino acid (31kD) lipoprotein, is expressed poorly in Escherichia coli and is associated with the insoluble membrane fraction. In contrast, a truncated form of OspA lacking the amino-terminal signal sequence which normally would direct localization of the protein to the outer membrane is expressed at very high levels (less than or equal to 100 mg/liter) and is soluble. The truncated protein was purified to homogeneity and is being tested to see if it will be useful as an immunogen in a vaccine against Lyme disease. Circular dichroism and fluorescence spectroscopy was used to characterize the secondary structure and study conformational changes in the protein. Studies underway with other surface proteins from B burgdorferi and a related spirochete, B. hermsii, which causes relapsing fever, leads us to conclude that a strategy similar to that used to express the truncated OspA can provide a facile method for producing variations of Borrelia lipoproteins which are highly expressed in E. coli and soluble without exposure to detergents.

  14. The host outer membrane proteins OmpA and OmpC are associated with the Shigella phage Sf6 virion

    Zhao Haiyan; Sequeira, Reuben D.; Galeva, Nadezhda A.; Tang Liang

    2011-01-01

    Assembly of dsDNA bacteriophage is a precisely programmed process. Potential roles of host cell components in phage assembly haven't been well understood. It was previously reported that two unidentified proteins were present in bacteriophage Sf6 virion (Casjens et al, 2004, J.Mol.Biol. 339, 379-394, Fig. 2A). Using tandem mass spectrometry, we have identified the two proteins as outer membrane proteins (OMPs) OmpA and OmpC from its host Shigella flexneri. The transmission electron cryo-microscopy structure of Sf6 shows significant density at specific sites at the phage capsid inner surface. This density fit well with the characteristic beta-barrel domains of OMPs, thus may be due to the two host proteins. Locations of this density suggest a role in Sf6 morphogenesis reminiscent of phage-encoded cementing proteins. These data indicate a new, OMP-related phage:host linkage, adding to previous knowledge that some lambdoid bacteriophage genomes contain OmpC-like genes that express phage-encoded porins in the lysogenic state.

  15. Differential Permeabilization Effects of Ca2+ and Valinomycin on the Inner and Outer Mitochondrial Membranes as Revealed by Proteomics Analysis of Proteins Released from Mitochondria*S⃞

    Yamada, Akiko; Yamamoto, Takenori; Yamazaki, Naoshi; Yamashita, Kikuji; Kataoka, Masatoshi; Nagata, Toshihiko; Terada, Hiroshi; Shinohara, Yasuo

    2009-01-01

    It is well established that cytochrome c is released from mitochondria when the permeability transition (PT) of this organelle is induced by Ca2+. Our previous study showed that valinomycin also caused the release of cytochrome c from mitochondria but without inducing this PT (Shinohara, Y., Almofti, M. R., Yamamoto, T., Ishida, T., Kita, F., Kanzaki, H., Ohnishi, M., Yamashita, K., Shimizu, S., and Terada, H. (2002) Permeability transition-independent release of mitochondrial cytochrome c induced by valinomycin. Eur. J. Biochem. 269, 5224–5230). These results indicate that cytochrome c may be released from mitochondria with or without the induction of PT. In the present study, we examined the protein species released from valinomycin- and Ca2+-treated mitochondria by LC-MS/MS analysis. As a result, the proteins located in the intermembrane space were found to be specifically released from valinomycin-treated mitochondria, whereas those in the intermembrane space and in the matrix were released from Ca2+-treated mitochondria. These results were confirmed by Western analysis. Furthermore to examine how the protein release occurred, we examined the correlation between the species of released proteins and those of the abundant proteins in mitochondria. Consequently most of the proteins released from mitochondria treated with either agent were highly expressed proteins in mitochondria, indicating that the release occurred not selectively but in a manner dependent on the concentration of the proteins. Based on these results, the permeabilization effects of Ca2+ and valinomycin on the inner and outer mitochondrial membranes are discussed. PMID:19218587

  16. Cell envelope of Bordetella pertussis: immunological and biochemical analyses and characterization of a major outer membrane porin protein

    Armstrong, S.K.

    1986-01-01

    Surface molecules of Bordetella pertussis which may be important in metabolism, pathogenesis, and immunity to whooping cough were examined using cell fractionation and 125 I cell surface labeling. Antigenic envelope proteins were examined by immunofluorescence microscopy and Western blotting procedures using monoclonal antibodies and convalescent sera. A surface protein with a high M/sub r/, missing in a mutant lacking the filamentous hemagglutinin, was identified in virulent Bordetella pertussis but was absent in virulent B. pertussis strains. At least three envelope proteins were found only in virulent B. pertussis strains and were absent or diminished in avirulent and most phenotypically modulated strains. Transposon-induced mutants unable to produce hemolysin, dermonecrotic toxin, pertussis toxin, and filamentous hemagglutinin also lacked these three envelope proteins, confirming that virulence-associated envelope proteins were genetically regulated with other virulence-associated traits. Two dimensional gel electrophoresis revealed at least five heat modifiable proteins which migrated as higher or lower M/sub r/ moieties if solubilized at 25 0 C instead of 100 0 C

  17. Structure analysis of OmpC, one of the major proteins in the outer membrane of E. coli, by high resolution electron microscopy

    Chang, C.F.

    1983-07-01

    This dissertation is concerned with the structure analysis of a pore-forming membrane protein, OmpC, which is one of the major proteins in the outer membrane of Escherichia coli. In order to obtain structural information it was necessary to develop a suitable technique for preparing two-dimensional crystalline arrays of this membrane protein in an unfixed, unstained and hydrated condition. Electron micrographs were recorded at exposures of less than 5 electrons/A 2 in order to avoid severe radiation damage. The resulting images were crystallographically averaged, in order to overcome the statistical limitations associated with the low electron exposures. The resulting images, which extend to a resolution of approx. 13.5 A, lend themselves to a natural interpretation that is consistent with the mass density of protein, water and lipid, prior data from 2-D and 3-D structure studies of negatively stained specimens at approx. = 20 A resolution, and published spectroscopic data on the peptide chain secondary structure

  18. In-vivo identification of direct electron transfer from Shewanella oneidensis MR-1 to electrodes via outer-membrane OmcA-MtrCAB protein complexes

    Okamoto, Akihiro [Department of Applied Chemistry, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Nakamura, Ryuhei, E-mail: nakamura@light.t.u-tokyo.ac.jp [Department of Applied Chemistry, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Hashimoto, Kazuhito, E-mail: hashimoto@light.t.u-tokyo.ac.jp [Department of Applied Chemistry, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); ERATO/JST, HASHIMOTO Light Energy Conversion Project (Japan)

    2011-06-30

    Graphical abstract: . Display Omitted Highlights: > Monolayer biofilm of Shewanella cells was prepared on an ITO electrode. > Extracellular electron transfer (EET) process was examined with series of mutants. > Direct ET was confirmed with outer-membrane-bound OmcA-MtrCAB complex. > The EET process was not prominently influenced by capsular polysaccharide. - Abstract: The direct electron-transfer (DET) property of Shewanella bacteria has not been resolved in detail due to the complexity of in vivo electrochemistry in whole-cell systems. Here, we report the in vivo assignment of the redox signal indicative of the DET property in biofilms of Shewanella oneidensis MR-1 by cyclic voltammetry (CV) with a series of mutants and a chemical marking technique. The CV measurements of monolayer biofilms formed by deletion mutants of c-type cytochromes ({Delta}mtrA, {Delta}mtrB, {Delta}mtrC/{Delta}omcA, and {Delta}cymA), and pilin ({Delta}pilD), capsular polysaccharide ({Delta}SO3177) and menaquinone ({Delta}menD) biosynthetic proteins demonstrated that the electrochemical redox signal with a midpoint potential at 50 mV (vs. SHE) was due to an outer-membrane-bound OmcA-MtrCAB protein complex of decaheme cytochromes, and did not involve either inner-membrane-bound CymA protein or secreted menaquinone. Using the specific binding affinity of nitric monoxide for the heme groups of c-type cytochromes, we further confirmed this conclusion. The heterogeneous standard rate constant for the DET process was estimated to be 300 {+-} 10 s{sup -1}, which was two orders of magnitude higher than that previously reported for the electron shuttling process via riboflavin. Experiments using a mutant unable to produce capsular polysaccharide ({Delta}SO3177) revealed that the DET property of the OmcA-MtrCAB complex was not influenced by insulating and hydrophilic extracellular polysaccharide. Accordingly, under physiological conditions, S. oneidensis MR-1 utilizes a high density of outer

  19. Engineering of the E. coli outer membrane protein FhuA to overcome the hydrophobic mismatch in thick polymeric membranes.

    Muhammad, Noor; Dworeck, Tamara; Fioroni, Marco; Schwaneberg, Ulrich

    2011-03-17

    Channel proteins like the engineered FhuA Δ1-159 often cannot insert into thick polymeric membranes due to a mismatch between the hydrophobic surface of the protein and the hydrophobic surface of the polymer membrane. To address this problem usually specific block copolymers are synthesized to facilitate protein insertion. Within this study in a reverse approach we match the protein to the polymer instead of matching the polymer to the protein. To increase the FhuA Δ1-159 hydrophobic surface by 1 nm, the last 5 amino acids of each of the 22 β-sheets, prior to the more regular periplasmatic β-turns, were doubled leading to an extended FhuA Δ1-159 (FhuA Δ1-159 Ext). The secondary structure prediction and CD spectroscopy indicate the β-barrel folding of FhuA Δ1-159 Ext. The FhuA Δ1-159 Ext insertion and functionality within a nanocontainer polymeric membrane based on the triblock copolymer PIB(1000)-PEG(6000)-PIB(1000) (PIB = polyisobutylene, PEG = polyethyleneglycol) has been proven by kinetic analysis using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine). Identical experiments with the unmodified FhuA Δ1-159 report no kinetics and presumably no insertion into the PIB(1000)-PEG(6000)-PIB(1000) membrane. Furthermore labeling of the Lys-NH(2) groups present in the FhuA Δ1-159 Ext channel, leads to controllability of in/out flux of substrates and products from the nanocontainer. Using a simple "semi rational" approach the protein's hydrophobic transmembrane region was increased by 1 nm, leading to a predicted lower hydrophobic mismatch between the protein and polymer membrane, minimizing the insertion energy penalty. The strategy of adding amino acids to the FhuA Δ1-159 Ext hydrophobic part can be further expanded to increase the protein's hydrophobicity, promoting the efficient embedding into thicker/more hydrophobic block copolymer membranes.

  20. Engineering of the E. coli Outer Membrane Protein FhuA to overcome the Hydrophobic Mismatch in Thick Polymeric Membranes

    Fioroni Marco

    2011-03-01

    Full Text Available Abstract Background Channel proteins like the engineered FhuA Δ1-159 often cannot insert into thick polymeric membranes due to a mismatch between the hydrophobic surface of the protein and the hydrophobic surface of the polymer membrane. To address this problem usually specific block copolymers are synthesized to facilitate protein insertion. Within this study in a reverse approach we match the protein to the polymer instead of matching the polymer to the protein. Results To increase the FhuA Δ1-159 hydrophobic surface by 1 nm, the last 5 amino acids of each of the 22 β-sheets, prior to the more regular periplasmatic β-turns, were doubled leading to an extended FhuA Δ1-159 (FhuA Δ1-159 Ext. The secondary structure prediction and CD spectroscopy indicate the β-barrel folding of FhuA Δ1-159 Ext. The FhuA Δ1-159 Ext insertion and functionality within a nanocontainer polymeric membrane based on the triblock copolymer PIB1000-PEG6000-PIB1000 (PIB = polyisobutylene, PEG = polyethyleneglycol has been proven by kinetic analysis using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine. Identical experiments with the unmodified FhuA Δ1-159 report no kinetics and presumably no insertion into the PIB1000-PEG6000-PIB1000 membrane. Furthermore labeling of the Lys-NH2 groups present in the FhuA Δ1-159 Ext channel, leads to controllability of in/out flux of substrates and products from the nanocontainer. Conclusion Using a simple "semi rational" approach the protein's hydrophobic transmembrane region was increased by 1 nm, leading to a predicted lower hydrophobic mismatch between the protein and polymer membrane, minimizing the insertion energy penalty. The strategy of adding amino acids to the FhuA Δ1-159 Ext hydrophobic part can be further expanded to increase the protein's hydrophobicity, promoting the efficient embedding into thicker/more hydrophobic block copolymer membranes.

  1. Outer membrane protein P4 is not required for virulence in the human challenge model of Haemophilus ducreyi infection.

    Janowicz, Diane M; Zwickl, Beth W; Fortney, Kate R; Katz, Barry P; Bauer, Margaret E

    2014-06-24

    Bacterial lipoproteins often play important roles in pathogenesis and can stimulate protective immune responses. Such lipoproteins are viable vaccine candidates. Haemophilus ducreyi, which causes the sexually transmitted disease chancroid, expresses a number of lipoproteins during human infection. One such lipoprotein, OmpP4, is homologous to the outer membrane lipoprotein e (P4) of H. influenzae. In H. influenzae, e (P4) stimulates production of bactericidal and protective antibodies and contributes to pathogenesis by facilitating acquisition of the essential nutrients heme and nicotinamide adenine dinucleotide (NAD). Here, we tested the hypothesis that, like its homolog, H. ducreyi OmpP4 contributes to virulence and stimulates production of bactericidal antibodies. We determined that OmpP4 is broadly conserved among clinical isolates of H. ducreyi. We next constructed and characterized an isogenic ompP4 mutant, designated 35000HPompP4, in H. ducreyi strain 35000HP. To test whether OmpP4 was necessary for virulence in humans, eight healthy adults were experimentally infected. Each subject was inoculated with a fixed dose of 35000HP on one arm and three doses of 35000HPompP4 on the other arm. The overall parent and mutant pustule formation rates were 52.4% and 47.6%, respectively (P = 0.74). These results indicate that expression of OmpP4 in not necessary for H. ducreyi to initiate disease or progress to pustule formation in humans. Hyperimmune mouse serum raised against purified, recombinant OmpP4 did not promote bactericidal killing of 35000HP or phagocytosis by J774A.1 mouse macrophages in serum bactericidal and phagocytosis assays, respectively. Our data suggest that, unlike e (P4), H. ducreyi OmpP4 is not a suitable vaccine candidate. OmpP4 may be dispensable for virulence because of redundant mechanisms in H. ducreyi for heme acquisition and NAD utilization.

  2. Evaluation of immunogenicity and protective efficacy of recombinant outer membrane proteins of Haemophilus parasuis serovar 5 in a murine model.

    Li, Miao; Cai, Ru-Jian; Song, Shuai; Jiang, Zhi-Yong; Li, Yan; Gou, Hong-Chao; Chu, Pin-Pin; Li, Chun-Ling; Qiu, Hua-Ji

    2017-01-01

    Glässer's disease is an economically important infectious disease of pigs caused by Haemophilus parasuis. Few vaccines are currently available that could provide effective cross-protection against various serovars of H. parasuis. In this study, five OMPs (OppA, TolC, HxuC, LppC, and HAPS_0926) identified by bioinformatic approaches, were cloned and expressed as recombinant proteins. Antigenicity of the purified proteins was verified through Western blotting, and primary screening for protective potential was evaluated in vivo. Recombinant TolC (rTolC), rLppC, and rHAPS_0926 proteins showing marked protection of mice against H. parasuis infection, and were further evaluated individually or in combination. Mice treated with these three OMPs produced humoral and host cell-mediated responses, with a significant rise in antigen-specific IgG titer and lymphoproliferative response in contrast with the mock-immunized group. Significant increases were noted in CD4+, CD8+ T cells, and three cytokines (IL-2, IL-4, and IFN-γ) in vaccinated animals. The antisera against candidate antigens could efficiently impede bacterial survival in whole blood bactericidal assay against H. parasuis infection. The multi-protein vaccine induced more pronounced immune responses and offered better protection than individual vaccines. Our findings indicate that these three OMPs are promising antigens for the development of multi-component subunit vaccines against Glässer's disease.

  3. Functional assay of Salmonella typhi OmpC using reconstituted large unilamellar vesicles: a general method for characterization of outer membrane proteins.

    Sundara Baalaji, N; Mathew, M K; Krishnaswamy, S

    2006-10-01

    The immunodominant trimeric beta-barrel outer membrane protein OmpC from Salmonella typhi, the causative agent of typhoid, has been functionally characterized here. The activity in the vesicle environment was studied in vitro using OmpC reconstituted into proteoliposomes. Passage of polysaccharides and polyethyleneglycols through OmpC has been examined to determine the permeability properties. The relative rate of neutral solute flux yields a radius of 1.1 nm for the S. typhi OmpC pore. This is almost double the pore size of Escherichia coli. This provides an example of large pore size present in the porins that form trimers as in the general bacterial porin family. The method used in this study provides a good membrane model for functional studies of porins.

  4. Recombinant major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis as antigens to distinguish chlamydial species-specific antibodies in animal sera.

    Hoelzle, Ludwig E; Hoelzle, Katharina; Wittenbrink, Max M

    2004-10-05

    Recombinant major outer membrane proteins (rMOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were used as antigens to distinguish chlamydial species-specific antibodies in (i) immune sera from six rabbits and three pigs raised against native purified elementary bodies, (ii) serum samples from 25 sows vaccinated with Ch. abortus, and (iii) 40 serum samples from four heifers experimentally infected with Ch. abortus. All post-exposition sera contained chlamydial antibodies as confirmed by strong ELISA seroreactivities against the chlamydial LPS. For the rMOMP ELISA mean IgG antibody levels were at least 5.8-fold higher with the particular rMOMP homologous to the chlamydial species used for immunisation or infection than with heterologous rMOMPs (P <0.001). Preferential rMOMP ELISA reactivities of sera were confirmed by Western blotting. The results suggest that the entire chlamydial rMOMP could provide a species-specific serodiagnostic antigen.

  5. [Construction of the eukaryotic recombinant vector and expression of the outer membrane protein LipL32 gene from Leptospira serovar Lai].

    Huang, Bi; Bao, Lang; Zhong, Qi; Shang, Zheng-ling; Zhang, Hui-dong; Zhang, Ying

    2008-02-01

    To construct the eukaryotic experssion vector of LipL32 gene from Leptospira serovar Lai and express the recombinant plasmid in COS-7 cell. The LipL32 gene was amplified from Leptospira strain 017 genomic DNA by PCR and cloned into pcDNA3.1, through restriction nuclease enzyme digestion. Then the recombinant plasmid was transformed into E.coli DH5alpha. After identified by nuclease digestion, PCR and sequencing analysis, the recombinant vector was transfected into COS-7 cell with lipsome. The expression of the target gene was detected by RT-PCR and Western blot. The eukaryotic experssion vector pcDNA3.1-LipL32 was successfully constructed and stably expressed in COS-7 cell. The eukaryotic recombinant vector of outer membrane protein LipL32 gene from Leptospira serovar Lai can be expressed in mammalian cell, which provides an experimental basis for the application of the Leptospira DNA vaccine.

  6. DipA, a pore-forming protein in the outer membrane of Lyme disease spirochetes exhibits specificity for the permeation of dicarboxylates.

    Marcus Thein

    Full Text Available Lyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a single-channel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl were obtained among the 11 tested anions for oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species.

  7. Oral Vaccination with the Porcine Rotavirus VP4 Outer Capsid Protein Expressed by Lactococcus lactis Induces Specific Antibody Production

    Yi-jing Li

    2010-01-01

    Full Text Available The objective of this study to design a delivery system resistant to the gastrointestinal environment for oral vaccine against porcine rotavirus. Lactococcus lactis NZ9000 was transformed with segments of vP4 of the porcine rotavirus inserted into the pNZ8112 surface-expression vector, and a recombinant L. lactis expressing VP4 protein was constructed. An approximately 27 kDa VP4 protein was confirmed by SDS-PAGE , Western blot and immunostaining analysis. BALB/c mice were immunized orally with VP4-expression recombinant L. lactis and cellular, mucosal and systemic humoral immune responses were examined. Specific anti-VP4 secretory IgA and IgG were found in feces, ophthalmic and vaginal washes and in serum. The induced antibodies demonstrated neutralizing effects on porcine rotavirus infection on MA104 cells. Our findings suggest that oral immunization with VP4-expressing L. lactis induced both specific local and systemic humoral and cellular immune responses in mice.

  8. Expression and characterization of recombinant leptospiral outer membrane protein LipL32 from Leptospira interrogans serovar autumnalis.

    Boonsathorn, Naphatsawan; Konghom, Ganokrot; Mongkolsiri, Kaveewan; Jirapongwattana, Chanin; Balachandra, Kruavon; Naigowit, Pimjai; Sawanpanyalert, Pathom

    2009-01-01

    Leptospira interrogans serovar autumnalis, a causative agent of leptospirosis in Thailand, was isolated from a patient for DNA extraction and amplification of LipL32 gene by polymerase chain reaction (PCR). The 782 bp PCR product was obtained, which was inserted into pAE plasmid with polyhistidine (His6 tag) to construct pAE-LipL32. This recombinant plasmid was transfected into E. coli BL21 (DE3). His6-LipL32 was purified by Ni-NTA affinity chromatography. The recombinant protein was used as antigen for testing with sera from leptospirosis and syphilis patients by dot-ELISA technique. It reacted positively with leptospirosis patient sera and negatively with syphilis and healthy sera.

  9. Three-dimensional structure of Wza, the protein required for translocation of group 1 capsular polysaccharide across the outer membrane of Escherichia coli.

    Beis, Konstantinos; Collins, Richard F; Ford, Robert C; Kamis, Alhaji B; Whitfield, Chris; Naismith, James H

    2004-07-02

    Wza is a highly conserved multimeric outer membrane protein complex required for the surface expression of the serotype K30 group 1 capsular polysaccharide in Escherichia coli. Here we present the first three-dimensional structure of this type of polysaccharide exporter at a 15.5-A resolution obtained using single particle averaging on a dataset of cryo-negatively stained protein. Previous structural studies on purified Wza have revealed a homo-oligomeric ring structure that is most probably composed of eight subunits. Symmetry analysis of the three-dimensional structure combined with biochemical two- and three-dimensional crystallographic data strongly suggest that Wza is an octameric complex with a C4 quasi-rotational symmetry and is organized as a tetramer of dimeric subunits. Wza is best described as a stack of two 4-A high rings with differing diameters providing a mushroom-like aspect from the side. The larger ring has a distinctive square shape with a diameter of 115 A, whereas the smaller is almost circular with a diameter of 90 A. In the center of the complex and enclosed by the four symmetrical arms is a small elliptical cagelike cavity of approximately 40 A in diameter. The central cavity is effectively sealed at the top and bottom of the complex but has small inter-arm holes when viewed from the side. We discuss the structure of this complex and implications in the surface translocation of cell-surface polysaccharide.

  10. Antibody and Cytokine Responses of Koalas (Phascolarctos cinereus) Vaccinated with Recombinant Chlamydial Major Outer Membrane Protein (MOMP) with Two Different Adjuvants.

    Khan, Shahneaz Ali; Desclozeaux, Marion; Waugh, Courtney; Hanger, Jon; Loader, Jo; Gerdts, Volker; Potter, Andrew; Polkinghorne, Adam; Beagley, Kenneth; Timms, Peter

    2016-01-01

    Developing a vaccine against Chlamydia is key to combating widespread mortalities and morbidities associated with this infection in koalas (Phascolarctos cinereus). In previous studies, we have shown that two or three doses of a Recombinant Major Outer Membrane Protein (rMOMP) antigen-based vaccine, combined with immune stimulating complex (ISC) adjuvant, results in strong cellular and humoral immune responses in koalas. We have also separately evaluated a single dose vaccine, utilising a tri-adjuvant formula that comprises polyphosphazine based poly I: C and host defense peptides, with the same antigen. This formulation also produced strong cellular and humoral immune responses in captive koalas. In this current study, we directly compared the host immune responses of two sub-groups of wild Chlamydia negative koalas in one population vaccinated with the rMOMP protein antigen and adjuvanted with either the ISC or tri-adjuvant formula. Overall, both adjuvants produced strong Chlamydia-specific cellular (IFN-γ and IL-17A) responses in circulating PBMCs as well as MOMP-specific and functional, in vitro neutralising antibodies. While the immune responses were similar, there were adjuvant-specific immune differences between the two adjuvants, particularly in relation to the specificity of the MOMP epitope antibody responses.

  11. Antibody and Cytokine Responses of Koalas (Phascolarctos cinereus Vaccinated with Recombinant Chlamydial Major Outer Membrane Protein (MOMP with Two Different Adjuvants.

    Shahneaz Ali Khan

    Full Text Available Developing a vaccine against Chlamydia is key to combating widespread mortalities and morbidities associated with this infection in koalas (Phascolarctos cinereus. In previous studies, we have shown that two or three doses of a Recombinant Major Outer Membrane Protein (rMOMP antigen-based vaccine, combined with immune stimulating complex (ISC adjuvant, results in strong cellular and humoral immune responses in koalas. We have also separately evaluated a single dose vaccine, utilising a tri-adjuvant formula that comprises polyphosphazine based poly I: C and host defense peptides, with the same antigen. This formulation also produced strong cellular and humoral immune responses in captive koalas. In this current study, we directly compared the host immune responses of two sub-groups of wild Chlamydia negative koalas in one population vaccinated with the rMOMP protein antigen and adjuvanted with either the ISC or tri-adjuvant formula. Overall, both adjuvants produced strong Chlamydia-specific cellular (IFN-γ and IL-17A responses in circulating PBMCs as well as MOMP-specific and functional, in vitro neutralising antibodies. While the immune responses were similar, there were adjuvant-specific immune differences between the two adjuvants, particularly in relation to the specificity of the MOMP epitope antibody responses.

  12. Roles of outer capsid proteins as determinants of pathogenicity and host range restriction of avian rotaviruses in a suckling mouse model

    Mori, Yoshio; Borgan, Mohammed Ali; Takayama, Mutsuyo; Ito, Naoto; Sugiyama, Makoto; Minamoto, Nobuyuki

    2003-01-01

    We previously demonstrated that a pigeon rotavirus, PO-13, but not turkey strains Ty-3 and Ty-1 and a chicken strain, Ch-1, induced diarrhea in heterologous suckling mice. In this study, it was suggested that these avirulent strains, but not PO-13, were inactivated immediately in gastrointestinal tracts of suckling mice when they were orally inoculated. To determine which viral proteins contribute to the differences between the pathogenicitiy and the inactivation of PO-13 and Ty-3 in suckling mice, six PO-13 x Ty-3 reassortant strains that had the genes of the outer capsid proteins, VP4 and VP7, derived from the opposite strain were prepared and were orally inoculated to suckling mice. A single strain that had both PO-13 VP4 and VP7 with the genetic background of Ty-3 had an intermediate virulence for suckling mice. Three strains with Ty-3 VP7, regardless of the origin of VP4, rapidly disappeared from gastrointestinal tracts of suckling mice. These results indicated that the difference between the pathogenicity of PO-13 and that of Ty-3 was mainly dependent on both their VP4 and VP7. In particular, VP7 was found to be related to the inactivation of Ty-3 in gastrointestinal tracts of suckling mice

  13. Competitive advantage of Borrelia burgdorferi with outer surface protein BBA03 during tick-mediated infection of the mammalian host.

    Bestor, Aaron; Rego, Ryan O M; Tilly, Kit; Rosa, Patricia A

    2012-10-01

    Linear plasmid lp54 is one of the most highly conserved and differentially expressed elements of the segmented genome of the Lyme disease spirochete Borrelia burgdorferi. We previously reported that deletion of a 4.1-kb region of lp54 (bba01 to bba07 [bba01-bba07]) led to a slight attenuation of tick-transmitted infection in mice following challenge with a large number of infected ticks. In the current study, we reduced the number of ticks in the challenge to more closely mimic the natural dose and found a profound defect in tick-transmitted infection of the bba01-bba07 mutant relative to wild-type B. burgdorferi. We next focused on deletion of bba03 as the most likely cause of this mutant phenotype, as previous studies have shown that expression of bba03 is increased by culture conditions that simulate tick feeding. Consistent with this hypothesis, we demonstrated increased expression of bba03 by spirochetes in fed relative to unfed ticks. We also observed that a bba03 deletion mutant, although fully competent by itself, did not efficiently infect mice when transmitted by ticks that were simultaneously coinfected with wild-type B. burgdorferi. These results suggest that BBA03 provides a competitive advantage to spirochetes carrying this protein during tick transmission to a mammalian host in the natural infectious cycle.

  14. Cloning and Expression of 31kDa Outer Membrane Protein of Brucella melitansis in E.coli

    Sayeneh Khodadadi

    2012-04-01

    Full Text Available Background & Objectives: The identification of Brucella spp. antigens with the capacity to elicit a protective immune response is of the great interest for the researchers. So, characterization and assessment of diverse antigens of Brucella need to be evaluated. In this study, we report the cloning and expression of the gene coding for 31 KDa OMP (OMP31 of Brucella melitensis 16M.   Methods: Brucella melitensis Omp31 gene was amplified with specific primers, cloned into pJET1/2 and subsequently subcloned in pET28a (+ vector. Both these recombinant plasmids were sequenced and then after, expression of recombinant protein was induced by 1mM IPTG. Western blot analysis was also performed by polyclonal rabbit antiserum.   Results: Omp31 successfully was cloned in both plasmid vectors. The recombinant Omp31 was expressed in E.coli host and purified with significant yield. Western blot results along with those of sequencing ensured accurate production of recombinant omp31 and retaining of its partial epitopes.   Conclusion: Our results show that, an expression host such as E. coli is suitable for omp31 production.

  15. Differential distribution of proteins and lipids in detergent-resistant and detergent-soluble domains in rod outer segment plasma membranes and disks.

    Elliott, Michael H; Nash, Zack A; Takemori, Nobuaki; Fliesler, Steven J; McClellan, Mark E; Naash, Muna I

    2008-01-01

    Membrane heterogeneity plays a significant role in regulating signal transduction and other cellular activities. We examined the protein and lipid components associated with the detergent-resistant membrane (DRM) fractions from retinal rod outer segment (ROS) disk and plasma membrane-enriched preparations. Proteomics and correlative western blot analysis revealed the presence of alpha and beta subunits of the rod cGMP-gated ion channel and glucose transporter type 1, among other proteins. The glucose transporter was present exclusively in ROS plasma membrane (not disks) and was highly enriched in DRMs, as was the cGMP-gated channel beta-subunit. In contrast, the majority of rod opsin and ATP-binding cassette transporter A4 was localized to detergent-soluble domains in disks. As expected, the cholesterol : fatty acid mole ratio was higher in DRMs than in the corresponding parent membranes (disk and plasma membranes, respectively) and was also higher in disks compared to plasma membranes. Furthermore, the ratio of saturated : polyunsaturated fatty acids was also higher in DRMs compared to their respective parent membranes (disk and plasma membranes). These results confirm that DRMs prepared from both disks and plasma membranes are enriched in cholesterol and in saturated fatty acids compared to their parent membranes. The dominant fatty acids in DRMs were 16 : 0 and 18 : 0; 22 : 6n3 and 18 : 1 levels were threefold higher and twofold lower, respectively, in disk-derived DRMs compared to plasma membrane-derived DRMs. We estimate, based on fatty acid recovery that DRMs account for only approximately 8% of disks and approximately 12% of ROS plasma membrane.

  16. The actin-binding proteins eps8 and gelsolin have complementary roles in regulating the growth and stability of mechanosensory hair bundles of mammalian cochlear outer hair cells.

    Jennifer Olt

    Full Text Available Sound transduction depends upon mechanosensitive channels localized on the hair-like bundles that project from the apical surface of cochlear hair cells. Hair bundles show a stair-case structure composed of rows of stereocilia, and each stereocilium contains a core of tightly-packed and uniformly-polarized actin filaments. The growth and maintenance of the stereociliary actin core are dynamically regulated. Recently, it was shown that the actin-binding protein gelsolin is expressed in the stereocilia of outer hair cells (OHCs and in its absence they become long and straggly. Gelsolin is part of a whirlin scaffolding protein complex at the stereocilia tip, which has been shown to interact with other actin regulatory molecules such as Eps8. Here we investigated the physiological effects associated with the absence of gelsolin and its possible overlapping role with Eps8. We found that, in contrast to Eps8, gelsolin does not affect mechanoelectrical transduction during immature stages of development. Moreover, OHCs from gelsolin knockout mice were able to mature into fully functional sensory receptors as judged by the normal resting membrane potential and basolateral membrane currents. Mechanoelectrical transducer current in gelsolin-Eps8 double knockout mice showed a profile similar to that observed in the single mutants for Eps8. We propose that gelsolin has a non-overlapping role with Eps8. While Eps8 is mainly involved in the initial growth of stereocilia in both inner hair cells (IHCs and OHCs, gelsolin is required for the maintenance of mature hair bundles of low-frequency OHCs after the onset of hearing.

  17. Expression of Haemophilus ducreyi collagen binding outer membrane protein NcaA is required for virulence in swine and human challenge models of chancroid.

    Fulcher, Robert A; Cole, Leah E; Janowicz, Diane M; Toffer, Kristen L; Fortney, Kate R; Katz, Barry P; Orndorff, Paul E; Spinola, Stanley M; Kawula, Thomas H

    2006-05-01

    Haemophilus ducreyi, the etiologic agent of the sexually transmitted genital ulcer disease chancroid, has been shown to associate with dermal collagen fibers within infected skin lesions. Here we describe NcaA, a previously uncharacterized outer membrane protein that is important for H. ducreyi collagen binding and host colonization. An H. ducreyi strain lacking the ncaA gene was impaired in adherence to type I collagen but not fibronectin (plasma or cellular form) or heparin. The mutation had no effect on serum resistance or binding to HaCaT keratinocytes or human foreskin fibroblasts in vitro. Escherichia coli expressing H. ducreyi NcaA bound to type I collagen, demonstrating that NcaA is sufficient to confer collagen attachment. The importance of NcaA in H. ducreyi pathogenesis was assessed using both swine and human experimental models of chancroid. In the swine model, 20% of lesions from sites inoculated with the ncaA mutant were culture positive for H. ducreyi 7 days after inoculation, compared to 73% of wild-type-inoculated sites. The average number of CFU recovered from mutant-inoculated lesions was also significantly reduced compared to that recovered from wild-type-inoculated sites at both 2 and 7 days after inoculation. In the human challenge model, 8 of 30 sites inoculated with wild-type H. ducreyi progressed to the pustular stage, compared to 0 of 30 sites inoculated with the ncaA mutant. Together these results demonstrate that the collagen binding protein NcaA is required for H. ducreyi infection.

  18. The outer membrane protein Omp35 affects the reduction of Fe(III, nitrate, and fumarate by Shewanella oneidensis MR-1

    Myers Charles R

    2004-06-01

    Full Text Available Abstract Background Shewanella oneidensis MR-1 uses several electron acceptors to support anaerobic respiration including insoluble species such as iron(III and manganese(IV oxides, and soluble species such as nitrate, fumarate, dimethylsulfoxide and many others. MR-1 has complex branched electron transport chains that include components in the cytoplasmic membrane, periplasm, and outer membrane (OM. Previous studies have implicated a role for anaerobically upregulated OM electron transport components in the use of insoluble electron acceptors, and have suggested that other OM components may also contribute to insoluble electron acceptor use. In this study, the role for an anaerobically upregulated 35-kDa OM protein (Omp35 in the use of anaerobic electron acceptors was explored. Results Omp35 was purified from the OM of anaerobically grown cells, the gene encoding Omp35 was identified, and an omp35 null mutant (OMP35-1 was isolated and characterized. Although OMP35-1 grew on all electron acceptors tested, a significant lag was seen when grown on fumarate, nitrate, and Fe(III. Complementation studies confirmed that the phenotype of OMP35-1 was due to the loss of Omp35. Despite its requirement for wild-type rates of electron acceptor use, analysis of Omp35 protein and predicted sequence did not identify any electron transport moieties or predicted motifs. OMP35-1 had normal levels and distribution of known electron transport components including quinones, cytochromes, and fumarate reductase. Omp35 is related to putative porins from MR-1 and S. frigidimarina as well as to the PorA porin from Neisseria meningitidis. Subcellular fraction analysis confirmed that Omp35 is an OM protein. The seven-fold anaerobic upregulation of Omp35 is mediated post-transcriptionally. Conclusion Omp35 is a putative porin in the OM of MR-1 that is markedly upregulated anaerobically by a post-transcriptional mechanism. Omp35 is required for normal rates of growth on Fe

  19. Application of Nanotrap technology for high sensitivity measurement of urinary outer surface protein A carboxyl-terminus domain in early stage Lyme borreliosis.

    Magni, Ruben; Espina, Benjamin H; Shah, Ketul; Lepene, Benjamin; Mayuga, Christine; Douglas, Temple A; Espina, Virginia; Rucker, Sally; Dunlap, Ross; Petricoin, Emanuel F Iii; Kilavos, Mary Frekko; Poretz, Donald M; Irwin, Gilbert R; Shor, Samuel M; Liotta, Lance A; Luchini, Alessandra

    2015-11-04

    Prompt antibiotic treatment of early stage Lyme borreliosis (LB) prevents progression to severe multisystem disease. There is a clinical need to improve the diagnostic specificity of early stage Lyme assays in the period prior to the mounting of a robust serology response. Using a novel analyte harvesting nanotechnology, Nanotrap particles, we evaluated urinary Borrelia Outer surface protein A (OspA) C-terminus peptide in early stage LB before and after treatment, and in patients suspected of late stage disseminated LB. We employed Nanotrap particles to concentrate urinary OspA and used a highly specific anti-OspA monoclonal antibody (mAb) as a detector of the C-terminus peptides. We mapped the mAb epitope to a narrow specific OspA C-terminal domain OspA236-239 conserved across infectious Borrelia species but with no homology to human proteins and no cross-reactivity with relevant viral and non-Borrelia bacterial proteins. 268 urine samples from patients being evaluated for all categories of LB were collected in a LB endemic area. The urinary OspA assay, blinded to outcome, utilized Nanotrap particle pre-processing, western blotting to evaluate the OspA molecular size, and OspA peptide competition for confirmation. OspA test characteristics: sensitivity 1.7 pg/mL (lowest limit of detection), % coefficient of variation (CV) = 8 %, dynamic range 1.7-30 pg/mL. Pre-treatment, 24/24 newly diagnosed patients with an erythema migrans (EM) rash were positive for urinary OspA while false positives for asymptomatic patients were 0/117 (Chi squared p antibiotic therapy, 10/10 were positive for urinary OspA. Urinary OspA of 8/8 patients switched from detectable to undetectable following symptom resolution post-treatment. Specificity of the urinary OspA test for the clinical symptoms was 40/40. Specificity of the urinary OspA antigen test for later serology outcome was 87.5 % (21 urinary OspA positive/24 serology positive, Chi squared p = 4.072e(-15)). 41 of 100 patients under

  20. BvrR/BvrS-Controlled Outer Membrane Proteins Omp3a and Omp3b Are Not Essential for Brucella abortus Virulence▿

    Manterola, Lorea; Guzmán-Verri, Caterina; Chaves-Olarte, Esteban; Barquero-Calvo, Elías; de Miguel, María-Jesús; Moriyón, Ignacio; Grilló, María-Jesús; López-Goñi, Ignacio; Moreno, Edgardo

    2007-01-01

    The Brucella abortus two-component regulatory system BvrR/BvrS controls the expression of outer membrane proteins (Omp) Omp3a (Omp25) and Omp3b (Omp22). Disruption of bvrS or bvrR generates avirulent mutants with altered cell permeability, higher sensitivity to microbicidal peptides, and complement. Consequently, the role of Omp3a and Omp3b in virulence was examined. Similar to bvrS or bvrR mutants, omp3a and omp3b mutants displayed increased attachment to cells, indicating surface alterations. However, they showed unaltered permeability; normal expression of Omp10, Omp16, Omp19, Omp2b, and Omp1; native hapten polysaccharide; and lipopolysaccharide and were resistant to complement and polymyxin B at ranges similar to those of the wild-type (WT) counterpart. Likewise, omp3a and omp3b mutants were able to replicate in murine macrophages and in HeLa cells, were resistant to the killing action of human neutrophils, and persisted in mice, like the WT strain. Murine macrophages infected with the omp3a mutant generated slightly higher levels of tumor necrosis factor alpha than the WT, whereas the bvrS mutant induced lower levels of this cytokine. Since the absence of Omp3a or Omp3b does not result in attenuation, it can be concluded that BvrR/BvrS influences additional Brucella properties involved in virulence. Our results are discussed in the light of previous works suggesting that disruption of omp3a generates attenuated Brucella strains, and we speculate on the role of group 3 Omps. PMID:17664262

  1. Roles of the outer membrane protein AsmA of Salmonella enterica in the control of marRAB expression and invasion of epithelial cells.

    Prieto, Ana I; Hernández, Sara B; Cota, Ignacio; Pucciarelli, M Graciela; Orlov, Yuri; Ramos-Morales, Francisco; García-del Portillo, Francisco; Casadesús, Josep

    2009-06-01

    A genetic screen for suppressors of bile sensitivity in DNA adenine methylase (dam) mutants of Salmonella enterica serovar Typhimurium yielded insertions in an uncharacterized locus homologous to the Escherichia coli asmA gene. Disruption of asmA suppressed bile sensitivity also in phoP and wec mutants of S. enterica and increased the MIC of sodium deoxycholate for the parental strain ATCC 14028. Increased levels of marA mRNA were found in asmA, asmA dam, asmA phoP, and asmA wec strains of S. enterica, suggesting that lack of AsmA activates expression of the marRAB operon. Hence, asmA mutations may enhance bile resistance by inducing gene expression changes in the marRAB-controlled Mar regulon. In silico analysis of AsmA structure predicted the existence of one transmembrane domain. Biochemical analysis of subcellular fractions revealed that the asmA gene of S. enterica encodes a protein of approximately 70 kDa located in the outer membrane. Because AsmA is unrelated to known transport and/or efflux systems, we propose that activation of marRAB in asmA mutants may be a consequence of envelope reorganization. Competitive infection of BALB/c mice with asmA(+) and asmA isogenic strains indicated that lack of AsmA attenuates Salmonella virulence by the oral route but not by the intraperitoneal route. Furthermore, asmA mutants showed a reduced ability to invade epithelial cells in vitro.

  2. Ectopic expression of TaOEP16-2-5B, a wheat plastid outer envelope protein gene, enhances heat and drought stress tolerance in transgenic Arabidopsis plants.

    Zang, Xinshan; Geng, Xiaoli; Liu, Kelu; Wang, Fei; Liu, Zhenshan; Zhang, Liyuan; Zhao, Yue; Tian, Xuejun; Hu, Zhaorong; Yao, Yingyin; Ni, Zhongfu; Xin, Mingming; Sun, Qixin; Peng, Huiru

    2017-05-01

    Abiotic stresses, such as heat and drought, are major environmental factors restricting crop productivity and quality worldwide. A plastid outer envelope protein gene, TaOEP16-2, was identified from our previous transcriptome analysis [1,2]. In this study, the isolation and functional characterization of the TaOEP16-2 gene was reported. Three homoeologous sequences of TaOEP16-2 were isolated from hexaploid wheat, which were localized on the chromosomes 5A, 5B and 5D, respectively. These three homoeologues exhibited different expression patterns under heat stress conditions, TaOEP16-2-5B was the dominant one, and TaOEP16-2-5B was selected for further analysis. Compared with wild type (WT) plants, transgenic Arabidopsis plants overexpressing the TaOEP16-2-5B gene exhibited enhanced tolerance to heat stress, which was supported by improved survival rate, strengthened cell membrane stability and increased sucrose content. It was also found that TaOEP16-2 was induced by drought stress and involved in drought stress tolerance. TaOEP16-2-5B has the same function in ABA-controlled seed germination as AtOEP16-2. Our results suggest that TaOEP16-2-5B plays an important role in heat and drought stress tolerance, and could be utilized in transgenic breeding of wheat and other crop plants. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Dissection of β-barrel Outer Membrane Protein Assembly Pathways through Characterizing BamA POTRA 1 Mutants of Escherichia coli

    Bennion, Drew; Charlson, Emily S.; Coon, Eric; Misra, Rajeev

    2010-01-01

    Summary BamA of Escherichia coli is an essential component of the hetero-oligomeric machinery that mediates β-barrel outer membrane protein (OMP) assembly. The C- and N-termini of BamA fold into trans-membrane β-barrel and five soluble POTRA domains, respectively. Detailed characterization of BamA POTRA 1 missense and deletion mutants revealed two competing OMP assembly pathways, one of which is followed by the archetypal trimeric β-barrel OMPs, OmpF and LamB, and is dependent on POTRA 1. Interestingly, our data suggest that BamA also requires its POTRA 1 domain for proper assembly. The second pathway is independent of POTRA 1 and is exemplified by TolC. Site-specific cross-linking analysis revealed that the POTRA 1 domain of BamA interacts with SurA, a periplasmic chaperone required for the assembly of OmpF and LamB, but not that of TolC and BamA. The data suggest that SurA and BamA POTRA 1 domain function in concert to assist folding and assembly of most β-barrel OMPs except for TolC, which folds into a unique soluble α-helical barrel and an OM-anchored β-barrel. The two assembly pathways finally merge at some step beyond POTRA 1 but presumably before membrane insertion, which is thought to be catalyzed by the trans-membrane β-barrel domain of Bam A. PMID:20598079

  4. A novel recombinant bivalent outer membrane protein of Vibrio vulnificus and Aeromonas hydrophila as a vaccine antigen of American eel (Anguilla rostrata).

    SongLin, Guo; PanPan, Lu; JianJun, Feng; JinPing, Zhao; Peng, Lin; LiHua, Duan

    2015-04-01

    The immogenicity of a novel vaccine antigen was evaluated after immunized American eels (Anguilla rostrata) with a recombinant bivalent expressed outer membrane protein (OMP) of Vibrio vulnificus and Aeromonas hydrophila. Three groups of eels were intraperitoneal (i.p) injected with phosphate-buffered saline (PBS group), formaline-killed-whole-cell (FKC) of A. hydrophila and V. vulnificus (FKC group) or the bivalent OMP (OMP group). On 14, 21, 28 and 42 days post-vaccination respectively, proliferation of the whole blood cells, titers of specific antibody and lysozyme activities of experimental eels were detected. On 28 day post-vaccination, eels from three groups were challenged by i.p injection of live A. hydrophila or V. vulnificus. The results showed that, compared with the PBS group, proliferation of whole blood cells in OMP group was significant enhanced on 28 days, and the serum titers of anti-A.hydrophila and anti-V. vulnificus antibody in eels of FKC and OMP group were significant increased on 14, 21 and 28d. Lysozyme Activities in serum, skin mucus, liver and kidney were significant changed between the three groups. Relative Percent Survival (RPS) after challenged A. hydrophila in KFC vs. PBS group and OMP vs. PBS group were 62.5% and 50% respectively, and the RPS challenged V. vulnificus in FKC and OMP vs. PBS group were 37.5% and 50% respectively. These results suggest that American eels immunized with the bivalent OMP would positively affect specific as well as non-specific immune parameters and protect against infection by the two pathogens in fresh water farming. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Comparison of clinical performance of antigen basedenzyme immunoassay (EIA and major outer membrane protein (MOMP-PCR for detection of genital Chlamydia trachomatis infection

    Mahmoud Nateghi Rostami

    2016-06-01

    Full Text Available Background: Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Early detection and treatment of C.trachomatis genital infection prevent serious reproductive complications. Objective: Performances of enzyme immunoassay (EIA and major outer membrane protein (MOMP-polymerase chain reaction (PCR for diagnosis of genital C.trachomatis infection in women were compared. Materials and Methods: In this cross sectional study a total of 518 women volunteers were included (33.67±8.3 yrs who had been referred to Gynecology clinics of Qom province, Iran, were included. Endocervical swab specimens were collected to detect lipopolysaccharide (LPS antigen in EIA and to amplify MOMP gene of C.trachomatis in PCR. Results were confirmed using ompI nested-PCR. Sensitivity, specificity, positive (PPV and negative predictive values (NPV were calculated for performance of the tests. Odds ratios were determined using binary logistic regression analysis. Results: In total, 37 (7.14% cases were positive by EIA and/or MOMP-PCR. All discrepant results were confirmed by nested-PCR. Sensitivity, specificity, PPV and NPV values of EIA were 59.46%, 100%, 100% and 96.98%, and those of MOMPPCR were 97.30%, 100%, 100%, 99.79%, respectively. Reproductive complications including 2.7% ectopic pregnancy, 5.4% stillbirth, 5.4% infertility, and 10.8% PROM were recorded. The risk of developing chlamydiosis was increased 4.8-fold in volunteers with cervicitis (p<0.05; OR 4.80; 95% CI 1.25-18.48. Conclusion: C.trachomatis infection should be regarded in women of reproductive ages especially those with cervicitis. Primary screening of women by using the low cost antigen-EIA is recommended; however, due to the low sensitivity of Ag-EIA, verification of the negative results by a DNA amplification method is needed.

  6. Comparison of clinical performance of antigen based-enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-PCR for detection of genital Chlamydia trachomatis infection.

    Nateghi Rostami, Mahmoud; Hossein Rashidi, Batool; Aghsaghloo, Fatemeh; Nazari, Razieh

    2016-06-01

    Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Early detection and treatment of C.trachomatis genital infection prevent serious reproductive complications. Performances of enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-polymerase chain reaction (PCR) for diagnosis of genital C.trachomatis infection in women were compared. In this cross sectional study a total of 518 women volunteers were included (33.67±8.3 yrs) who had been referred to Gynecology clinics of Qom province, Iran, were included. Endocervical swab specimens were collected to detect lipopolysaccharide (LPS) antigen in EIA and to amplify MOMP gene of C.trachomatis in PCR. Results were confirmed using ompI nested-PCR. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated for performance of the tests. Odds ratios were determined using binary logistic regression analysis. In total, 37 (7.14%) cases were positive by EIA and/or MOMP-PCR. All discrepant results were confirmed by nested-PCR. Sensitivity, specificity, PPV and NPV values of EIA were 59.46%, 100%, 100% and 96.98%, and those of MOMP-PCR were 97.30%, 100%, 100%, 99.79%, respectively. Reproductive complications including 2.7% ectopic pregnancy, 5.4% stillbirth, 5.4% infertility, and 10.8% PROM were recorded. The risk of developing chlamydiosis was increased 4.8-fold in volunteers with cervicitis (p<0.05; OR 4.80; 95% CI 1.25-18.48). C.trachomatis infection should be regarded in women of reproductive ages especially those with cervicitis. Primary screening of women by using the low cost antigen-EIA is recommended; however, due to the low sensitivity of Ag-EIA, verification of the negative results by a DNA amplification method is needed.

  7. Humoral immune responses in koalas (Phascolarctos cinereus) either naturally infected with Chlamydia pecorum or following administration of a recombinant chlamydial major outer membrane protein vaccine.

    Khan, Shahneaz Ali; Polkinghorne, Adam; Waugh, Courtney; Hanger, Jon; Loader, Jo; Beagley, Kenneth; Timms, Peter

    2016-02-03

    The development of a vaccine is a key strategy to combat the widespread and debilitating effects of chlamydial infection in koalas. One such vaccine in development uses recombinant chlamydial major outer membrane protein (rMOMP) as an antigen and has shown promising results in several koala trials. Previous chlamydial vaccine studies, primarily in the mouse model, suggest that both cell-mediated and antibody responses will be required for adequate protection. Recently, the important protective role of antibodies has been highlighted. In our current study, we conducted a detailed analysis of the antibody-mediated immune response in koalas that are either (a) naturally-infected, and/or (b) had received an rMOMP vaccine. Firstly, we observed that naturally-infected koalas had very low levels of Chlamydia pecorum-specific neutralising antibodies. A strong correlation between low IgG total titers/neutralising antibody levels, and higher C. pecorum infection load was also observed in these naturally-infected animals. In vaccinated koalas, we showed that the vaccine was able to boost the humoral immune response by inducing strong levels of C. pecorum-specific neutralising antibodies. A detailed characterisation of the MOMP epitope response was also performed in naturally-infected and vaccinated koalas using a PepScan epitope approach. This analysis identified unique sets of MOMP epitope antibodies between naturally-infected non-protected and diseased koalas, versus vaccinated koalas, with the latter group of animals producing a unique set of specific epitope-directed antibodies that we demonstrated were responsible for the in vitro neutralisation activity. Together, these results show the importance of antibodies in chlamydial infection and immunity following vaccination in the koala. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Immunization with r-Lactococcus lactis expressing outer membrane protein A of Shigella dysenteriae type-1: evaluation of oral and intranasal route of administration.

    Yagnik, B; Sharma, D; Padh, H; Desai, P

    2017-02-01

    To evaluate the comparative immunogenic potential of food grade Lactococcus lactis expressing outer membrane protein A (OmpA) of Shigella dysenteriae type-1 (SD-1) when administered either orally or intranasally. OmpA of SD-1 was cloned and expressed first in Escherichia coli and then in L. lactis. Presence of recombinant gene was confirmed by restriction enzyme digestion and immunoblot analysis. Using immobilized metal affinity chromatography, OmpA was purified from recombinant E. coliBL21 (DE3) and subcutaneously administered in BALB/c mice. Detection of OmpA-specific IgG antibodies by enzyme-linked immunosorbent assay (ELISA) confirmed the immunogenicity of OmpA. In order to establish r-L. lactis as a mucosal delivery vehicle, it was administered orally and nasally in BALB/c mice. Serum IgG and faecal IgA were assessed through ELISA to compare the relative potential of immunization routes and immunogenic potential of r-L. lactis. Immunization via the oral route proved superior to intranasal exposure. Recombinant L. lactis expressing OmpA of SD-1 was found to be immunogenic. Oral administration of r-L. lactis elicited higher systemic and mucosal immune response when compared with the nasal route. Using food grade recombinant L. lactis has implications in the development of a prophylactic against multidrug-resistant Shigella, which can be used as a prospective vaccine candidate. Evaluating mucosal routes of immunization demonstrated that the oral route of administration elicited better immune response against OmpA of Shigella. © 2016 The Society for Applied Microbiology.

  9. The effect of human factor H on immunogenicity of meningococcal native outer membrane vesicle vaccines with over-expressed factor H binding protein.

    Peter T Beernink

    Full Text Available The binding of human complement inhibitors to vaccine antigens in vivo could diminish their immunogenicity. A meningococcal ligand for the complement down-regulator, factor H (fH, is fH-binding protein (fHbp, which is specific for human fH. Vaccines containing recombinant fHbp or native outer membrane vesicles (NOMV from mutant strains with over-expressed fHbp are in clinical development. In a previous study in transgenic mice, the presence of human fH impaired the immunogenicity of a recombinant fHbp vaccine. In the present study, we prepared two NOMV vaccines from mutant group B strains with over-expressed wild-type fHbp or an R41S mutant fHbp with no detectable fH binding. In wild-type mice in which mouse fH did not bind to fHbp in either vaccine, the NOMV vaccine with wild-type fHbp elicited 2-fold higher serum IgG anti-fHbp titers (P = 0.001 and 4-fold higher complement-mediated bactericidal titers against a PorA-heterologous strain than the NOMV with the mutant fHbp (P = 0.003. By adsorption, the bactericidal antibodies were shown to be directed at fHbp. In transgenic mice in which human fH bound to the wild-type fHbp but not to the R41S fHbp, the NOMV vaccine with the mutant fHbp elicited 5-fold higher serum IgG anti-fHbp titers (P = 0.002, and 19-fold higher bactericidal titers than the NOMV vaccine with wild-type fHbp (P = 0.001. Thus, in mice that differed only by the presence of human fH, the respective results with the two vaccines were opposite. The enhanced bactericidal activity elicited by the mutant fHbp vaccine in the presence of human fH far outweighed the loss of immunogenicity of the mutant protein in wild-type animals. Engineering fHbp not to bind to its cognate complement inhibitor, therefore, may increase vaccine immunogenicity in humans.

  10. Regulatory protein OmpR influences the serum resistance of Yersinia enterocolitica O:9 by modifying the structure of the outer membrane.

    Karolina Skorek

    Full Text Available The EnvZ/OmpR two-component system constitutes a regulatory pathway involved in bacterial adaptive responses to environmental cues. Our previous findings indicated that the OmpR regulator in Yersinia enterocolitica O:9 positively regulates the expression of FlhDC, the master flagellar activator, which influences adhesion/invasion properties and biofilm formation. Here we show that a strain lacking OmpR grown at 37°C exhibits extremely high resistance to the bactericidal activity of normal human serum (NHS compared with the wild-type strain. Analysis of OMP expression in the ompR mutant revealed that OmpR reciprocally regulates Ail and OmpX, two homologous OMPs of Y. enterocolitica, without causing significant changes in the level of YadA, the major serum resistance factor. Analysis of mutants in individual genes belonging to the OmpR regulon (ail, ompX, ompC and flhDC and strains lacking plasmid pYV, expressing YadA, demonstrated the contribution of the respective proteins to serum resistance. We show that Ail and OmpC act in an opposite way to the OmpX protein to confer serum resistance to the wild-type strain, but are not responsible for the high resistance of the ompR mutant. The serum resistance phenotype of ompR seems to be multifactorial and mainly attributable to alterations that potentiate the function of YadA. Our results indicate that a decreased level of FlhDC in the ompR mutant cells is partly responsible for the serum resistance and this effect can be suppressed by overexpression of flhDC in trans. The observation that the loss of FlhDC enhances the survival of wild-type cells in NHS supports the involvement of FlhDC regulator in this phenotype. In addition, the ompR mutant exhibited a lower level of LPS, but this was not correlated with changes in the level of FlhDC. We propose that OmpR might alter the susceptibility of Y. enterocolitica O:9 to complement-mediated killing through remodeling of the outer membrane.

  11. Small RNAs controlling outer membrane porins

    Valentin-Hansen, Poul; Johansen, Jesper; Rasmussen, Anders A

    2007-01-01

    are key regulators of environmental stress. Recent work has revealed an intimate interplay between small RNA regulation of outer membrane proteins and the stress-induced sigmaE-signalling system, which has an essential role in the maintenance of the integrity of the outer membrane.......Gene regulation by small non-coding RNAs has been recognized as an important post-transcriptional regulatory mechanism for several years. In Gram-negative bacteria such as Escherichia coli and Salmonella, these RNAs control stress response and translation of outer membrane proteins and therefore...

  12. Phase 1 testing of detoxified LPS/group B meningococcal outer membrane protein vaccine with and without synthetic CPG 7909 adjuvant for the prevention and treatment of sepsis.

    Cross, Alan S; Greenberg, Nancy; Billington, Melissa; Zhang, Lei; DeFilippi, Christopher; May, Ryan C; Bajwa, Kanwaldeep K

    2015-11-27

    Gram-negative bacteria (GNB) are a leading cause of nosocomial infection and sepsis. Increasing multi-antibiotic resistance has left clinicians with fewer therapeutic options. Antibodies to GNB lipopolysaccharide (LPS, or endotoxin) have reduced morbidity and mortality as a result of infection and are not subject to the resistance mechanisms deployed by bacteria against antibiotics. In this phase 1 study, we administered a vaccine that elicits antibodies against a highly conserved portion of LPS with and without a CpG oligodeoxynucleotide (ODN) TLR9 agonist as adjuvant. A vaccine composed of the detoxified LPS (dLPS) from E. coli O111:B4 (J5 mutant) non-covalently complexed to group B meningococcal outer membrane protein (OMP). Twenty healthy adult subjects received three doses at 0, 29 and 59 days of antigen (10 μg dLPS) with or without CPG 7909 (250 or 500 μg). Subjects were evaluated for local and systemic adverse effects and laboratory findings. Anti-J5 LPS IgG and IgM antibody levels were measured by electrochemiluminesence. Due to premature study termination, not all subjects received all three doses. All vaccine formulations were well-tolerated with no local or systemic events of greater than moderate severity. The vaccine alone group achieved a ≥ 4-fold "responder" response in IgG and IgM antibody in only one of 6 subjects. In contrast, the vaccine plus CPG 7909 groups appeared to have earlier and more sustained (to 180 days) responses, greater mean-fold increases, and a higher proportion of "responders" achieving ≥ 4-fold increases over baseline. Although the study was halted before all enrolled subjects received all three doses, the J5dLPS/OMP vaccine, with or without CpG adjuvant, was safe and well-tolerated. The inclusion of CpG increased the number of subjects with a ≥ 4-fold antibody response, evident even after the second of three planned doses. A vaccine comprising J5dLPS/OMP antigen with CpG adjuvant merits further investigation. Clinical

  13. Parenteral immunization of PLA/PLGA nanoparticle encapsulating outer membrane protein (Omp) from Aeromonas hydrophila: Evaluation of immunostimulatory action in Labeo rohita (rohu).

    Rauta, Pradipta Ranjan; Nayak, Bismita

    2015-05-01

    Advanced vaccine research approaches needs to explore on biodegradable nanoparticles (NPs) based vaccine carrier that can serve as antigen delivery systems as well as immuno-stimulatory action to induce both innate and adaptive immune response in fish. Immunogenicity of PLA and PLGA NPs encapsulating outer membrane protein (Omp) antigen of Aeromonas hydrophila were evaluated through intra-peritoneal injection in fish, Labeo rohita. Antigen loaded PLA-Omp (223.5 ± 13.19 nm) and PLGA-Omp (166.4 ± 21.23 nm) NPs were prepared using double emulsion method by efficiently encapsulating the antigen reaching the encapsulation efficiency 44 ± 4.58% and 59.33 ± 5.13% respectively. Our formulated PLA Omp and PLGA-Omp NPs were in nanometer range (PLA-Omp, it showed considerably slower antigen release in vitro than PLGA-Omp NPs. Other physical properties like zetapotential values and poly dispersity index (PDI) confirmed the stability as well as monodisperse nature of the formulated nanoparticles. The spherical and isolated nature of PLA-Omp and PLGA-Omp NPs were revealed by SEM analysis. Upon immunization of all antigenic formulations (PLA-Omp NP, PLGA-Omp NP, FIA-Omp, PLA NP, PLGA NP, PBS as control), significant higher bacterial agglutination titre and haemolytic activity were observed in case of PLA-Omp and PLGA-Omp immunized groups than rest groups at both 21 days and 42 days. The specific antibody response was significantly increased and persisted up to 42 days of post immunization by PLA-Omp, PLGA-Omp, FIA-Omp. PLA-Omp NPs showed better immune response (higher bacterial agglutination titre, haemolytic activity, specific antibody titre, higher percent survival upon A. hydrophila challenge) than PLGA-Omp in L. rohita confirming its better efficacy. Comparable antibody response of PLA-Omp and PLGA-Omp with FIA-Omp treated groups suggested that PLA and PLGA could be replacement for Freund's adjuvant (for stimulating antibody response) to overcome many side effects

  14. Chloroform-Methanol Residue of Coxiella burnetii Markedly Potentiated the Specific Immunoprotection Elicited by a Recombinant Protein Fragment rOmpB-4 Derived from Outer Membrane Protein B of Rickettsia rickettsii in C3H/HeN Mice.

    Wenping Gong

    Full Text Available The obligate intracellular bacteria, Rickettsia rickettsii and Coxiella burnetii, are the potential agents of bio-warfare/bio-terrorism. Here C3H/HeN mice were immunized with a recombinant protein fragment rOmp-4 derived from outer membrane protein B, a major protective antigen of R. rickettsii, combined with chloroform-methanol residue (CMR extracted from phase I C. burnetii organisms, a safer Q fever vaccine. These immunized mice had significantly higher levels of IgG1 and IgG2a to rOmpB-4 and interferon-γ (IFN-γ and tumor necrosis factor-α (TNF-α, two crucial cytokines in resisting intracellular bacterial infection, as well as significantly lower rickettsial loads and slighter pathological lesions in organs after challenge with R. rickettsii, compared with mice immunized with rOmpB-4 or CMR alone. Additionally, after challenge with C. burnetii, the coxiella loads in the organs of these mice were significantly lower than those of mice immunized with rOmpB-4 alone. Our results prove that CMR could markedly potentiate enhance the rOmpB-4-specific immunoprotection by promoting specific and non-specific immunoresponses and the immunization with the protective antigen of R. rickettsii combined with CMR of C. burnetii could confer effective protection against infection of R. rickettsii or C. burnetii.

  15. Incorporation of squalene into rod outer segments

    Keller, R.K.; Fliesler, S.J.

    1990-01-01

    We have reported previously that squalene is the major radiolabeled nonsaponifiable lipid product derived from [ 3 H]acetate in short term incubations of frog retinas. In the present study, we demonstrate that newly synthesized squalene is incorporated into rod outer segments under similar in vitro conditions. We show further that squalene is an endogenous constituent of frog rod outer segment membranes; its concentration is approximately 9.5 nmol/mumol of phospholipid or about 9% of the level of cholesterol. Pulse-chase experiments with radiolabeled precursors revealed no metabolism of outer segment squalene to sterols in up to 20 h of chase. Taken together with our previous absolute rate studies, these results suggest that most, if not all, of the squalene synthesized by the frog retina is transported to rod outer segments. Synthesis of protein is not required for squalene transport since puromycin had no effect on squalene incorporation into outer segments. Conversely, inhibition of isoprenoid synthesis with mevinolin had no effect on the incorporation of opsin into the outer segment. These latter results support the conclusion that the de novo synthesis and subsequent intracellular trafficking of opsin and isoprenoid lipids destined for the outer segment occur via independent mechanisms

  16. The Outer Space Treaty

    Johnson, Christopher Daniel

    2018-01-01

    Negotiated at the United Nations and in force since 1967, the Outer Space Treaty has been ratified by over 100 countries and is the most important and foundational source of space law. The treaty, whose full title is "Treaty on Principles Governing the Activities of States in the Exploration and Use of Outer Space, Including the Moon and Other Celestial Bodies," governs all of humankind's activities in outer space, including activities on other celestial bodies and many activities on Earth related to outer space. All space exploration and human spaceflight, planetary sciences, and commercial uses of space—such as the global telecommunications industry and the use of space technologies such as position, navigation, and timing (PNT), take place against the backdrop of the general regulatory framework established in the Outer Space Treaty. A treaty is an international legal instrument which balances rights and obligations between states, and exists as a kind of mutual contract of shared understandings, rights, and responsibilities between them. Negotiated and drafted during the Cold War era of heightened political tensions, the Outer Space Treaty is largely the product of efforts by the United States and the USSR to agree on certain minimum standards and obligations to govern their competition in "conquering" space. Additionally, the Outer Space Treaty is similar to other treaties, including treaties governing the high seas, international airspace, and the Antarctic, all of which govern the behavior of states outside of their national borders. The treaty is brief in nature and only contains 17 articles, and is not comprehensive in addressing and regulating every possible scenario. The negotiating states knew that the Outer Space Treaty could only establish certain foundational concepts such as freedom of access, state responsibility and liability, non-weaponization of space, the treatment of astronauts in distress, and the prohibition of non-appropriation of

  17. Transcriptional Modulation of Penicillin-Binding Protein 1b, Outer Membrane Protein P2 and Efflux Pump (AcrAB-TolC during Heat Stress Is Correlated to Enhanced Bactericidal Action of Imipenem on Non-typeable Haemophilus influenzae

    Abdessalam Cherkaoui

    2018-01-01

    Full Text Available Objective: The purpose of the present study was to investigate the penicillin binding proteins (PBPs, drug influx and efflux modulations during heat stress and their effects on the bactericidal action of imipenem on non-typeable Haemophilus influenzae (NTHi.Methods: The two NTHi clinical isolates (GE47 and GE88, imipenem MICs by E-test > 32 μg/mL examined in this study were collected at Geneva University Hospitals. The imipenem killing activity was assessed after incubation of the NTHi strains at either 37 or 42°C for 3 h with increasing concentrations of imipenem. The detection of PBPs was carried out by Bocillin-FL. Global transcriptional changes were monitored by RNA-seq after pre-incubation of bacterial cells at either 37 or 42°C, and the expression levels of relevant target genes were confirmed by qRT-PCR.Results: Quantitation of NTHi viable cells after incubation with 0.25 μg/mL of imipenem for 3 h revealed more than a twofold decrease in GE47 and GE88 viable cells at 42°C as compared to 37°C. Transcriptome analysis showed that under heat stress conditions, there were 141 differentially expressed genes with a | log2(fold change| > 1, including 67 up-regulated and 74 down-regulated genes. The expression levels of ponB (encoding PBP1b and acrR (regulator of AcrAB-TolC efflux pump were significantly increased at 42°C. In contrast, the transcript levels of ompP2 (encoding the outer membrane protein P2 and acrB gene (encoding AcrB were significantly lower under heat stress condition.Conclusion: This study shows that the transcriptional modulation of ponB, ompP2, acrR, and acrB in the heat stress response is correlated to enhanced antimicrobial effects of imipenem on non-typeable H. influenzae.

  18. Outer membrane protein A (OmpA of Shigella flexneri 2a induces TLR2-mediated activation of B cells: involvement of protein tyrosine kinase, ERK and NF-κB.

    Rajsekhar Bhowmick

    Full Text Available B cells are critically important in combating bacterial infections and their differentiation into plasma cells and memory cells aids bacterial clearance and long-lasting immunity conferred by essentially all vaccines. Outer membrane protein A (OmpA of Shigella flexneri 2a has been demonstrated to induce the production of IgG and IgA in vivo following immunization of mice through intranasal route, but the direct involvement of B cells in OmpA-mediated immune regulation was not determined. Consequently, we investigated whether OmpA can modulate B cell functions and identified the molecular events involved in OmpA-induced B cell immune response in vitro. We show that OmpA of S. flexneri 2a activates B cells to produce protective cytokines, IL-6 and IL-10 as well as facilitates their differentiation into antibody secreting cells (ASCs. The immunostimulatory properties of OmpA are attributed to the increased surface expression of MHCII and CD86 on B cells. We also report here that B cell activation by OmpA is mediated strictly through recognition by TLR2, resulting in initiation of cascades of signal transduction events, involving increased phosphorylation of protein tyrosine kinases (PTKs, ERK and IκBα, leading to nuclear translocation of NF-κB. Importantly, a TLR2 antibody diminishes OmpA-induced upregulation of MHCII and CD86 on B cell surface as well as significantly inhibits B cell differentiation and cytokine secretion. Furthermore, we illustrate that B cell differentiation into ASCs and induction of cytokine secretion by OmpA are dependent on PTKs activity. Moreover, we identify that OmpA-induced B cell differentiation is entirely dependent on ERK pathway, whereas both NF-κB and ERK are essential for cytokine secretion by B cells. Overall, our data demonstrate that OmpA of S. flexneri 2a amplifies TLR signaling in B cells and triggers B cell immune response, which is critical for the development of an effective adaptive immunity to an

  19. Saturn's outer magnetosphere

    Schardt, A. W.; Behannon, K. W.; Carbary, J. F.; Eviatar, A.; Lepping, R. P.; Siscoe, G. L.

    1983-01-01

    Similarities between the Saturnian and terrestrial outer magnetosphere are examined. Saturn, like Earth, has a fully developed magnetic tail, 80 to 100 RS in diameter. One major difference between the two outer magnetospheres is the hydrogen and nitrogen torus produced by Titan. This plasma is, in general, convected in the corotation direction at nearly the rigid corotation speed. Energies of magnetospheric particles extend to above 500 keV. In contrast, interplanetary protons and ions above 2 MeV have free access to the outer magnetosphere to distances well below the Stormer cutoff. This access presumably occurs through the magnetotail. In addition to the H+, H2+, and H3+ ions primarily of local origin, energetic He, C, N, and O ions are found with solar composition. Their flux can be substantially enhanced over that of interplanetary ions at energies of 0.2 to 0.4 MeV/nuc.

  20. The HtrA-like serine protease PepD interacts with and modulates the Mycobacterium tuberculosis 35-kDa antigen outer envelope protein.

    Mark J White

    2011-03-01

    Full Text Available Mycobacterium tuberculosis remains a significant global health concern largely due to its ability to persist for extended periods within the granuloma of the host. While residing within the granuloma, the tubercle bacilli are likely to be exposed to stress that can result in formation of aberrant proteins with altered structures. Bacteria encode stress responsive determinants such as proteases and chaperones to deal with misfolded or unfolded proteins. pepD encodes an HtrA-like serine protease and is thought to process proteins altered following exposure of M. tuberculosis to extra-cytoplasmic stress. PepD functions both as a protease and chaperone in vitro, and is required for aspects of M. tuberculosis virulence in vivo. pepD is directly regulated by the stress-responsive two-component signal transduction system MprAB and indirectly by extracytoplasmic function (ECF sigma factor SigE. Loss of PepD also impacts expression of other stress-responsive determinants in M. tuberculosis. To further understand the role of PepD in stress adaptation by M. tuberculosis, a proteomics approach was taken to identify binding proteins and possible substrates of this protein. Using subcellular fractionation, the cellular localization of wild-type and PepD variants was determined. Purified fractions as well as whole cell lysates from Mycobacterium smegmatis or M. tuberculosis strains expressing a catalytically compromised PepD variant were immunoprecipitated for PepD and subjected to LC-MS/MS analyses. Using this strategy, the 35-kDa antigen encoding a homolog of the PspA phage shock protein was identified as a predominant binding partner and substrate of PepD. We postulate that proteolytic cleavage of the 35-kDa antigen by PepD helps maintain cell wall homeostasis in Mycobacterium and regulates specific stress response pathways during periods of extracytoplasmic stress.

  1. The mitochondrial outer membrane protein mitoNEET is a redox enzyme catalyzing electron transfer from FMNH2 to oxygen or ubiquinone.

    Wang, Yiming; Landry, Aaron P; Ding, Huangen

    2017-06-16

    Increasing evidence suggests that mitoNEET, a target of the type II diabetes drug pioglitazone, is a key regulator of energy metabolism in mitochondria. MitoNEET is anchored to the mitochondrial outer membrane via its N-terminal α helix domain and hosts a redox-active [2Fe-2S] cluster in its C-terminal cytosolic region. The mechanism by which mitoNEET regulates energy metabolism in mitochondria, however, is not fully understood. Previous studies have shown that mitoNEET specifically interacts with the reduced flavin mononucleotide (FMNH 2 ) and that FMNH 2 can quickly reduce the mitoNEET [2Fe-2S] clusters. Here we report that the reduced mitoNEET [2Fe-2S] clusters can be readily oxidized by oxygen. In the presence of FMN, NADH, and flavin reductase, which reduces FMN to FMNH 2 using NADH as the electron donor, mitoNEET mediates oxidation of NADH with a concomitant reduction of oxygen. Ubiquinone-2, an analog of ubiquinone-10, can also oxidize the reduced mitoNEET [2Fe-2S] clusters under anaerobic or aerobic conditions. Compared with oxygen, ubiquinone-2 is more efficient in oxidizing the mitoNEET [2Fe-2S] clusters, suggesting that ubiquinone could be an intrinsic electron acceptor of the reduced mitoNEET [2Fe-2S] clusters in mitochondria. Pioglitazone or its analog NL-1 appears to inhibit the electron transfer activity of mitoNEET by forming a unique complex with mitoNEET and FMNH 2 The results suggest that mitoNEET is a redox enzyme that may promote oxidation of NADH to facilitate enhanced glycolysis in the cytosol and that pioglitazone may regulate energy metabolism in mitochondria by inhibiting the electron transfer activity of mitoNEET. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. TPS for Outer Planets

    Venkatapathy, Ethiraj; Ellerby, D.; Gage, P.; Gasch, M.; Hwang, H.; Prabhu, D.; Stackpoole, M.; Wercinski, Paul

    2018-01-01

    This invited talk will provide an assessment of the TPS needs for Outer Planet In-situ missions to destinations with atmosphere. The talk will outline the drivers for TPS from destination, science, mission architecture and entry environment. An assessment of the readiness of the TPS, both currently available and under development, for Saturn, Titan, Uranus and Neptune are provided. The challenges related to sustainability of the TPS for future missions are discussed.

  3. The lissencephaly protein Lis1 is present in motile mammalian cilia and requires outer arm dynein for targeting to Chlamydomonas flagella

    Pedersen, Lotte B; Rompolas, Panteleimon; Christensen, Søren T

    2007-01-01

    Lissencephaly is a developmental brain disorder characterized by a smooth cerebral surface, thickened cortex and misplaced neurons. Classical lissencephaly is caused by mutations in LIS1, which encodes a WD-repeat protein involved in cytoplasmic dynein regulation, mitosis and nuclear migration. S...

  4. Outer Continental Shelf Lands Act

    National Oceanic and Atmospheric Administration, Department of Commerce — This data represents geographic terms used within the Outer Continental Shelf Lands Act (OCSLA or Act). The Act defines the United States outer continental shelf...

  5. A novel ELISA using a recombinant outer membrane protein, rTp0663, as the antigen for serological diagnosis of syphilis.

    Xu, Man; Xie, Yafeng; Jiang, Chuanhao; Xiao, Yongjian; Kuang, Xingxing; Zhao, Feijun; Zeng, Tiebing; Liu, Shuangquan; Liang, Mingxing; Li, Li; Wang, Chuan; Wu, Yimou

    2016-02-01

    The lack of Treponema pallidum-specific antigens with highly accurate diagnosis makes the diagnosis of syphilis challenging. A soluble recombinant version of a new diagnostic protein Tp0663 has been produced. The serodiagnostic potential of this protein was assessed by screening 3326 serum samples simultaneously evaluated by rapid plasma reagin and T. pallidum particle agglutination tests. Kappa (κ) coefficients were used to compare the concordance between clinical diagnosis and the Tp0663-based ELISA or the ARCHITECT Syphilis TP chemiluminescent immunoassay (Abbott GmbH and Co. KG). Using the results of clinical diagnosis as the gold standard, the sensitivity and specificity of Tp0663 were found to be 98.83% (95% confidence interval (CI) 96.61-99.60%) and 100% (95% CI 99.88-100%), respectively. In comparison, the ARCHITECT Syphilis TP assay was found to have a lower sensitivity (97.27%, 95% CI 94.46-98.67%) and specificity (99.61%, 95% CI 99.32-99.78%). In particular, the ARCHITECT Syphilis TP exhibited a false-positive rate of 0.39%. Moreover, the ELISA was in perfect agreement with the gold standard, with a κ value of 0.99, comparable to that of ARCHITECT Syphilis TP (0.96). These results identified Tp0663 as a novel serodiagnostic candidate with great potential for developing novel tests for the diagnosis of syphilis. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. Management of outer space

    Perek, Lubos

    1993-10-01

    Various aspects of space-environment management are discussed. Attention is called to the fact that, while space radio communications are already under an adequate management by the International Communications Union, the use of nuclear power sources is regulated by the recently adopted set of principles, and space debris will be discussed in the near future at the UN COPUOS, other aspects of management of outer space received little or no attention of the international community. These include the competency of crews and technical equipment of spacecraft launched by newcomers to space exploration; monitoring of locations and motions of space objects (now in national hands), with relevant data made accessible through a computer network; and the requirement to use space only for beneficial purposes and not for promoting narrow and debatable interests damaging the outer space environment and impeding on astronomical observations. It is suggested that some of these tasks would be best performed by an international space agency within the UN system of organizations.

  7. Modifications to the composition of the hyphal outer layer of Aspergillus fumigatus modulates HUVEC proteins related to inflammatory and stress responses.

    Neves, Gabriela Westerlund Peixoto; Curty, Nathália de Andrade; Kubitschek-Barreira, Paula Helena; Fontaine, Thierry; Souza, Gustavo Henrique Martins Ferreira; Cunha, Marcel Lyra; Goldman, Gustavo H; Beauvais, Anne; Latgé, Jean-Paul; Lopes-Bezerra, Leila M

    2017-01-16

    Aspergillus fumigatus, the main etiologic agent causing invasive aspergillosis, can induce an inflammatory response and a prothrombotic phenotype upon contact with human umbilical vein endothelial cells (HUVECs). However, the fungal molecules involved in this endothelial response remain unknown. A. fumigatus hyphae produce an extracellular matrix composed of galactomannan, galactosaminogalactan and α-(1,3)-glucan. In this study, we investigated the consequences of UGM1 gene deletion in A. fumigatus, which produces a mutant with increased galactosaminogalactan production. The ∆ugm1 mutant exhibited an HUVEC-hyperadhesive phenotype and induced increased endothelial TNF-α secretion and tissue factor mRNA overexpression in this "semi-professional" immune host cell. Using a shotgun proteomics approach, we show that the A. fumigatus ∆ugm1 strain can modulate the levels of proteins in important endothelial pathways related to the inflammatory response mediated by TNF-α and to stress response pathways. Furthermore, a purified galactosaminogalactan fraction was also able to induce TNF-α secretion and the coincident HUVEC pathways regulated by the ∆ugm1 mutant, which overexpresses this component, as demonstrated by fluorescence microscopy. This work contributes new data regarding endothelial mechanisms in response to A. fumigatus infection. Invasive aspergillosis is the main opportunistic fungal infection described in neutropenic hematologic patients. One important clinical aspect of this invasive fungal infection is vascular thrombosis, which could be related, at least in part, to the activation of endothelial cells, as shown in previous reports from our group. It is known that direct contact between the A. fumigatus hyphal cell wall and the HUVEC cell surface is necessary to induce an endothelial prothrombotic phenotype and secretion of pro-inflammatory cytokines, though the cell surface components of this angioinvasive fungus that trigger this endothelial

  8. Characterization of a Vibrio vulnificus LysR homologue, HupR, which regulates expression of the haem uptake outer membrane protein, HupA.

    Litwin, C M; Quackenbush, J

    2001-12-01

    In Vibrio vulnificus, the ability to acquire iron from the host has been shown to correlate with virulence. Here, we show that the DNA upstream of hupA (haem uptake receptor) in V. vulnificus encodes a protein in the inverse orientation to hupA (named hupR). HupR shares homology with the LysR family of positive transcriptional activators. A hupA-lacZ fusion contained on a plasmid was transformed into Fur(-), Fur(+)and HupR(-)strains of V. vulnificus. The beta-galactosidase assays and Northern blot analysis showed that transcription of hupA is negatively regulated by iron and the Fur repressor in V. vulnificus. Under low-iron conditions with added haemin, the expression of hupA in the hupR mutant was significantly lower than in the wild-type. This diminished response to haem was detected by both Northern blot and hupA-lacZ fusion analysis. The haem response of hupA in the hupR mutant was restored to wild-type levels when complemented with hupR in trans. These studies suggest that HupR may act as a positive regulator of hupA transcription under low-iron conditions in the presence of haemin. Copyright 2001 Academic Press.

  9. Outer atmospheric research

    Anderson, J.L.

    1988-01-01

    The region above the earth from about 90 km to 150 km is a major part of the upper or outer atmosphere. It is relatively unexplored, being too high for balloons or aircraft and too low for persistent orbiting spacecraft. However, the concept of a tethered subsatellite, deployed downward from an orbiting, more massive craft such as the Space Shuttle, opens the possibility of a research capability that could provide global mapping of this region. The need for research in this thick spherical shell above the earth falls into two major categories: (1) scientific data for understanding and modeling the global atmosphere and thereby determining its role in the earth system, and (2) engineering data for the design of future aerospace vehicles that will operate there. This paper presents an overview and synthesis of the currently perceived research needs and the state-of-the-art of the proposed tethered research capability. 16 references

  10. Efficient activation of T cells by human monocyte-derived dendritic cells (HMDCs pulsed with Coxiella burnetii outer membrane protein Com1 but not by HspB-pulsed HMDCs

    Wang Xile

    2011-09-01

    Full Text Available Abstract Background Coxiella burnetii is an obligate intracellular bacterium and the etiologic agent of Q fever; both coxiella outer membrane protein 1 (Com1 and heat shock protein B (HspB are its major immunodominant antigens. It is not clear whether Com1 and HspB have the ability to mount immune responses against C. burnetii infection. Results The recombinant proteins Com1 and HspB were applied to pulse human monocyte-derived dendritic cells (HMDCs, and the pulsed HMDCs were used to stimulate isogenic T cells. Com1-pulsed HMDCs expressed substantially higher levels of surface molecules (CD83, CD40, CD80, CD86, CD54, and CD58 and a higher level of interleukin-12 than HspB-pulsed HMDCs. Moreover, Com1-pulsed HMDCs induced high-level proliferation and activation of CD4+ and CD8+ cells, which expressed high levels of T-cell activation marker CD69 and inflammatory cytokines IFN-γ and TNF-α. In contrast, HspB-pulsed HMDCs were unable to induce efficient T-cell proliferation and activation. Conclusions Our results demonstrate that Com1-pulsed HMDCs are able to induce efficient T-cell proliferation and drive T cells toward Th1 and Tc1 polarization; however, HspB-pulsed HMDCs are unable to do so. Unlike HspB, Com1 is a protective antigen, which was demonstrated by the adoptive transfer of Com1-pulsed bone marrow dendritic cells into naive BALB/c mice.

  11. Vacuum Outer-Gap Structure in Pulsar Outer Magnetospheres

    Gui-Fang, Lin; Li, Zhang

    2009-01-01

    We study the vacuum outer-gap structure in the outer magnetosphere of rotation-powered pulsars by considering the limit of trans-field height through a pair production process. In this case, the trans-field height is limited by the photon-photon pair production process and the outer boundary of the outer gap can be extended outside the light cylinder. By solving self-consistently the Poisson equation for electrical potential and the Boltzmann equations of electrons/positrons and γ-rays in a vacuum outer gap for the parameters of Vela pulsar, we obtain an approximate geometry of the outer gap, i.e. the trans-field height is limited by the pair-production process and increases with the radial distance to the star and the width of the outer gap starts at the inner boundary (near the null charge surface) and ends at the outer boundary which locates inside or outside the light cylinder depending on the inclination angle. (geophysics, astronomy, and astrophysics)

  12. Experimental validation of the predicted binding site of Escherichia coli K1 outer membrane protein A to human brain microvascular endothelial cells: identification of critical mutations that prevent E. coli meningitis.

    Pascal, Tod A; Abrol, Ravinder; Mittal, Rahul; Wang, Ying; Prasadarao, Nemani V; Goddard, William A

    2010-11-26

    Escherichia coli K1, the most common cause of meningitis in neonates, has been shown to interact with GlcNAc1-4GlcNAc epitopes of Ecgp96 on human brain microvascular endothelial cells (HBMECs) via OmpA (outer membrane protein A). However, the precise domains of extracellular loops of OmpA interacting with the chitobiose epitopes have not been elucidated. We report the loop-barrel model of these OmpA interactions with the carbohydrate moieties of Ecgp96 predicted from molecular modeling. To test this model experimentally, we generated E. coli K1 strains expressing OmpA with mutations of residues predicted to be critical for interaction with the HBMEC and tested E. coli invasion efficiency. For these same mutations, we predicted the interaction free energies (including explicit calculation of the entropy) from molecular dynamics (MD), finding excellent correlation (R(2) = 90%) with experimental invasion efficiency. Particularly important is that mutating specific residues in loops 1, 2, and 4 to alanines resulted in significant inhibition of E. coli K1 invasion in HBMECs, which is consistent with the complete lack of binding found in the MD simulations for these two cases. These studies suggest that inhibition of the interactions of these residues of Loop 1, 2, and 4 with Ecgp96 could provide a therapeutic strategy to prevent neonatal meningitis due to E. coli K1.

  13. Vaccination of koalas (Phascolarctos cinereus) with a recombinant chlamydial major outer membrane protein adjuvanted with poly I:C, a host defense peptide and polyphosphazine, elicits strong and long lasting cellular and humoral immune responses.

    Khan, Shahneaz Ali; Waugh, Courtney; Rawlinson, Galit; Brumm, Jacqui; Nilsson, Karen; Gerdts, Volker; Potter, Andrew; Polkinghorne, Adam; Beagley, Kenneth; Timms, Peter

    2014-10-07

    Chlamydial infections are wide spread in koalas across their range and a solution to this debilitating disease has been sought for over a decade. Antibiotics are the currently accepted therapeutic measure, but are not an effective treatment due to the asymptomatic nature of some infections and a low efficacy rate. Thus, a vaccine would be an ideal way to address this infectious disease threat in the wild. Previous vaccine trials have used a three-dose regimen; however this is very difficult to apply in the field as it would require multiple capture events, which are stressful and invasive processes for the koala. In addition, it requires skilled koala handlers and a significant monetary investment. To overcome these challenges, in this study we utilized a polyphosphazine based poly I:C and a host defense peptide adjuvant combined with recombinant chlamydial major outer membrane protein (rMOMP) antigen to induce long lasting (54 weeks) cellular and humoral immunity in female koalas with a novel single immunizing dose. Immunized koalas produced a strong IgG response in plasma, as well as at mucosal sites. Moreover, they showed high levels of C. pecorum specific neutralizing antibodies in the plasma as well as vaginal and conjunctival secretions. Lastly, Chlamydia-specific lymphocyte proliferation responses were produced against both whole chlamydial elementary bodies and rMOMP protein, over the 12-month period. The results of this study suggest that a single dose rMOMP vaccine incorporating a poly I:C, host defense peptide and polyphosphazine adjuvant is able to stimulate both arms of the immune system in koalas, thereby providing an alternative to antibiotic treatment and/or a three-dose vaccine regime. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. THE OUTER MEMBRANE OF PATHOGENIC REPRESENTATIVES OF THE LEPTOSPIRA GENIUS

    A. N. Vaganova

    2011-01-01

    Full Text Available Abstract. Pathogenic leptospires can infect wide spectrum of hosts and they can survive in the environment long time. The outer membrane is the cellular component participated in interaction of microorganisms and environment. In present time several proteins located in the outer membrane of leptospires which are responsible for colonization of host organism, protection from influence of immune system of host, transport of substances in to the cell and other processes have been described. The outer membrane contains proteins and lipopolysaccharide molecules which have citotoxic effect. It was shown that regulation of protein composition of membranes depends on several factors of environment such as temperature, osmolarity, presence of certain substances in environment. Lipopolysaccharide and protein molecules of outer membranes have antigenic properties. These molecules can be used in practice as the components of vaccine against leptospiroses and diagnostic tools. Current review summarize information concerning structural organization of the outer membrane of leptospires, diversities of incoming parts of molecules and regulation of their synthesis. Moreover, perspectives of practical using of the outer membrane components in diagnostics and prevention of leptospiroses are presented.

  15. Profiling the outer membrane proteome during growth and development of the social bacterium Myxococcus xanthus by selective biotinylation and analyses of outer membrane vesicles.

    Kahnt, Jörg; Aguiluz, Kryssia; Koch, Jürgen; Treuner-Lange, Anke; Konovalova, Anna; Huntley, Stuart; Hoppert, Michael; Søgaard-Andersen, Lotte; Hedderich, Reiner

    2010-10-01

    Social behavior in the bacterium Myxococcus xanthus relies on contact-dependent activities involving cell-cell and cell-substratum interactions. To identify outer membrane proteins that have a role in these activities, we profiled the outer membrane proteome of growing and starving cells using two strategies. First, outer membrane proteins were enriched by biotinylation of intact cells using the reagent NHS (N-hydroxysuccinimide)-PEO(12) (polyethylene oxide)-biotin with subsequent membrane solubilization and affinity chromatography. Second, the proteome of outer membrane vesicles (OMV) was determined. Comparisons of detected proteins show that these methods have different detection profiles and together provide a comprehensive view of the outer membrane proteome. From 362 proteins identified, 274 (76%) were cell envelope proteins including 64 integral outer membrane proteins and 85 lipoproteins. The majority of these proteins were of unknown function. Among integral outer membrane proteins with homologues of known function, TonB-dependent transporters comprise the largest group. Our data suggest novel functions for these transporters. Among lipoproteins with homologues of known function, proteins with hydrolytic functions comprise the largest group. The luminal load of OMV was enriched for proteins with hydrolytic functions. Our data suggest that OMV have functions in predation and possibly in transfer of intercellular signaling molecules between cells.

  16. Phylogenetic and bioinformatic analysis of gap junction-related proteins, innexins, pannexins and connexins.

    Fushiki, Daisuke; Hamada, Yasuo; Yoshimura, Ryoichi; Endo, Yasuhisa

    2010-04-01

    All multi-cellular animals, including hydra, insects and vertebrates, develop gap junctions, which communicate directly with neighboring cells. Gap junctions consist of protein families called connexins in vertebrates and innexins in invertebrates. Connexins and innexins have no homology in their amino acid sequence, but both are thought to have some similar characteristics, such as a tetra-membrane-spanning structure, formation of a channel by hexamer, and transmission of small molecules (e.g. ions) to neighboring cells. Pannexins were recently identified as a homolog of innexins in vertebrate genomes. Although pannexins are thought to share the function of intercellular communication with connexins and innexins, there is little information about the relationship among these three protein families of gap junctions. We phylgenetically and bioinformatically examined these protein families and other tetra-membrane-spanning proteins using a database and three analytical softwares. The clades formed by pannexin families do not belong to the species classification but do to paralogs of each member of pannexins. Amino acid sequences of pannexins are closely related to those of innexins but less to those of connexins. These data suggest that innexins and pannexins have a common origin, but the relationship between innexins/pannexins and connexins is as slight as that of other tetra-membrane-spanning members.

  17. Turbine airfoil with outer wall thickness indicators

    Marra, John J; James, Allister W; Merrill, Gary B

    2013-08-06

    A turbine airfoil usable in a turbine engine and including a depth indicator for determining outer wall blade thickness. The airfoil may include an outer wall having a plurality of grooves in the outer surface of the outer wall. The grooves may have a depth that represents a desired outer surface and wall thickness of the outer wall. The material forming an outer surface of the outer wall may be removed to be flush with an innermost point in each groove, thereby reducing the wall thickness and increasing efficiency. The plurality of grooves may be positioned in a radially outer region of the airfoil proximate to the tip.

  18. Analysis of putative chemoreceptor proteins of Campylobacter jejuni

    Vegge, Christina Skovgaard; Brøndsted, Lone; Bang, Dang D.

    Campylobacter jejuni is the primary food borne bacterial pathogen in the developed world. A very important reservoir for C. jejuni is the gut of chickens, which are colonized efficiently and commensally by this organism. Predominantly the mucus filled crypts of the lower gastrointestinal tract...... are being analyzed in adherence and invasion assays with both human and chicken cells to explore the possibility that these membrane spanning proteins interact with host cells rather than operating as chemoreceptors....

  19. NMR structural studies of peptides and proteins in membranes

    Opella, S J [Pennsylvania Univ., Philadelphia, PA (United States). Dept. of Chemistry

    1994-12-31

    The use of NMR methodology in structural studies is described as applicable to larger proteins, considering that the majority of membrane proteins is constructed from a limited repertoire of structural and dynamic elements. The membrane associated domains of these proteins are made up of long hydrophobic membrane spanning helices, shorter amphipathic bridging helices in the plane of the bilayer, connecting loops with varying degrees of mobility, and mobile N- and C- terminal sections. NMR studies have been successful in identifying all of these elements and their orientations relative to each other and the membrane bilayer 19 refs., 9 figs.

  20. Protection by meningococcal outer membrane protein PorA-specific antibodies and a serogroup B capsular polysaccharide-specific antibody in complement-sufficient and C6-deficient infant rats.

    Toropainen, Maija; Saarinen, Leena; Vidarsson, Gestur; Käyhty, Helena

    2006-05-01

    The relative contributions of antibody-induced complement-mediated bacterial lysis and antibody/complement-mediated phagocytosis to host immunity against meningococcal infections are currently unclear. Further, the in vivo effector functions of antibodies may vary depending on their specificity and Fc heavy-chain isotype. In this study, a mouse immunoglobulin G2a (mIgG2a) monoclonal antibody (MN12H2) to meningococcal outer membrane protein PorA (P1.16), its human IgG subclass derivatives (hIgG1 to hIgG4), and an mIgG2a monoclonal antibody (Nmb735) to serogroup B capsular polysaccharide (B-PS) were evaluated for passive protection against meningococcal serogroup B strain 44/76-SL (B:15:P1.7,16) in an infant rat infection model. Complement component C6-deficient (PVG/c-) rats were used to assess the importance of complement-mediated bacterial lysis for protection. The PorA-specific parental mIgG2a and the hIgG1 to hIgG3 derivatives all induced efficient bactericidal activity in vitro in the presence of human or infant rat complement and augmented bacterial clearance in complement-sufficient HsdBrlHan:WIST rats, while the hIgG4 was unable to do so. In C6-deficient PVG/c- rats, lacking complement-mediated bacterial lysis, the augmentation of bacterial clearance by PorA-specific mIgG2a and hIgG1 antibodies was impaired compared to that in the syngeneic complement-sufficient PVG/c+ rat strain. This was in contrast to the case for B-PS-specific mIgG2a, which conferred similar protective activity in both rat strains. These data suggest that while anti-B-PS antibody can provide protection in the infant rats without membrane attack complex formation, the protection afforded by anti-PorA antibody is more dependent on the activation of the whole complement pathway and subsequent bacterial lysis.

  1. Unlipidated Outer Membrane Protein Omp16 (U-Omp16) from Brucella spp. as Nasal Adjuvant Induces a Th1 Immune Response and Modulates the Th2 Allergic Response to Cow’s Milk Proteins

    Ibañez, Andrés E.; Smaldini, Paola; Coria, Lorena M.; Delpino, María V.; Pacífico, Lucila G. G.; Oliveira, Sergio C.; Risso, Gabriela S.; Pasquevich, Karina A.; Fossati, Carlos Alberto; Giambartolomei, Guillermo H.; Docena, Guillermo H.; Cassataro, Juliana

    2013-01-01

    The discovery of novel mucosal adjuvants will help to develop new formulations to control infectious and allergic diseases. In this work we demonstrate that U-Omp16 from Brucella spp. delivered by the nasal route (i.n.) induced an inflammatory immune response in bronchoalveolar lavage (BAL) and lung tissues. Nasal co-administration of U-Omp16 with the model antigen (Ag) ovalbumin (OVA) increased the amount of Ag in lung tissues and induced OVA-specific systemic IgG and T helper (Th) 1 immune responses. The usefulness of U-Omp16 was also assessed in a mouse model of food allergy. U-Omp16 i.n. administration during sensitization ameliorated the hypersensitivity responses of sensitized mice upon oral exposure to Cow’s Milk Protein (CMP), decreased clinical signs, reduced anti-CMP IgE serum antibodies and modulated the Th2 response in favor of Th1 immunity. Thus, U-Omp16 could be used as a broad Th1 mucosal adjuvant for different Ag formulations. PMID:23861971

  2. The CMS Outer Hadron Calorimeter

    Acharya, Bannaje Sripathi; Banerjee, Sunanda; Banerjee, Sudeshna; Bawa, Harinder Singh; Beri, Suman Bala; Bhandari, Virender; Bhatnagar, Vipin; Chendvankar, Sanjay; Deshpande, Pandurang Vishnu; Dugad, Shashikant; Ganguli, Som N; Guchait, Monoranjan; Gurtu, Atul; Kalmani, Suresh Devendrappa; Kaur, Manjit; Kohli, Jatinder Mohan; Krishnaswamy, Marthi Ramaswamy; Kumar, Arun; Maity, Manas; Majumder, Gobinda; Mazumdar, Kajari; Mondal, Naba Kumar; Nagaraj, P; Narasimham, Vemuri Syamala; Patil, Mandakini Ravindra; Reddy, L V; Satyanarayana, B; Sharma, Seema; Singh, B; Singh, Jas Bir; Sudhakar, Katta; Tonwar, Suresh C; Verma, Piyush

    2006-01-01

    The CMS hadron calorimeter is a sampling calorimeter with brass absorber and plastic scintillator tiles with wavelength shifting fibres for carrying the light to the readout device. The barrel hadron calorimeter is complemented with a outer calorimeter to ensure high energy shower containment in CMS and thus working as a tail catcher. Fabrication, testing and calibrations of the outer hadron calorimeter are carried out keeping in mind its importance in the energy measurement of jets in view of linearity and resolution. It will provide a net improvement in missing $\\et$ measurements at LHC energies. The outer hadron calorimeter has a very good signal to background ratio even for a minimum ionising particle and can hence be used in coincidence with the Resistive Plate Chambers of the CMS detector for the muon trigger.

  3. Origin of Outer Solar System

    Holman, Matthew J.; Lindstrom, David (Technical Monitor)

    2005-01-01

    Our ongoing research program combines extensive deep and wide-field observations using a variety of observational platforms with numerical studies of the dynamics of small bodies in the outer solar system in order to advance the main scientific goals of the community studying the Kuiper belt and the outer solar system. These include: (1) determining the relative populations of the known classes of KBOs as well as other possible classes; ( 2 ) determining the size distributions or luminosity function of the individual populations or the Kuiper belt as a whole; (3) determining the inclinations distributions of these populations; (4) establishing the radial extent of the Kuiper belt; ( 5 ) measuring and relating the physical properties of different types of KBOs to those of other solar system bodies; and, (6) completing our systematic inventory of the satellites of the outer planets.

  4. Nuclear fuel grid outer strap

    Duncan, R.; Craver, J.E.

    1989-01-01

    This patent describes a nuclear reactor fuel assembly grid. It comprises a first outer grip strap segment end. The first end having a first tab arranged in substantially the same plane as the plane defined by the first end; a second outer grip strap end. The second end having a second slot arranged in substantially the same plane as the plane defined by the second end, with the tab being substantially disposed in the slot, defining a socket therebetween; and a fort tine interposed substantially perpendicularly in the socket

  5. Getting Sloshed in Outer Space

    Home; Journals; Resonance – Journal of Science Education; Volume 12; Issue 12. Getting Sloshed in Outer Space - Liquid Behavior in Microgravity. N Ananthkrishnan. General Article Volume 12 Issue 12 December 2007 pp 40-45. Fulltext. Click here to view fulltext PDF. Permanent link:

  6. Outer space structure and development

    Zeldovich, J.; Novikov, I.

    1975-01-01

    A brief account is presented answering the question of what in fact the outer space we observe consists of. The principle of spatial homogeneity of the universe and the idea of non-stationary cosmology are discussed. The origin and the future development of the universe are explained using the two above mentioned and some other hypotheses. (J.K.)

  7. Outer space structure and development

    Zeldovich, J; Novikov, I

    1975-10-01

    A brief account is presented answering the question of what in fact the outer space we observe consists of. The principle of spatial homogeneity of the universe and the idea of non-stationary cosmology are discussed. The origin and the future development of the universe are explained using the two above mentioned and some other hypotheses.

  8. Plasmas in the outer heliosphere

    Belcher, J. W.; Richardson, J. D.; Lazarus, A. J.; Gazis, P. R.; Barnes, A.

    1995-01-01

    We review the observed properties of the solar wind in the outer heliosphere, including observations from Voyager and the Pioneers, as well as from inner heliospheric probes as appropriate. These observations are crucial to modeling of the heliosphere and its interactions with the interstellar medium, since the wind ram pressure and its temporal variations are important in understanding the distance to the termination shock and heliopause and how those boundaries might vary in time. We focus on results since Solar Wind 7. Among the issues we will discuss are: (1) the time scales for and statistical properties of variations in the ram pressure in the outer heliosphere, and how those variations might affect the morphology of the heliospheric/interstellar medium interface; (2) the question of possible solar wind slowing in the outer heliosphere due to the pick-up of interstellar ions; (3) the issue of whether there is bulk heating of the solar wind associated either with interstellar ion pick-up or with continued heating due to stream-stream interactions; (4) evidence for latitudinal variations in solar wind properties; and (5) the 1.3 year periodicities apparent in the outer heliosphere, and the close correspondence with similar variations seen with inner heliospheric probes.

  9. Outer Synchronization of Complex Networks by Impulse

    Sun Wen; Yan Zizong; Chen Shihua; Lü Jinhu

    2011-01-01

    This paper investigates outer synchronization of complex networks, especially, outer complete synchronization and outer anti-synchronization between the driving network and the response network. Employing the impulsive control method which is uncontinuous, simple, efficient, low-cost and easy to implement in practical applications, we obtain some sufficient conditions of outer complete synchronization and outer anti-synchronization between two complex networks. Numerical simulations demonstrate the effectiveness of the proposed impulsive control scheme. (general)

  10. Generation of a genetically encoded marker of rod photoreceptor outer segment growth and renewal

    John J. Willoughby

    2011-10-01

    Vertebrate photoreceptors are specialized light sensing neurons. The photoreceptor outer segment is a highly modified cilium where photons of light are transduced into a chemical and electrical signal. The outer segment has the typical cilary axoneme but, in addition, it has a large number of densely packed, stacked, intramembranous discs. The molecular and cellular mechanisms that contribute to vertebrate photoreceptor outer segment morphogenesis are still largely unknown. Unlike typical cilia, the outer segment is continuously regenerated or renewed throughout the life of the animal through the combined process of distal outer segment shedding and proximal outer segment growth. The process of outer segment renewal was discovered over forty years ago, but we still lack an understanding of how photoreceptors renew their outer segments and few, if any, molecular mechanisms that regulate outer segment growth or shedding have been described. Our lack of progress in understanding how photoreceptors renew their outer segments has been hampered by the difficulty in measuring rates of renewal. We have created a new method that uses heat-shock induction of a fluorescent protein that can be used to rapidly measure outer segment growth rates. We describe this method, the stable transgenic line we created, and the growth rates observed in larval and adult rod photoreceptors using this new method. This new method will allow us to begin to define the genetic and molecular mechanisms that regulate rod outer segment renewal, a crucial aspect of photoreceptor function and, possibly, viability.

  11. Exploring bacterial outer membrane barrier to combat bad bugs

    Ghai I

    2017-08-01

    Full Text Available Ishan Ghai,1 Shashank Ghai2 1School of Engineering and Life Sciences, Jacobs University, Bremen, 2Leibniz University, Hannover, Germany Abstract: One of the main fundamental mechanisms of antibiotic resistance in Gram-negative bacteria comprises an effective change in the membrane permeability to antibiotics. The Gram-negative bacterial complex cell envelope comprises an outer membrane that delimits the periplasm from the exterior environment. The outer membrane contains numerous protein channels, termed as porins or nanopores, which are mainly involved in the influx of hydrophilic compounds, including antibiotics. Bacterial adaptation to reduce influx through these outer membrane proteins (Omps is one of the crucial mechanisms behind antibiotic resistance. Thus to interpret the molecular basis of the outer membrane permeability is the current challenge. This review attempts to develop a state of knowledge pertinent to Omps and their effective role in antibiotic influx. Further, it aims to study the bacterial response to antibiotic membrane permeability and hopefully provoke a discussion toward understanding and further exploration of prospects to improve our knowledge on physicochemical parameters that direct the translocation of antibiotics through the bacterial membrane protein channels. Keywords: antibiotics, Gram-negative bacteria, cell envelope, protein channels, nanopores, influx, antibiotic resistance

  12. Turbine airfoil with a compliant outer wall

    Campbell, Christian X [Oviedo, FL; Morrison, Jay A [Oviedo, FL

    2012-04-03

    A turbine airfoil usable in a turbine engine with a cooling system and a compliant dual wall configuration configured to enable thermal expansion between inner and outer layers while eliminating stress formation in the outer layer is disclosed. The compliant dual wall configuration may be formed a dual wall formed from inner and outer layers separated by a support structure. The outer layer may be a compliant layer configured such that the outer layer may thermally expand and thereby reduce the stress within the outer layer. The outer layer may be formed from a nonplanar surface configured to thermally expand. In another embodiment, the outer layer may be planar and include a plurality of slots enabling unrestricted thermal expansion in a direction aligned with the outer layer.

  13. Topology of Legionella pneumophila DotA: an inner membrane protein required for replication in macrophages.

    Roy, C R; Isberg, R R

    1997-01-01

    The Legionella pneumophila dotA gene is required for intracellular growth of the bacterium in macrophages. In this study, a structure-function analysis of the DotA protein was conducted to elucidate the role of this protein in L. pneumophila pathogenesis. Translational fusions of dotA to the Escherichia coli phoA and lacZ genes indicated that DotA is an integral cytoplasmic membrane protein with eight membrane-spanning domains. DotA contains two large periplasmic domains of approximately 503 ...

  14. Exploring bacterial outer membrane barrier to combat bad bugs.

    Ghai, Ishan; Ghai, Shashank

    2017-01-01

    One of the main fundamental mechanisms of antibiotic resistance in Gram-negative bacteria comprises an effective change in the membrane permeability to antibiotics. The Gram-negative bacterial complex cell envelope comprises an outer membrane that delimits the periplasm from the exterior environment. The outer membrane contains numerous protein channels, termed as porins or nanopores, which are mainly involved in the influx of hydrophilic compounds, including antibiotics. Bacterial adaptation to reduce influx through these outer membrane proteins (Omps) is one of the crucial mechanisms behind antibiotic resistance. Thus to interpret the molecular basis of the outer membrane permeability is the current challenge. This review attempts to develop a state of knowledge pertinent to Omps and their effective role in antibiotic influx. Further, it aims to study the bacterial response to antibiotic membrane permeability and hopefully provoke a discussion toward understanding and further exploration of prospects to improve our knowledge on physicochemical parameters that direct the translocation of antibiotics through the bacterial membrane protein channels.

  15. Mitochondrial dysfunction underlying outer retinal diseases

    Lefevere, Evy; Toft-Kehler, Anne Katrine; Vohra, Rupali

    2017-01-01

    Dysfunction of photoreceptors, retinal pigment epithelium (RPE) or both contribute to the initiation and progression of several outer retinal disorders. Disrupted Müller glia function might additionally subsidize to these diseases. Mitochondrial malfunctioning is importantly associated with outer...

  16. Substrate specificity within a family of outer membrane carboxylate channels.

    Elif Eren

    2012-01-01

    Full Text Available Many Gram-negative bacteria, including human pathogens such as Pseudomonas aeruginosa, do not have large-channel porins. This results in an outer membrane (OM that is highly impermeable to small polar molecules, making the bacteria intrinsically resistant towards many antibiotics. In such microorganisms, the majority of small molecules are taken up by members of the OprD outer membrane protein family. Here we show that OprD channels require a carboxyl group in the substrate for efficient transport, and based on this we have renamed the family Occ, for outer membrane carboxylate channels. We further show that Occ channels can be divided into two subfamilies, based on their very different substrate specificities. Our results rationalize how certain bacteria can efficiently take up a variety of substrates under nutrient-poor conditions without compromising membrane permeability. In addition, they explain how channel inactivation in response to antibiotics can cause resistance but does not lead to decreased fitness.

  17. WORKSHOP: Inner space - outer space

    Anon.

    1984-01-01

    During the first week of May, the Fermilab theoretical astrophysics group hosted an international conference on science at the interface of particle physics and cosmology/astrophysics. The conference (Inner Space-Outer Space) was attended by a very diverse group of more than 200 physical scientists, including astronomers, astrophysicists, cosmologists, low-temperature physicists, and elementary particle theorists and experimentalists. The common interest which brought this diverse group to gether is the connection between physics on the smallest scale probed by man - the realm of elementary particle physics - and physics on the largest scale imaginable (the entire Universe) - the realm of cosmology

  18. WORKSHOP: Inner space - outer space

    Anon.

    1984-09-15

    During the first week of May, the Fermilab theoretical astrophysics group hosted an international conference on science at the interface of particle physics and cosmology/astrophysics. The conference (Inner Space-Outer Space) was attended by a very diverse group of more than 200 physical scientists, including astronomers, astrophysicists, cosmologists, low-temperature physicists, and elementary particle theorists and experimentalists. The common interest which brought this diverse group to gether is the connection between physics on the smallest scale probed by man - the realm of elementary particle physics - and physics on the largest scale imaginable (the entire Universe) - the realm of cosmology.

  19. Outer scale of atmospheric turbulence

    Lukin, Vladimir P.

    2005-10-01

    In the early 70's, the scientists in Italy (A.Consortini, M.Bertolotti, L.Ronchi), USA (R.Buser, Ochs, S.Clifford) and USSR (V.Pokasov, V.Lukin) almost simultaneously discovered the phenomenon of deviation from the power law and the effect of saturation for the structure phase function. During a period of 35 years we have performed successively the investigations of the effect of low-frequency spectral range of atmospheric turbulence on the optical characteristics. The influence of the turbulence models as well as a outer scale of turbulence on the characteristics of telescopes and systems of laser beam formations has been determined too.

  20. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.

    Thomas Kieselbach

    Full Text Available Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT and leukotoxin (LtxA into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs. To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e using liquid chromatography-tandem mass spectrometry (LC-MS/MS. This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease.

  1. The Effects of Urethane on Rat Outer Hair Cells

    Mingyu Fu

    2016-01-01

    Full Text Available The cochlea converts sound vibration into electrical impulses and amplifies the low-level sound signal. Urethane, a widely used anesthetic in animal research, has been shown to reduce the neural responses to auditory stimuli. However, the effects of urethane on cochlea, especially on the function of outer hair cells, remain largely unknown. In the present study, we compared the cochlear microphonic responses between awake and urethane-anesthetized rats. The results revealed that the amplitude of the cochlear microphonic was decreased by urethane, resulting in an increase in the threshold at all of the sound frequencies examined. To deduce the possible mechanism underlying the urethane-induced decrease in cochlear sensitivity, we examined the electrical response properties of isolated outer hair cells using whole-cell patch-clamp recording. We found that urethane hyperpolarizes the outer hair cell membrane potential in a dose-dependent manner and elicits larger outward current. This urethane-induced outward current was blocked by strychnine, an antagonist of the α9 subunit of the nicotinic acetylcholine receptor. Meanwhile, the function of the outer hair cell motor protein, prestin, was not affected. These results suggest that urethane anesthesia is expected to decrease the responses of outer hair cells, whereas the frequency selectivity of cochlea remains unchanged.

  2. Outer grid strap protruding spring repair apparatus

    Widener, W.H.

    1987-01-01

    This patent describes a nuclear fuel assembly grid spring repair apparatus for repairing a spring formed on an outer strap of a fuel assembly grid and having a portion protruding outwardly beyond the strap, the apparatus comprising: (a) a support frame defining an opening and having means defining a guide channel extending along the opening in a first direction; (b) means mounted on the frame and being adjustable for attaching the frame to the outer strap of the support grid so that the frame opening is aligned with the outwardly protruding spring on the outer strap; (c) an outer slide having a passageway defined therethrough and being mounted in the guide channel for reciprocable movement along the frame opening in the first direction for aligning the passageway with the outwardly protruding portion of the spring on the outer strap. The outer slide also has means defining a guide way extending along the passageway in a second direction generally orthogonal to the first direction; (d) a spring reset mechanism being operable for resetting the protruding spring to a nonprotruding position relative to the outer strap when the mechanism is aligned with the protruding portion of the spring; and (e) an inner slide supporting the spring reset mechanism and being mounted to the guide way for reciprocable movement along the passageway of the outer slide in the second direction for aligning the spring reset mechanism with the protruding portion of the spring on the outer strap

  3. Sorting of bacterial lipoproteins to the outer membrane by the Lol system.

    Narita, Shin-ichiro; Tokuda, Hajime

    2010-01-01

    Bacterial lipoproteins comprise a subset of membrane proteins with a lipid-modified cysteine residue at their amino termini through which they are anchored to the membrane. In Gram-negative bacteria, lipoproteins are localized on either the inner or the outer membrane. The Lol system is responsible for the transport of lipoproteins to the outer membrane.The Lol system comprises an inner-membrane ABC transporter LolCDE complex, a periplasmic carrier protein, LolA, and an outer membrane receptor protein, LolB. Lipoproteins are synthesized as precursors in the cytosol and then translocated across the inner membrane by the Sec translocon to the outer leaflet of the inner membrane, where lipoprotein precursors are processed to mature lipoproteins. The LolCDE complex then mediates the release of outer membrane-specific lipoproteins from the inner membrane while the inner membrane-specific lipoproteins possessing Asp at position 2 are not released by LolCDE because it functions as a LolCDE avoidance signal, causing the retention of these lipoproteins in the inner membrane. A water-soluble lipoprotein-LolA complex is formed as a result of the release reaction mediated by LolCDE. This complex traverses the hydrophilic periplasm to reach the outer membrane, where LolB accepts a lipoprotein from LolA and then catalyzes its incorporation into the inner leaflet of the outer membrane.

  4. Improved method of measurement for outer leak

    Xu Guang

    2012-01-01

    Pneumatic pipeline is installed for the airborne radioactivity measurement equipment, air tightness and outer leak rate are essential for the testing of the characteristics, both in the national criteria and ISO standards, an improved practical method is available for the measurement of the outer air leak rate based on the engineering experiences for the equipment acceptance and testing procedure. (authors)

  5. Reverse Genetics for Fusogenic Bat-Borne Orthoreovirus Associated with Acute Respiratory Tract Infections in Humans: Role of Outer Capsid Protein σC in Viral Replication and Pathogenesis.

    Takahiro Kawagishi

    2016-02-01

    Full Text Available Nelson Bay orthoreoviruses (NBVs are members of the fusogenic orthoreoviruses and possess 10-segmented double-stranded RNA genomes. NBV was first isolated from a fruit bat in Australia more than 40 years ago, but it was not associated with any disease. However, several NBV strains have been recently identified as causative agents for respiratory tract infections in humans. Isolation of these pathogenic bat reoviruses from patients suggests that NBVs have evolved to propagate in humans in the form of zoonosis. To date, no strategy has been developed to rescue infectious viruses from cloned cDNA for any member of the fusogenic orthoreoviruses. In this study, we report the development of a plasmid-based reverse genetics system free of helper viruses and independent of any selection for NBV isolated from humans with acute respiratory infection. cDNAs corresponding to each of the 10 full-length RNA gene segments of NBV were cotransfected into culture cells expressing T7 RNA polymerase, and viable NBV was isolated using a plaque assay. The growth kinetics and cell-to-cell fusion activity of recombinant strains, rescued using the reverse genetics system, were indistinguishable from those of native strains. We used the reverse genetics system to generate viruses deficient in the cell attachment protein σC to define the biological function of this protein in the viral life cycle. Our results with σC-deficient viruses demonstrated that σC is dispensable for cell attachment in several cell lines, including murine fibroblast L929 cells but not in human lung epithelial A549 cells, and plays a critical role in viral pathogenesis. We also used the system to rescue a virus that expresses a yellow fluorescent protein. The reverse genetics system developed in this study can be applied to study the propagation and pathogenesis of pathogenic NBVs and in the generation of recombinant NBVs for future vaccines and therapeutics.

  6. Spheres of influence: Porphyromonas gingivalis outer membrane vesicles.

    Gui, M J; Dashper, S G; Slakeski, N; Chen, Y-Y; Reynolds, E C

    2016-10-01

    Outer membrane vesicles (OMVs) are asymmetrical single bilayer membranous nanostructures produced by Gram-negative bacteria important for bacterial interaction with the environment. Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces OMVs that act as a virulence factor secretion system contributing to its pathogenicity. Despite their biological importance, the mechanisms of OMV biogenesis have not been fully elucidated. The ~14 times more curvature of the OMV membrane than cell outer membrane (OM) indicates that OMV biogenesis requires energy expenditure for significant curvature of the OMV membrane. In P. gingivalis, we propose that this may be achieved by upregulating the production of certain inner or outer leaflet lipids, which causes localized outward curvature of the OM. This results in selection of anionic lipopolysaccharide (A-LPS) and associated C-terminal domain (CTD) -family proteins on the outer surface due to their ability to accommodate the curvature. Deacylation of A-LPS may further enable increased curvature leading to OMV formation. Porphyromonas gingivalis OMVs that are selectively enriched in CTD-family proteins, largely the gingipains, can support bacterial coaggregation, promote biofilm development and act as an intercessor for the transport of non-motile bacteria by motile bacteria. The P. gingivalis OMVs are also believed to contribute to host interaction and colonization, evasion of immune defense mechanisms, and destruction of periodontal tissues. They may be crucial for both micro- and macronutrient capture, especially heme and probably other assimilable compounds for its own benefit and that of the wider biofilm community. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Structural basis for alginate secretion across the bacterial outer membrane

    Whitney, J.C.; Robinson, H.; Hay, I. D.; Li, C.; Eckford, P. D. W.; Amaya, M. F.; Wood, L. F.; Ohman, D. E.; Bear, C. E.; Rehm, B. H.; Howell, P. L.

    2011-08-09

    Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate. Secretion of newly synthesized alginate across the outer membrane is believed to occur through the outer membrane protein AlgE. Here we report the 2.3 {angstrom} crystal structure of AlgE, which reveals a monomeric 18-stranded {beta}-barrel characterized by a highly electropositive pore constriction formed by an arginine-rich conduit that likely acts as a selectivity filter for the negatively charged alginate polymer. Interestingly, the pore constriction is occluded on either side by extracellular loop L2 and an unusually long periplasmic loop, T8. In halide efflux assays, deletion of loop T8 ({Delta}T8-AlgE) resulted in a threefold increase in anion flux compared to the wild-type or {Delta}L2-AlgE supporting the idea that AlgE forms a transport pathway through the membrane and suggesting that transport is regulated by T8. This model is further supported by in vivo experiments showing that complementation of an algE deletion mutant with {Delta}T8-AlgE impairs alginate production. Taken together, these studies support a mechanism for exopolysaccharide export across the outer membrane that is distinct from the Wza-mediated translocation observed in canonical capsular polysaccharide export systems.

  8. Structural Basis for Alginate Secretion Across the Bacterial Outer Membrane

    J Whitney; I Hay; C Li; P Eckford; H Robinson; M Amaya; L Wood; D Ohman; C Bear; et al.

    2011-12-31

    Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate. Secretion of newly synthesized alginate across the outer membrane is believed to occur through the outer membrane protein AlgE. Here we report the 2.3 {angstrom} crystal structure of AlgE, which reveals a monomeric 18-stranded {beta}-barrel characterized by a highly electropositive pore constriction formed by an arginine-rich conduit that likely acts as a selectivity filter for the negatively charged alginate polymer. Interestingly, the pore constriction is occluded on either side by extracellular loop L2 and an unusually long periplasmic loop, T8. In halide efflux assays, deletion of loop T8 ({Delta}T8-AlgE) resulted in a threefold increase in anion flux compared to the wild-type or {Delta}L2-AlgE supporting the idea that AlgE forms a transport pathway through the membrane and suggesting that transport is regulated by T8. This model is further supported by in vivo experiments showing that complementation of an algE deletion mutant with {Delta}T8-AlgE impairs alginate production. Taken together, these studies support a mechanism for exopolysaccharide export across the outer membrane that is distinct from the Wza-mediated translocation observed in canonical capsular polysaccharide export systems.

  9. Redefining the essential trafficking pathway for outer membrane lipoproteins

    Grabowicz, Marcin; Silhavy, Thomas J.

    2017-01-01

    In Gram-negative bacteria, most lipoproteins synthesized in the inner membrane (IM) are trafficked to the outer membrane (OM). The Lol pathway is the trafficking paradigm: LolCDE releases lipoproteins from the IM; LolA shuttles them between membranes to LolB in the OM. Several OM lipoproteins are essential for viability. In apparent concordance, the Lol proteins are each essential in wild-type cells. However, we show that Escherichia coli grows well without LolA and LolB in the absence of one...

  10. Oscillations of the Outer Boundary of the Outer Radiation Belt During Sawtooth Oscillations

    Jae-Hun Kim

    2006-09-01

    Full Text Available We report three sawtooth oscillation events observed at geosynchronous orbit where we find quasi-periodic (every 2-3 hours sudden flux increases followed by slow flux decreases at the energy levels of ˜50-400 keV. For these three sawtooth events, we have examined variations of the outer boundary of the outer radiation belt. In order to determine L values of the outer boundary, we have used data of relativistic electron flux observed by the SAMPEX satellite. We find that the outer boundary of the outer radiation belt oscillates periodically being consistent with sawtooth oscillation phases. Specifically, the outer boundary of the outer radiation belt expands (namely, the boundary L value increases following the sawtooth particle flux enhancement of each tooth, and then contracts (namely, the boundary L value decreases while the sawtooth flux decreases gradually until the next flux enhancement. On the other hand, it is repeatedly seen that the asymmetry of the magnetic field intensity between dayside and nightside decreases (increases due to the dipolarization (the stretching on the nightside as the sawtooth flux increases (decreases. This implies that the periodic magnetic field variations during the sawtooth oscillations are likely responsible for the expansion-contraction oscillations of the outer boundary of the outer radiation belt.

  11. Protein diffusion in plant cell plasma membranes: The cell-wall corral

    Alexandre eMartinière

    2013-12-01

    Full Text Available Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.

  12. Protein diffusion in plant cell plasma membranes: the cell-wall corral.

    Martinière, Alexandre; Runions, John

    2013-01-01

    Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.

  13. Outer planet probe cost estimates: First impressions

    Niehoff, J.

    1974-01-01

    An examination was made of early estimates of outer planetary atmospheric probe cost by comparing the estimates with past planetary projects. Of particular interest is identification of project elements which are likely cost drivers for future probe missions. Data are divided into two parts: first, the description of a cost model developed by SAI for the Planetary Programs Office of NASA, and second, use of this model and its data base to evaluate estimates of probe costs. Several observations are offered in conclusion regarding the credibility of current estimates and specific areas of the outer planet probe concept most vulnerable to cost escalation.

  14. A phase 1 study of a meningococcal native outer membrane vesicle vaccine made from a group B strain with deleted lpxL1 and synX, over-expressed factor H binding protein, two PorAs and stabilized OpcA expression.

    Keiser, P B; Biggs-Cicatelli, S; Moran, E E; Schmiel, D H; Pinto, V B; Burden, R E; Miller, L B; Moon, J E; Bowden, R A; Cummings, J F; Zollinger, W D

    2011-02-04

    This phase I clinical trial assessed the safety and immunogenicity of a native outer membrane vesicle (NOMV) vaccine prepared from an lpxL1(-) synX(-) mutant of strain 8570(B:4:P1.19,15:L8-5) of Neisseria meningitidis. Additional mutations enhance the expression of factor H binding protein variant 1 (fHbp v.1), stabilize expression of OpcA and introduce a second PorA (P1.22,14). Thirty-six volunteers were assigned to one of four dose groups (10, 25, 50 and 75 mcg, based on protein content) to receive three intramuscular injections at six week intervals with aluminum hydroxide adjuvant. Specific local and systemic adverse events were solicited by diary and at visits on days 2, 7, and 14 after each vaccination. Blood chemistries, complete blood count, and coagulation studies were measured on each vaccination day and again 2 and 14 days later. Blood for ELISA and serum bactericidal assays was drawn two and six weeks after each vaccination. The proportion of volunteers who developed a fourfold or greater increase in bactericidal activity to the wild type parent of the vaccine strain at two weeks after the third dose was 27 out of 34 (0.79, 95% C.I. 0.65-0.93). Against four other group B strains the response rate ranged from 41% to 82% indicating a good cross reactive antibody response. Depletion assays show contributions to bactericidal activity from antibodies to lipooligosaccharide (LOS), fHbp v.1 and OpcA. Published by Elsevier Ltd.

  15. Proteomic characterization of the outer membrane vesicle of the halophilic marine bacterium Novosphingobium pentaromativorans US6-1.

    Yun, Sung Ho; Lee, Sang-Yeop; Choi, Chi-Won; Lee, Hayoung; Ro, Hyun-Joo; Jun, Sangmi; Kwon, Yong Min; Kwon, Kae Kyoung; Kim, Sang-Jin; Kim, Gun-Hwa; Kim, Seung Il

    2017-01-01

    Novosphingobium pentaromativorans US6-1 is a Gram-negative halophilic marine bacterium able to utilize several polycyclic aromatic hydrocarbons such as phenanthrene, pyrene, and benzo[a]pyrene. In this study, using transmission electron microscopy, we confirmed that N. pentaromativorans US6-1 produces outer membrane vesicles (OMVs). N. pentaromativorans OMVs (hereafter OMV Novo ) are spherical in shape, and the average diameter of OMV Novo is 25-70 nm. Proteomic analysis revealed that outer membrane proteins and periplasmic proteins of N. pentaromativorans are the major protein components of OMV Novo . Comparative proteomic analysis with the membrane-associated protein fraction and correlation analysis demonstrated that the outer membrane proteins of OMV Novo originated from the membrane- associated protein fraction. To the best of our knowledge, this study is the first to characterize OMV purified from halophilic marine bacteria.

  16. Usher syndrome type 1-associated cadherins shape the photoreceptor outer segment.

    Schietroma, Cataldo; Parain, Karine; Estivalet, Amrit; Aghaie, Asadollah; Boutet de Monvel, Jacques; Picaud, Serge; Sahel, José-Alain; Perron, Muriel; El-Amraoui, Aziz; Petit, Christine

    2017-06-05

    Usher syndrome type 1 (USH1) causes combined hearing and sight defects, but how mutations in USH1 genes lead to retinal dystrophy in patients remains elusive. The USH1 protein complex is associated with calyceal processes, which are microvilli of unknown function surrounding the base of the photoreceptor outer segment. We show that in Xenopus tropicalis , these processes are connected to the outer-segment membrane by links composed of protocadherin-15 (USH1F protein). Protocadherin-15 deficiency, obtained by a knockdown approach, leads to impaired photoreceptor function and abnormally shaped photoreceptor outer segments. Rod basal outer disks displayed excessive outgrowth, and cone outer segments were curved, with lamellae of heterogeneous sizes, defects also observed upon knockdown of Cdh23 , encoding cadherin-23 (USH1D protein). The calyceal processes were virtually absent in cones and displayed markedly reduced F-actin content in rods, suggesting that protocadherin-15-containing links are essential for their development and/or maintenance. We propose that calyceal processes, together with their associated links, control the sizing of rod disks and cone lamellae throughout their daily renewal. © 2017 Schietroma et al.

  17. Usher syndrome type 1–associated cadherins shape the photoreceptor outer segment

    Parain, Karine; Aghaie, Asadollah; Picaud, Serge

    2017-01-01

    Usher syndrome type 1 (USH1) causes combined hearing and sight defects, but how mutations in USH1 genes lead to retinal dystrophy in patients remains elusive. The USH1 protein complex is associated with calyceal processes, which are microvilli of unknown function surrounding the base of the photoreceptor outer segment. We show that in Xenopus tropicalis, these processes are connected to the outer-segment membrane by links composed of protocadherin-15 (USH1F protein). Protocadherin-15 deficiency, obtained by a knockdown approach, leads to impaired photoreceptor function and abnormally shaped photoreceptor outer segments. Rod basal outer disks displayed excessive outgrowth, and cone outer segments were curved, with lamellae of heterogeneous sizes, defects also observed upon knockdown of Cdh23, encoding cadherin-23 (USH1D protein). The calyceal processes were virtually absent in cones and displayed markedly reduced F-actin content in rods, suggesting that protocadherin-15–containing links are essential for their development and/or maintenance. We propose that calyceal processes, together with their associated links, control the sizing of rod disks and cone lamellae throughout their daily renewal. PMID:28495838

  18. 27 CFR 9.207 - Outer Coastal Plain.

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Outer Coastal Plain. 9.207... Outer Coastal Plain. (a) Name. The name of the viticultural area described in this section is “Outer Coastal Plain”. For purposes of part 4 of this chapter, “Outer Coastal Plain” is a term of viticultural...

  19. Outer-2-independent domination in graphs

    independent dominating set of a graph is a set of vertices of such that every vertex of ()\\ has a neighbor in and the maximum vertex degree of the subgraph induced by ()\\ is at most one. The outer-2-independent domination ...

  20. Intershell correlations in photoionization of outer shells

    Amusia, M.Ya. [The Racah Institute of Physics, The Hebrew University of Jerusalem, Jerusalem 91904 (Israel); A.F. Ioffe Physical-Technical Institute, St. Petersburg 194021 (Russian Federation); Chernysheva, L.V. [A.F. Ioffe Physical-Technical Institute, St. Petersburg 194021 (Russian Federation); Drukarev, E.G. [National Research Center “Kurchatov Institute”, Konstantinov Petersburg Nuclear Physics Institute, St. Petersburg 188300 (Russian Federation)

    2016-02-15

    We demonstrate that the cross sections for photoionization of the outer shells are noticeably modified at the photon energies close to the thresholds of ionization of the inner shells due to correlations with the latter. The correlations may lead to increase or to decrease of the cross sections just above the ionization thresholds.

  1. Outer-2-independent domination in graphs

    Outer-2-independent domination in graphs. MARCIN KRZYWKOWSKI1,2,∗, DOOST ALI MOJDEH3 and MARYEM RAOOFI4. 1Department of Pure and Applied Mathematics, University of Johannesburg,. Johannesburg, South Africa. 2Faculty of Electronics, Telecommunications and Informatics, Gdansk University.

  2. Intershell correlations in photoionization of outer shells

    Amusia, M.Ya.; Chernysheva, L.V.; Drukarev, E.G.

    2016-01-01

    We demonstrate that the cross sections for photoionization of the outer shells are noticeably modified at the photon energies close to the thresholds of ionization of the inner shells due to correlations with the latter. The correlations may lead to increase or to decrease of the cross sections just above the ionization thresholds.

  3. Differential Response to Heat Stress in Outer and Inner Onion Bulb Scales.

    Galsurker, Ortal; Doron-Faigenboim, Adi; Teper-Bamnolker, Paula; Daus, Avinoam; Lers, Amnon; Eshel, Dani

    2018-05-18

    Brown protective skin formation in onion bulbs can be induced by rapid postharvest heat treatment. Onions that were peeled to different depths and were exposed to heat stress showed that only the outer scale formed dry brown skin, whereas the inner scales maintained high water content and did not change color. Our results reveal that browning of the outer scale during heat treatment is due to an enzymatic process that is associated with high levels of oxidation components, such as peroxidase and quercetin glucoside. De-novo transcriptome analysis revealed differential molecular responses of the outer and inner scales to the heat stress. Genes involved in lipid metabolism, oxidation pathways and cell-wall modification were highly expressed in the outer scale during heating. Defense-response-related genes such as those encoding heat-shock proteins, antioxidative stress defense or production of osmoprotectant metabolites were mostly induced in the inner scale in response to the heat exposure. These transcriptomic data led to a conceptual model that suggests sequential processes for browning development and desiccation of the outer scales versus processes associated with defense response and heat tolerance in the inner scale. Thus, the observed physiological differences between the outer and inner scales is supported by the identified molecular differences.

  4. Acute Zonal Cone Photoreceptor Outer Segment Loss.

    Aleman, Tomas S; Sandhu, Harpal S; Serrano, Leona W; Traband, Anastasia; Lau, Marisa K; Adamus, Grazyna; Avery, Robert A

    2017-05-01

    The diagnostic path presented narrows down the cause of acute vision loss to the cone photoreceptor outer segment and will refocus the search for the cause of similar currently idiopathic conditions. To describe the structural and functional associations found in a patient with acute zonal occult photoreceptor loss. A case report of an adolescent boy with acute visual field loss despite a normal fundus examination performed at a university teaching hospital. Results of a complete ophthalmic examination, full-field flash electroretinography (ERG) and multifocal ERG, light-adapted achromatic and 2-color dark-adapted perimetry, and microperimetry. Imaging was performed with spectral-domain optical coherence tomography (SD-OCT), near-infrared (NIR) and short-wavelength (SW) fundus autofluorescence (FAF), and NIR reflectance (REF). The patient was evaluated within a week of the onset of a scotoma in the nasal field of his left eye. Visual acuity was 20/20 OU, and color vision was normal in both eyes. Results of the fundus examination and of SW-FAF and NIR-FAF imaging were normal in both eyes, whereas NIR-REF imaging showed a region of hyporeflectance temporal to the fovea that corresponded with a dense relative scotoma noted on light-adapted static perimetry in the left eye. Loss in the photoreceptor outer segment detected by SD-OCT co-localized with an area of dense cone dysfunction detected on light-adapted perimetry and multifocal ERG but with near-normal rod-mediated vision according to results of 2-color dark-adapted perimetry. Full-field flash ERG findings were normal in both eyes. The outer nuclear layer and inner retinal thicknesses were normal. Localized, isolated cone dysfunction may represent the earliest photoreceptor abnormality or a distinct entity within the acute zonal occult outer retinopathy complex. Acute zonal occult outer retinopathy should be considered in patients with acute vision loss and abnormalities on NIR-REF imaging, especially if

  5. Protecting peroxidase activity of multilayer enzyme-polyion films using outer catalase layers.

    Lu, Haiyun; Rusling, James F; Hu, Naifei

    2007-12-27

    Films constructed layer-by-layer on electrodes with architecture {protein/hyaluronic acid (HA)}n containing myoglobin (Mb) or horseradish peroxidase (HRP) were protected against protein damage by H2O2 by using outer catalase layers. Peroxidase activity for substrate oxidation requires activation by H2O2, but {protein/HA}n films without outer catalase layers are damaged slowly and irreversibly by H2O2. The rate and extent of damage were decreased dramatically by adding outer catalase layers to decompose H2O2. Comparative studies suggest that protection results from catalase decomposing a fraction of the H2O2 as it enters the film, rather than by an in-film diffusion barrier. The outer catalase layers controlled the rate of H2O2 entry into inner regions of the film, and they biased the system to favor electrocatalytic peroxide reduction over enzyme damage. Catalase-protected {protein/HA}n films had an increased linear concentration range for H2O2 detection. This approach offers an effective way to protect biosensors from damage by H2O2.

  6. Genomic analysis indicates the presence of an asymmetric bilayer outer membrane in Planctomycetes and Verrucomicrobia

    Daan R Speth

    2012-08-01

    Full Text Available Bacteria of the phylum Planctomycetes are of special interest for the study of compartmental cellular organization. Members of this phylum share a very unusual prokaryotic cell plan, featuring several membrane-bound compartments. Recently, it was shown that this cellular organization might extend to certain members of the phylum Verrucomicrobia. The Planctomycete cell plan has been defined as featuring a proteinaceous cell wall, a cytoplasmic membrane surrounding the paryphoplasm and an intracytoplasmic membrane defining the riboplasm. So far it was presumed that Planctomycetes did not have an asymmetric bilayer outer membrane as observed in Gram-negative bacteria. However, recent work on outer membrane biogenesis has provided several marker genes in the outer membrane protein (OMP assembly and the lipopolysaccharide (LPS insertion complexes. Additionally, advances in computational prediction of OMPs provided new tools to perform more accurate genomic screening for such proteins.Here we searched all 22 Planctomycetes and Verrucomicrobia genomes available in Genbank, plus the recently published genome of ‘Candidatus Scalindua profunda’, for markers of outer membrane biogenesis and OMPs. We were able to identify the key components of LPS insertion, OMP assembly and at least eight OMPs in all genomes tested. Additionally, we have analyzed the transcriptome and proteome data of the Planctomycetes ‘Candidatus Kuenenia stuttgartiensis’ and ‘Ca. S. profunda’ and could confirm high expression of several predicted OMPs, including the biomarkers of outer membrane biogenesis.

  7. The properties of the outer membrane localized Lipid A transporter LptD

    Haarmann, Raimund; Ibrahim, Mohamed; Stevanovic, Mara; Bredemeier, Rolf; Schleiff, Enrico

    2010-01-01

    Gram-negative bacteria are surrounded by a cell wall including the outer membrane. The outer membrane is composed of two distinct monolayers where the outer layer contains lipopolysaccharides (LPS) with the non-phospholipid Lipid A as the core. The synthesis of Lipid A is initiated in the cytosol and thereby the molecule has to be transported across the inner and outer membranes. The β-barrel lipopolysaccharide-assembly protein D (LptD) was discovered to be involved in the transfer of Lipid A into the outer membrane of Gram-negative bacteria. At present the molecular procedure of lipid transfer across the outer membrane remains unknown. Here we approached the functionality of the transfer system by an electrophysiological analysis of the outer membrane protein from Escherichia coli named ecLptD. In vitro the protein shows cation selectivity and has an estimated pore diameter of about 1.8 nm. Addition of Lipid A induces a transition of the open state to a sub-conductance state with two independent off-rates, which might suggest that LptD is able to bind and transport the molecule in vitro. To generalize our findings with respect to the Lipid A transport system of other Gram-negative bacteria we have explored the existence of the proteins involved in this pathway by bioinformatic means. We were able to identify the membrane-inserted components of the Lipid A transport system in all Gram-negative bacteria, whereas the periplasmic components appear to be species-specific. The LptD proteins of different bacteria are characterized by their periplasmic N-terminal domain and a C-terminal barrel region. The latter shows distinct sequence properties, particularly in LptD proteins of cyanobacteria, and this specific domain can be found in plant proteins as well. By electrophysiological experiments on LptD from Anabaena sp. PCC 7120 we are able to confirm the functional relation of anaLptD to Lipid A transport.

  8. Outer membrane lipoprotein biogenesis: Lol is not the end.

    Konovalova, Anna; Silhavy, Thomas J

    2015-10-05

    Bacterial lipoproteins are lipid-anchored proteins that contain acyl groups covalently attached to the N-terminal cysteine residue of the mature protein. Lipoproteins are synthesized in precursor form with an N-terminal signal sequence (SS) that targets translocation across the cytoplasmic or inner membrane (IM). Lipid modification and SS processing take place at the periplasmic face of the IM. Outer membrane (OM) lipoproteins take the localization of lipoproteins (Lol) export pathway, which ends with the insertion of the N-terminal lipid moiety into the inner leaflet of the OM. For many lipoproteins, the biogenesis pathway ends here. We provide examples of lipoproteins that adopt complex topologies in the OM that include transmembrane and surface-exposed domains. Biogenesis of such lipoproteins requires additional steps beyond the Lol pathway. In at least one case, lipoprotein sequences reach the cell surface by being threaded through the lumen of a beta-barrel protein in an assembly reaction that requires the heteropentomeric Bam complex. The inability to predict surface exposure reinforces the importance of experimental verification of lipoprotein topology and we will discuss some of the methods used to study OM protein topology. © 2015 The Author(s).

  9. Performance of the LHCb Outer Tracker

    Arink, R; Bachmann, S.; Bagaturia, Y.; Band, H.; Bauer, Th.; Berkien, A.; Farber, Ch.; Bien, A.; Blouw, J.; Ceelie, L.; Coco, V.; Deckenhoff, M.; Deng, Z.; Dettori, F.; van Eijk, D.; Ekelhof, R.; Gersabeck, E.; Grillo, L.; Hulsbergen, W.D.; Karbach, T.M.; Koopman, R.; Kozlinskiy, A.; Langenbruch, Ch.; Lavrentyev, V.; Linn, Ch.; Merk, M.; Merkel, J.; Meissner, M.; Michalowski, J.; Morawski, P.; Nawrot, A.; Nedos, M.; Pellegrino, A.; Polok, G.; van Petten, O.; Rovekamp, J.; Schimmel, F.; Schuylenburg, H.; Schwemmer, R.; Seyfert, P.; Serra, N.; Sluijk, T.; Spaan, B.; Spelt, J.; Storaci, B.; Szczekowski, M.; Swientek, S.; Tolk, S.; Tuning, N.; Uwer, U.; Wiedner, D.; Witek, M.; Zeng, M.; Zwart, A.

    2014-01-01

    The LHCb Outer Tracker is a gaseous detector covering an area of 5x6 m2 with 12 double layers of straw tubes. The detector with its services are described together with the commissioning and calibration procedures. Based on data of the first LHC running period from 2010 to 2012, the performance of the readout electronics and the single hit resolution and efficiency are presented. The efficiency to detect a hit in the central half of the straw is estimated to be 99.2%, and the position resolution is determined to be approximately 200 um. The Outer Tracker received a dose in the hottest region corresponding to 0.12 C/cm, and no signs of gain deterioration or other ageing effects are observed.

  10. The Outer Space as an Educational Motivation

    Pérez-Pérez, Melquíades; Hernández-López, Montserrat

    2017-06-01

    STEAM is an educational approach to learning that uses Science, Technology, Engineering, the Arts and Mathematics as access points for guiding student inquiry, dialogue, and critical thinking. The end results are students who take thoughtful risks, engage in experiential learning, persist in problem-solving, embrace collaboration, and work through the creative process. The Outer Space is a window to the past and the future of our travel around the history of the Universe and can be used as a educational tool in primary and secondary education. This paper talks about the integration of the resources of European Space Agency, Space Awareness, Nuclio, Scientix and Schoolnet as motivation to integrate STEAM methodology in secondary education. Keywords: STEAM, outer space, motivation, methodology

  11. The Outer Banks of North Carolina

    Dolan, Robert; Lins, Harry F.; Smith, Jodi Jones

    2016-12-27

    The Outer Banks of North Carolina are excellent examples of the nearly 300 barrier islands rimming the Atlantic and Gulf coasts of the United States. These low, sandy islands are among the most dynamic natural landscapes occupied by man. Beach sands move offshore, onshore, and along the shore in the direction of the prevailing longshore currents. In this way, sandy coasts continuously adjust to different tide, wave, and current conditions and to rising sea level that causes the islands to migrate landward.Despite such changes, barrier islands are of considerable environmental importance. The Outer Banks are home to diverse natural ecosystems that are adapted to the harsh coastal environment. Native species tend to be robust and many are specifically adapted to withstand salt spray, periodic saltwater flooding, and the islands’ well-drained sandy soil. The Outer Banks provide an important stopover for birds on the Atlantic flyway, and many species inhabit the islands year round. In addition, Outer Banks beaches provide an important nesting habitat for five endangered or threatened sea turtle species.European explorers discovered North Carolina’s barrier islands in the 16th century, although the islands were not permanently settled until the middle 17th century. By the early 19th century, shipbuilding and lumber industries were among the most successful, until forest resources were depleted. Commercial fishing eventually followed, and it expanded considerably after the Civil War. By the Great Depression, however, little industry existed on the Outer Banks. In response to the effects of a severe hurricane in 1933, the National Park Service and the Civilian Conservation Corps proposed a massive sand-fixation program to stabilize the moving sand and prevent storm waves from sweeping across the entire width of some sections of the islands. Between 1933 and 1940, this program constructed sand fencing on 185 kilometers (115 miles) of beach and planted grass seedlings

  12. Cryovolcanism in the outer solar system

    Geissler, Paul E.

    2015-01-01

    Cryovolcanism is defined as the extrusion of liquids and vapors of materials that would be frozen solid at the planetary surface temperatures of the icy bodies of the outer solar system. Active cryovolcanism is now known to occur on Saturn's moon Enceladus and on Neptune's moon Triton and is suspected on Jupiter's moon Europa, while evidence for past cryovolcanic activity is widespread throughout the outer solar system. This chapter examines the mechanisms and manifestations of cryovolcanism, beginning with a review of the materials that make up these unusual ‘‘magmas’’ and the means by which they might erupt and concluding with a volcanologist's tour of the farthest reaches of the solar system.

  13. Protection of nuclear facilities against outer aggressions

    Aussourd, P.; Candes, P.; Le Quinio, R.

    1976-01-01

    The various types of outer aggressions envisaged in safety analysis for nuclear facilities are reviewed. These outer aggressions are classified as natural and non-natural phenomena, the latter depending on the human activities in the vicinity of nuclear sites. The principal natural phenomena able to constitute aggressions are atmospheric phenomena (strong winds, snow storms, hail, frosting mists), hydrologie phenomena such as tides, surges, flood, low waters, and geologic phenomena such as earthquakes. Artificial phenomena are concerned with aircraft crashes, projectiles, fire, possible ruptures of dams, and intentional human aggressions. The protection against intentional human aggressions is of two sorts: first, the possibility of access to the installations mostly sensitive to sabotage are to be prevented or reduced, secondly redundant circuits and functions must be separated for preventing their simultaneous destruction in the case when sabotage actors have reach the core of the facility [fr

  14. Outer crust of nonaccreting cold neutron stars

    Ruester, Stefan B.; Hempel, Matthias; Schaffner-Bielich, Juergen

    2006-01-01

    The properties of the outer crust of nonaccreting cold neutron stars are studied by using modern nuclear data and theoretical mass tables, updating in particular the classic work of Baym, Pethick, and Sutherland. Experimental data from the atomic mass table from Audi, Wapstra, and Thibault of 2003 are used and a thorough comparison of many modern theoretical nuclear models, both relativistic and nonrelativistic, is performed for the first time. In addition, the influences of pairing and deformation are investigated. State-of-the-art theoretical nuclear mass tables are compared to check their differences concerning the neutron drip line, magic neutron numbers, the equation of state, and the sequence of neutron-rich nuclei up to the drip line in the outer crust of nonaccreting cold neutron stars

  15. Boundary layers of the earth's outer magnetosphere

    Eastman, T. E.; Frank, L. A.

    1984-01-01

    The magnetospheric boundary layer and the plasma-sheet boundary layer are the primary boundary layers of the earth's outer magnetosphere. Recent satellite observations indicate that they provide for more than 50 percent of the plasma and energy transport in the outer magnetosphere although they constitute less than 5 percent by volume. Relative to the energy density in the source regions, plasma in the magnetospheric boundary layer is predominantly deenergized whereas plasma in the plasma-sheet boundary layer has been accelerated. The reconnection hypothesis continues to provide a useful framework for comparing data sampled in the highly dynamic magnetospheric environment. Observations of 'flux transfer events' and other detailed features near the boundaries have been recently interpreted in terms of nonsteady-state reconnection. Alternative hypotheses are also being investigated. More work needs to be done, both in theory and observation, to determine whether reconnection actually occurs in the magnetosphere and, if so, whether it is important for overall magnetospheric dynamics.

  16. Boundary layers of the earth's outer magnetosphere

    Eastman, T.E.; Frank, L.A.

    1984-01-01

    The magnetospheric boundary layer and the plasma-sheet boundary layer are the primary boundary layers of the earth's outer magnetosphere. Recent satellite observations indicate that they provide for more than 50 percent of the plasma and energy transport in the outer magnetosphere although they constitute less than 5 percent by volume. Relative to the energy density in the source regions, plasma in the magnetospheric boundary layer is predominantly deenergized whereas plasma in the plasma-sheet boundary layer has been accelerated. The reconnection hypothesis continues to provide a useful framework for comparing data sampled in the highly dynamic magnetospheric environment. Observations of flux transfer events and other detailed features near the boundaries have been recently interpreted in terms of nonsteady-state reconnection. Alternative hypotheses are also being investigated. More work needs to be done, both in theory and observation, to determine whether reconnection actually occurs in the magnetosphere and, if so, whether it is important for overall magnetospheric dynamics. 30 references

  17. Differential Rotation within the Earth's Outer Core

    Hide, R.; Boggs, D. H.; Dickey, J. O.

    1998-01-01

    Non-steady differential rotation drive by bouyancy forces within the Earth's liquid outer core (OC) plays a key role not only in the generation of the main geomagnetic field by the magnetohydrodynamic (MHD) dynamo process but also in the excitation of irregular fluctuations in the angular speed of rotation of the overlying solid mantle, as evidenced by changes in the length of the day (LOD) on decadal and longer timescales (1-8).

  18. Fluxgate magnetometers for outer planets exploration

    Acuna, M. H.

    1974-01-01

    The exploration of the interplanetary medium and the magnetospheres of the outer planets requires the implementation of magnetic field measuring instrumentation with wide dynamic range, high stability, and reliability. The fluxgate magnetometers developed for the Pioneer 11 and Mariner-Jupiter-Saturn missions are presented. These instruments cover the range of .01 nT to 2 million nT with optimum performance characteristics and low power consumption.

  19. Outer Limits of Biotechnologies: A Jewish Perspective

    John D. Loike

    2018-01-01

    Full Text Available A great deal of biomedical research focuses on new biotechnologies such as gene editing, stem cell biology, and reproductive medicine, which have created a scientific revolution. While the potential medical benefits of this research may be far-reaching, ethical issues related to non-medical applications of these technologies are demanding. We analyze, from a Jewish legal perspective, some of the ethical conundrums that society faces in pushing the outer limits in researching these new biotechnologies.

  20. Outer membrane biogenesis in Helicobacter pylori: A deviation from the paradigm

    George W. Liechti

    2012-04-01

    Full Text Available The bacterial pathogen Helicobacter pylori is capable of colonizing the gastric mucosa of the human stomach using a variety of factors associated with or secreted from its outer membrane (OM. Lipopolysaccharide (LPS and numerous outer membrane proteins have been shown to be involved in adhesion and immune stimulation/evasion. Many of these factors are essential for colonization and/or pathogenesis in a variety of animal models. Despite this wide array of potential targets present on the bacterial surface, the ability of H. pylori to vary its outer membrane profile limits the effectiveness of vaccines that use any single one of these components. However, it has become evident that the proteins comprising the complexes that transport the majority of these molecules to the OM are highly conserved and often essential. The field of membrane biogenesis has progressed remarkably in the last few years, and the possibility now exists for targeting the mechanisms by which β-barrel proteins, lipoproteins, and LPS are transported to the OM, resulting in loss of bacterial fitness and significant altering of membrane permeability. In this review, the OM transport machinery for LPS, lipoproteins, and outer membrane proteins are discussed. While the principal investigations of these transport mechanisms have been conducted in Escherichia coli and Neisseria meningitidis, here these systems will be presented in the genetic context of ε- proteobacteria. Bioinformatic analysis reveals that minimalist genomes, such as that of Helicobacter pylori, offer insight into the smallest number of components required for these essential pathways to function. Interestingly, in the majority of ε-proteobacteria, while the inner and outer membrane associated apparatus of LPS, lipoprotein, and OM protein transport pathways appear to all be intact, most of the components associated with the periplasmic compartment are either missing or are almost unrecognizable when compared to

  1. Chlamydomonas Outer Arm Dynein Alters Conformation in Response to Ca2+

    Sakato, Miho; Sakakibara, Hitoshi; King, Stephen M.

    2007-01-01

    We have previously shown that Ca2+ directly activates ATP-sensitive microtubule binding by a Chlamydomonas outer arm dynein subparticle containing the β and γ heavy chains (HCs). The γ HC–associated LC4 light chain is a member of the calmodulin family and binds 1-2 Ca2+ with KCa = 3 × 10−5 M in vitro, suggesting it may act as a Ca2+ sensor for outer arm dynein. Here we investigate interactions between the LC4 light chain and γ HC. Two IQ consensus motifs for binding calmodulin-like proteins a...

  2. 75 FR 1076 - Outer Continental Shelf Civil Penalties

    2010-01-08

    ... initiate civil penalty proceedings; however, violations that cause injury, death, or environmental damage... DEPARTMENT OF THE INTERIOR Minerals Management Service Outer Continental Shelf Civil Penalties... daily civil penalty assessment. SUMMARY: The Outer Continental Shelf Lands Act requires the MMS to...

  3. Cellular and molecular biology of orphan G protein-coupled receptors.

    Oh, Da Young; Kim, Kyungjin; Kwon, Hyuk Bang; Seong, Jae Young

    2006-01-01

    The superfamily of G protein-coupled receptors (GPCRs) is the largest and most diverse group of membrane-spanning proteins. It plays a variety of roles in pathophysiological processes by transmitting extracellular signals to cells via heterotrimeric G proteins. Completion of the human genome project revealed the presence of approximately 168 genes encoding established nonsensory GPCRs, as well as 207 genes predicted to encode novel GPCRs for which the natural ligands remained to be identified, the so-called orphan GPCRs. Eighty-six of these orphans have now been paired to novel or previously known molecules, and 121 remain to be deorphaned. A better understanding of the GPCR structures and classification; knowledge of the receptor activation mechanism, either dependent on or independent of an agonist; increased understanding of the control of GPCR-mediated signal transduction; and development of appropriate ligand screening systems may improve the probability of discovering novel ligands for the remaining orphan GPCRs.

  4. Issues concerning outer space investments in international law ...

    Issues concerning outer space investments in international law. ... Recent improvements in technology have in essence increased the viability of outer space as the next frontier for international investment and development. In addition to ... Key words: Outer Space, Investments, International Law, International Space Station ...

  5. Dark matter in the outer solar system

    Owen, T.; Cruikshank, D.; De Bergh, C.; Geballe, T.

    1994-01-01

    There are now a large number of small bodies in the outer solar system that are known to be covered with dark material. Attempts to identify that material have been thwarted by the absence of discrete absorption features in the reflection spectra of these planetesimals. An absorption at 2.2 micrometers that appeared to be present in several objects has not been confirmed by new observations. Three absorptions in the spectrum of the unusually red planetesimal 5145 Pholus are well-established, but their identity remains a mystery.

  6. Inner and Outer Life at Work

    Lundgaard Andersen, Linda

    2012-01-01

    involving people to people interactions offered by psychodynamic theories and methods take up a pivotal position. Psychoanalytic organisational and work life research explores how work, organisations and individuals are affected by psychic dynamics, the influence of the unconscious in the forms of human...... development and interaction situated in a societal context. Based on this substantial work I draw upon two influential psychoanalytical positions—the British Tavistock position and German psychoanalytic social psychology in order to situate and identify how to understand the inner and outer life at work...

  7. The urgency of outer territories anthropology

    Miloš Milenković

    2016-02-01

    Full Text Available In the context of transforming a part of Serbian anthropology into social theoretic management of identity, I suggest both comparative historiographic and ethnographic learning from societies with similar post-colonial experience, with the aim to include the discipline into an urgent defense of Serbia and Belgrade from further ethno-profiteering interests of elites in/from outer territories, left over on the ruins of our ill judged, resource incompatible, exaggerated or immoral twentieth century adventures. Serbian anthropology, written by anthropologists to whom Serbia and Belgrade are "homeland" by origin or civilized choice, should play the key role in the defense of Serbian citizens from the interest of elites in/from the outer "homelands", particularly by revealing the processes for which it is, as a discipline, most expert at – the professionalization of ethnicity, interactive and hybrid nature of identity, instrumental nature of tradition and the identity politics in general. Having in mind the latest attempt, a particularly successful one, conducted by the end of the 20th and the beginning of the 21st century that the lives, health, well-being, dignity and future of persons born in and loyal to the interest of Serbia and Belgrade, in large scale, thoroughly and long term be sacrificed and dedicated to the interests of ethno-profiteering elites in/from outer territories, in this article I point to the possibility to, along with the comparative learning from the above mentioned post-colonial experiences, delicate experiences of urgent anthropology be applied as well as the rich tradition of collective research. This text analyzes the results of first such research, that represenst the initial, praiseworthy and a brave step in the wise striving to engage social sciences and humanities in a search of expert and not mythical/daily-political solutions of the key problem of the Serbian nation – that of how to settle the interests of the

  8. Calcium sensors of ciliary outer arm dynein: functions and phylogenetic considerations for eukaryotic evolution.

    Inaba, Kazuo

    2015-01-01

    The motility of eukaryotic cilia and flagella is modulated in response to several extracellular stimuli. Ca(2+) is the most critical intracellular factor for these changes in motility, directly acting on the axonemes and altering flagellar asymmetry. Calaxin is an opisthokont-specific neuronal calcium sensor protein first described in the sperm of the ascidian Ciona intestinalis. It binds to a heavy chain of two-headed outer arm dynein in a Ca(2+)-dependent manner and regulates 'asymmetric' wave propagation at high concentrations of Ca(2+). A Ca(2+)-binding subunit of outer arm dynein in Chlamydomonas reinhardtii, the light chain 4 (LC4), which is a Ca(2+)-sensor phylogenetically different from calaxin, shows Ca(2+)-dependent binding to a heavy chain of three-headed outer arm dynein. However, LC4 appears to participate in 'symmetric' wave propagation at high concentrations of Ca(2+). LC4-type dynein light chain is present in bikonts, except for some subclasses of the Excavata. Thus, flagellar asymmetry-symmetry conversion in response to Ca(2+) concentration represents a 'mirror image' relationship between Ciona and Chlamydomonas. Phylogenetic analyses indicate the duplication, divergence, and loss of heavy chain and Ca(2+)-sensors of outer arm dynein among excavate species. These features imply a divergence point with respect to Ca(2+)-dependent regulation of outer arm dynein in cilia and flagella during the evolution of eukaryotic supergroups.

  9. Heating of the outer solar atmosphere

    Parker, E.N.

    1983-01-01

    The author discusses the idea that there must be a source of magnetic fields somewhere below the solar surface. He starts by considering present day ideas about the sun's internal structure. The sun has a radius of approximately 700,000 km, of which the outer 100,000 km or so is the convective zone, according to mixing-length models. The dynamo is believed to operate in the convective zone, across which there may be a 5-10% variation in the angular velocity. There are the stretched east-west fields similar to the ones in the earth's core. Associated with these are poloidal fields which contribute to a net dipole moment of the sun and are generated by a dynamo. The author shows that essentially no magnetic field configuration has an equilibrium; they dissipate quickly in spite of the high conductivity in fluid motions and heating. This is probably the major part of the heating of the sun's outer atmosphere. (Auth.)

  10. Cosmics in the LHCb Outer Tracker

    Aaij, Roel

    2010-01-01

    The LHCb experiment at the Large Hadron Collider studies the decay of B mesons to test the description of CP violation in the Standard Model and to search for new physics. The decay $B_s \\to \\mu^+ \\mu^-$ has been identified as very promising in the search for new physics. An excellent invariant mass resolution is required to suppress backgrounds to this decay. This in turn requires a momentum resolution of dp/p = 0.4%. The Outer Tracker is part of the LHCb tracking system and has been commissioned with cosmic muons. The noise in the Outer Tracker is shown to be less than 0.05%. To use drift time information in the reconstruction of cosmic tracks, the event time must be known. Four methods to obtain the event time are studied and compared. It is shown that the event time can be obtained with a resolution better than 2.6 ns. Using drift time information, tracks are reconstructed with a resolution of 344 $\\mu$m. Knowledge of the event time enables the calibration of electronic time offsets and the r(t)– relati...

  11. Gamma rays from pulsar outer gaps

    Chiang, J.; Romani, R.W.; Cheng Ho

    1993-01-01

    We describe a gamma ray pulsar code which computes the high energy photon emissivities from vacuum gaps in the outer magnetosphere, after the model outlined by Cheng, Ho and Ruderman (1986) and Ho (1989). Pair-production due to photon-photon interactions and radiation processes including curvature, synchrotron and inverse Compton processes are computed with an iterative scheme which converges to self-consistent photon and particle distributions for a sampling of locations in the outer magnetosphere. We follow the photons from these distributions as they propagate through the pulsar magnetosphere toward a distant observer. We include the effects of relativistic aberration, time-of-flight delays and reabsorption by photon-photon pair-production to determine an intensity map of the high energy pulsar emission on the sky. Using data from radio and optical observations to constrain the geometry of the magnetosphere as well as the possible observer viewing angles, we derive light curves and phase dependent spectra which can be directly compared to data from the Compton Observatory. Observations for Crab, Vela and the recently identified gamma ray pulsars Geminga, PSR1706-44 aNd PSR 1509-58 will provide important tests of our model calculations, help us to improve our picture of the relevant physics at work in pulsar magnetospheres and allow us to comment on the implications for future pulsar discoveries

  12. Residual Stress Testing of Outer 3013 Containers

    Dunn, K.

    2004-01-01

    A Gas Tungsten Arc Welded (GTAW) outer 3013 container and a laser welded outer 3013 container have been tested for residual stresses according to the American Society for Testing Materials (ASTM) Standard G-36-94 [1]. This ASTM standard describes a procedure for conducting stress-corrosion cracking tests in boiling magnesium chloride (MgCl2) solution. Container sections in both the as-fabricated condition as well as the closure welded condition were evaluated. Significantly large residual stresses were observed in the bottom half of the as-fabricated container, a result of the base to can fabrication weld because through wall cracks were observed perpendicular to the weld. This observation indicates that regardless of the closure weld technique, sufficient residual stresses exist in the as-fabricated container to provide the stress necessary for stress corrosion cracking of the container, at the base fabrication weld. Additionally, sufficiently high residual stresses were observed in both the lid and the body of the GTAW as well as the laser closure welded containers. The stresses are oriented perpendicular to the closure weld in both the container lid and the container body. Although the boiling MgCl2 test is not a quantitative test, a comparison of the test results from the closure welds shows that there are noticeably more through wall cracks in the laser closure welded container than in the GTAW closure welded container

  13. Redefining the essential trafficking pathway for outer membrane lipoproteins.

    Grabowicz, Marcin; Silhavy, Thomas J

    2017-05-02

    The outer membrane (OM) of Gram-negative bacteria is a permeability barrier and an intrinsic antibiotic resistance factor. Lipoproteins are OM components that function in cell wall synthesis, diverse secretion systems, and antibiotic efflux pumps. Moreover, each of the essential OM machines that assemble the barrier requires one or more lipoproteins. This dependence is thought to explain the essentiality of the periplasmic chaperone LolA and its OM receptor LolB that traffic lipoproteins to the OM. However, we show that in strains lacking substrates that are toxic when mislocalized, both LolA and LolB can be completely bypassed by activating an envelope stress response without compromising trafficking of essential lipoproteins. We identify the Cpx stress response as a monitor of lipoprotein trafficking tasked with protecting the cell from mislocalized lipoproteins. Moreover, our findings reveal that an alternate trafficking pathway exists that can, under certain conditions, bypass the functions of LolA and LolB, implying that these proteins do not perform any truly essential mechanistic steps in lipoprotein trafficking. Instead, these proteins' key function is to prevent lethal accumulation of mislocalized lipoproteins.

  14. Recurrent Neural Network for Computing Outer Inverse.

    Živković, Ivan S; Stanimirović, Predrag S; Wei, Yimin

    2016-05-01

    Two linear recurrent neural networks for generating outer inverses with prescribed range and null space are defined. Each of the proposed recurrent neural networks is based on the matrix-valued differential equation, a generalization of dynamic equations proposed earlier for the nonsingular matrix inversion, the Moore-Penrose inversion, as well as the Drazin inversion, under the condition of zero initial state. The application of the first approach is conditioned by the properties of the spectrum of a certain matrix; the second approach eliminates this drawback, though at the cost of increasing the number of matrix operations. The cases corresponding to the most common generalized inverses are defined. The conditions that ensure stability of the proposed neural network are presented. Illustrative examples present the results of numerical simulations.

  15. Ageing of the LHCb outer tracker

    Blom, M R; Tuning, N

    2009-01-01

    The modules of the LHCb outer tracker have shown to suffer severe gain loss under moderate irradiation. This process is called ageing. Ageing of the modules results from contamination of the gas system by glue, araldite AY 103-1, used in their construction. In this thesis the ageing process will be shown. The schemes known to reduce, reverse, or prevent ageing have been investigated to determine their effect on the detector performance. The addition of O2 to the gas mixture lowers the detector response by an acceptable amount and does not affect the gas transport properties significantly. The ageing rate is decreased after extensive flushing and HV training could eventually repair the irradiation damage. The risks of HV training have been assessed. Furthermore, several gaseous and aquatic additions have been tested for their capability to prevent, or moderate ageing, but none showed significant improvement.

  16. The fate of the outer plasmasphere

    Elphic, R.C.; Thomsen, M.F.; Borovsky, J.E.

    1997-01-01

    Both the solar wind and the ionosphere contribute to Earth close-quote s magnetospheric plasma environment. However, it is not widely appreciated that the plasmasphere is a large reservoir of ionospheric ions that can be tapped to populate the plasma sheet. We employ empirical models of high-latitude ionospheric convection and the geomagnetic field to describe the transport of outer plasmasphere flux tubes from the dayside, over the polar cap and into the magnetotail during the early phases of a geomagnetic storm. We calculate that this process can give rise to high densities of cold plasma in the magnetotail lobes and in the near-Earth plasma sheet during times of enhanced geomagnetic activity, and especially during storms. This model can help explain both polar cap ionization patches and the presence of cold flowing ions downtail.copyright 1997 American Geophysical Union

  17. Impulsive ion acceleration in earth's outer magnetosphere

    Baker, D.N.; Belian, R.D.

    1985-01-01

    Considerable observational evidence is found that ions are accelerated to high energies in the outer magnetosphere during geomagnetic disturbances. The acceleration often appears to be quite impulsive causing temporally brief (10's of seconds), very intense bursts of ions in the distant plasma sheet as well as in the near-tail region. These ion bursts extend in energy from 10's of keV to over 1 MeV and are closely associated with substorm expansive phase onsets. Although the very energetic ions are not of dominant importance for magnetotail plasma dynamics, they serve as an important tracer population. Their absolute intensity and brief temporal appearance bespeaks a strong and rapid acceleration process in the near-tail, very probably involving large induced electric fields substantially greater than those associated with cross-tail potential drops. Subsequent to their impulsive acceleration, these ions are injected into the outer trapping regions forming ion ''drift echo'' events, as well as streaming tailward away from their acceleration site in the near-earth plasma sheet. Most auroral ion acceleration processes occur (or are greatly enhanced) during the time that these global magnetospheric events are occurring in the magnetotail. A qualitative model relating energetic ion populations to near-tail magnetic reconnection at substorm onset followed by global redistribution is quite successful in explaining the primary observational features. Recent measurements of the elemental composition and charge-states have proven valuable for showing the source (solar wind or ionosphere) of the original plasma population from which the ions were accelerated

  18. Identification of membrane proteins by tandem mass spectrometry of protein ions

    Carroll, Joe; Altman, Matthew C.; Fearnley, Ian M.; Walker, John E.

    2007-01-01

    The most common way of identifying proteins in proteomic analyses is to use short segments of sequence (“tags”) determined by mass spectrometric analysis of proteolytic fragments. The approach is effective with globular proteins and with membrane proteins with significant polar segments between membrane-spanning α-helices, but it is ineffective with other hydrophobic proteins where protease cleavage sites are either infrequent or absent. By developing methods to purify hydrophobic proteins in organic solvents and by fragmenting ions of these proteins by collision induced dissociation with argon, we have shown that partial sequences of many membrane proteins can be deduced easily by manual inspection. The spectra from small proteolipids (1–4 transmembrane α-helices) are dominated usually by fragment ions arising from internal amide cleavages, from which internal sequences can be obtained, whereas the spectra from larger membrane proteins (5–18 transmembrane α-helices) often contain fragment ions from N- and/or C-terminal parts yielding sequences in those regions. With these techniques, we have, for example, identified an abundant protein of unknown function from inner membranes of mitochondria that to our knowledge has escaped detection in proteomic studies, and we have produced sequences from 10 of 13 proteins encoded in mitochondrial DNA. They include the ND6 subunit of complex I, the last of its 45 subunits to be analyzed. The procedures have the potential to be developed further, for example by using newly introduced methods for protein ion dissociation to induce fragmentation of internal regions of large membrane proteins, which may remain partially folded in the gas phase. PMID:17720804

  19. A tonoplast intrinsic protein in Gardenia jasminoides

    Gao, Lan; Li, Hao-Ming

    2017-08-01

    Physiological and molecular studies proved that plasma membrane intrinsic proteins (PIPs) and tonoplast intrinsic proteins (TIPs) subfamily of aquaporins play key functions in plant water homeostasis. Five specialized subgroups (TIP1-5) of TIPs have been found in higher plants, in which the TIP1 and TIP2 isoforms are the largest arbitrary groups. TIPs have high water-transport activity than PIPs, some TIPs can transport other small molecule such as urea, ammonia, hydrogen peroxide, and carbon dioxide. In this work, the structure of the putative tonoplast aquaporin from Gardenia jasminoides (GjTIP) was analyzed. Its transcript level has increased during fruit maturation. A phylogenetic analysis indicates that the protein belongs to TIP1 subfamily. A three-dimensional model structure of GjTIP was built based on crystal structure of an ammonia-permeable AtTIP2-1 from Arabidopsis thaliana. The model structure displayed as a homo-tetramer, each monomer has six trans-membrane and two half-membrane-spanning α helices. The data suggests that the GjTIP has tendency to be a mixed function aquaporin, might involve in water, urea and hydrogen peroxide transport, and the gating machanism founded in some AQPs involving pH and phosphorylation response have not been proved in GjTIP.

  20. Will Outer Tropical Cyclone Size Change due to Anthropogenic Warming?

    Schenkel, B. A.; Lin, N.; Chavas, D. R.; Vecchi, G. A.; Knutson, T. R.; Oppenheimer, M.

    2017-12-01

    Prior research has shown significant interbasin and intrabasin variability in outer tropical cyclone (TC) size. Moreover, outer TC size has even been shown to vary substantially over the lifetime of the majority of TCs. However, the factors responsible for both setting initial outer TC size and determining its evolution throughout the TC lifetime remain uncertain. Given these gaps in our physical understanding, there remains uncertainty in how outer TC size will change, if at all, due to anthropogenic warming. The present study seeks to quantify whether outer TC size will change significantly in response to anthropogenic warming using data from a high-resolution global climate model and a regional hurricane model. Similar to prior work, the outer TC size metric used in this study is the radius in which the azimuthal-mean surface azimuthal wind equals 8 m/s. The initial results from the high-resolution global climate model data suggest that the distribution of outer TC size shifts significantly towards larger values in each global TC basin during future climates, as revealed by 1) statistically significant increase of the median outer TC size by 5-10% (p<0.05) according to a 1,000-sample bootstrap resampling approach with replacement and 2) statistically significant differences between distributions of outer TC size from current and future climate simulations as shown using two-sample Kolmogorov Smirnov testing (p<<0.01). Additional analysis of the high-resolution global climate model data reveals that outer TC size does not uniformly increase within each basin in future climates, but rather shows substantial locational dependence. Future work will incorporate the regional mesoscale hurricane model data to help focus on identifying the source of the spatial variability in outer TC size increases within each basin during future climates and, more importantly, why outer TC size changes in response to anthropogenic warming.

  1. Hydrogen exchange kinetics in a membrane protein determined by 15N NMR spectroscopy: Use of the INEPT [insensitive nucleus enhancement by polarization transfer] experiment to follow individual amides in detergent-solubilized M13 coat protein

    Henry, G.D.; Sykes, B.D.

    1990-01-01

    The coat protein of the filamentous coliphage M13 is a 50-residue polypeptide which spans the inner membrane of the Escherichia coli host upon infection. Amide hydrogen exchange kinetics have been used to probe the structure and dynamics of M13 coat protein which has been solubilized in sodium dodecyl sulfate (SDS) micelles. In a previous 1 H nuclear magnetic resonance (NMR) study, multiple exponential analysis of the unresolved amide proton envelope revealed the existence of two slow kinetic sets containing a total of about 30 protons. The slower set (15-20 amides) originates from the hydrophobic membrane-spanning region and exchanges at least 10 5 -fold slower than the unstructured, non-H-bonded model polypeptide poly(DL-alanine). Herein the authors use 15 N NMR spectroscopy of biosynthetically labeled coat protein to follow individual, assigned, slowly exchanging amides in or near the hydrophobic segment. The INEPT (insensitive nucleus enhancement by polarization transfer) experiments can be used to transfer magnetization to the 15 N nucleus from a coupled proton; when 15 N-labeled protonated protein is dissolved in 2 H 2 O, the INEPT signal disappears with time as the amide protons are replaced by solvent deuterons. Amide hydrogen exchange is catalyzed by both H + and OH - ions. The time-dependent exchange-out experiment is suitable for slow exchange rates (k ex ). The INEPT experiment was also adapted to measure some of the more rapidly exchanging amides in the coat protein using either saturation transfer from water or exchange effects on the polarization transfer step itself. The results of all of these experiments are consistent with previous models of the coat protein in which a stable segment extends from the hydrophobic membrane-spanning region through to the C-terminus, whereas the N-terminal region is undergoing more extensive dynamic fluctuations

  2. Role of Outer Membrane Vesicles of Bacteria

    IAS Admin

    tion and protection of the bacterial cells from various stress factors. Recent ... role of a pathogen inside the body of plants and animals. The secretion of proteins ..... peroxide (which imparts oxidative stress inside the bacterial cell). Unlike what ...

  3. Analysis of long-chain fatty acid binding activity in vesicles of the outer membrane generated from Escherchia coli

    Black, P.N.

    1987-01-01

    Escherichia coli transports long-chain fatty acids across the dual membrane by a high affinity, saturable, energy-dependent process. The fadL gene codes for an outer membrane protein which appears to act specifically as a long-chain fatty acid binding protein when fatty acid utilization is blocked by mutation. In an effort to understand the function of the fadL gene product, FLP, membranes have been isolated from fadL + and fadL - strains following osmotic lysis. Following isolation, total membranes were separated into inner and outer membrane fractions and assayed for long-chain fatty acid binding activity. Outer membrane vesicles were incubated 2-5 min at 37 0 C with 3 H oleate (C/sub 18:1/), cooled to 0 0 C, and centrifuged through a Lipidex 100 column for 3 min to remove the unbound fatty acid. The level of fatty acid binding was quantitated by scintillation counting of the eluate. Outer membrane vesicles generated from a fadL + strain bind 325 pmol fatty acid/mg protein whereas vesicles generated for a mutant strain bind 175 pmol fatty acid/mg protein. These data suggest that FLP acts at least as a long-chain fatty acid binding protein on the surface of the cell

  4. 大黄鱼三种病原弧菌外膜蛋白交叉保护性抗原筛选%Selection of cross-protective antigens from outer membrane proteins of three pathogenic vibrios isolated from infected large yellow croaker (Pseudosciaena crocea)

    张崇文; 毛芝娟; 于涟

    2012-01-01

    . parahaemolyticus and one V. alginolyticus by physiological, biochemical and molecular biological methods. Their outer membrane proteins (OMPs) were extracted and the SDS-PAGE and Western blotting results show that three immuno-blots with common molecular weight presented at approximate 45 kDa, 35 kDa and 22 kDa on their OMP electrophoretogram, indicating the existence of antigens with cross-protection in their OMPs. With the aids of combination of two-dimensional electrophoresis (2-D) and Western blotting and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), a deduced porin (GenBank Accession No. ZP01260407) from V. alginolyticus and a maltoporin precursor (GenBank Accession No. NP801154) from V. parahaemolyticus were able to react with polyclonal antibody to whole V. harveyi, suggesting these two proteins could act as the cross-protective antigens and the vaccines prepared with these porins would be probable to bring cross protection to three different vibrios.

  5. Statistics of the outer radiation belt

    Rodgers, D.J.; Johnstone, A.D.

    1996-01-01

    The highly variable electron flux levels in the outer radiation belt come about by competition between time-dependent source and loss mechanisms. In order to identify some of the different mechanisms involved, we examine the statistics of the variability of fluxes at geostationary orbit. Data from the SEM-2 analyzer on Meteosat-3 and from GOES-7 are used. Correlation analysis is used to find time-delays between changes in flux at different energies. We see that low energy flux is added to this region during sub-storms and that higher energy fluxes appear after 2 or 3 days. Whilst the timescale for this process is brief compared to a complete cycle of the open-quote Recirculation close-quote energization process, it is consistent with the timescale of its final step endash outward radial diffusion. By isolating periods when no new injection of plasma occurs, we make an assessment of flux loss rates in a quiet magnetosphere. copyright 1996 American Institute of Physics

  6. Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids.

    Li, Guangtao; Kim, JiHyun; Huang, Zhen; St Clair, Johnna R; Brown, Deborah A; London, Erwin

    2016-12-06

    Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70-80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids.

  7. Identification and characterization of the pseudorabies virus UL43 protein

    Klupp, Barbara G.; Altenschmidt, Jan; Granzow, Harald; Fuchs, Walter; Mettenleiter, Thomas C.

    2005-01-01

    Among the least characterized herpesvirus membrane proteins are the homologs of UL43 of herpes simplex virus 1 (HSV-1). To identify and characterize the UL43 protein of pseudorabies virus (PrV), part of the open reading frame was expressed in Escherichia coli and used for immunization of a rabbit. The antiserum recognized in Western blots a 34-kDa protein in lysates of PrV infected cells and purified virions, demonstrating that the UL43 protein is a virion component. In indirect immunofluorescence analysis, the antiserum labeled vesicular structures in PrV infected cells which also contained glycoprotein B. To functionally analyze UL43, a deletion mutant was constructed lacking amino acids 23-332 of the 373aa protein. This mutant was only slightly impaired in replication as assayed by one-step growth kinetics, measurement of plaque sizes, and electron microscopy. Interestingly, the PrV UL43 protein was able to inhibit fusion induced by PrV glycoproteins in a transient expression-fusion assay to a similar extent as gM. Double mutant viruses lacking, in addition to UL43, the multiply membrane spanning glycoproteins K or M did not show a phenotype beyond that observed in the gK and gM single deletion mutants

  8. Outer membrane components of the Tad (tight adherence) secreton of Aggregatibacter actinomycetemcomitans.

    Clock, Sarah A; Planet, Paul J; Perez, Brenda A; Figurski, David H

    2008-02-01

    Prokaryotic secretion relies on proteins that are widely conserved, including NTPases and secretins, and on proteins that are system specific. The Tad secretion system in Aggregatibacter actinomycetemcomitans is dedicated to the assembly and export of Flp pili, which are needed for tight adherence. Consistent with predictions that RcpA forms the multimeric outer membrane secretion channel (secretin) of the Flp pilus biogenesis apparatus, we observed the RcpA protein in multimers that were stable in the presence of detergent and found that rcpA and its closely related homologs form a novel and distinct subfamily within a well-supported gene phylogeny of the entire secretin gene superfamily. We also found that rcpA-like genes were always linked to Aggregatibacter rcpB- or Caulobacter cpaD-like genes. Using antisera, we determined the localization and gross abundances of conserved (RcpA and TadC) and unique (RcpB, RcpC, and TadD) Tad proteins. The three Rcp proteins (RcpA, RcpB, and RcpC) and TadD, a putative lipoprotein, localized to the bacterial outer membrane. RcpA, RcpC, and TadD were also found in the inner membrane, while TadC localized exclusively to the inner membrane. The RcpA secretin was necessary for wild-type abundances of RcpB and RcpC, and TadC was required for normal levels of all three Rcp proteins. TadC abundance defects were observed in rcpA and rcpC mutants. TadD production was essential for wild-type RcpA and RcpB abundances, and RcpA did not multimerize or localize to the outer membrane without the expression of TadD. These data indicate that membrane proteins TadC and TadD may influence the assembly, transport, and/or function of individual outer membrane Rcp proteins.

  9. Redefining the essential trafficking pathway for outer membrane lipoproteins

    Grabowicz, Marcin; Silhavy, Thomas J.

    2017-01-01

    The outer membrane (OM) of Gram-negative bacteria is a permeability barrier and an intrinsic antibiotic resistance factor. Lipoproteins are OM components that function in cell wall synthesis, diverse secretion systems, and antibiotic efflux pumps. Moreover, each of the essential OM machines that assemble the barrier requires one or more lipoproteins. This dependence is thought to explain the essentiality of the periplasmic chaperone LolA and its OM receptor LolB that traffic lipoproteins to the OM. However, we show that in strains lacking substrates that are toxic when mislocalized, both LolA and LolB can be completely bypassed by activating an envelope stress response without compromising trafficking of essential lipoproteins. We identify the Cpx stress response as a monitor of lipoprotein trafficking tasked with protecting the cell from mislocalized lipoproteins. Moreover, our findings reveal that an alternate trafficking pathway exists that can, under certain conditions, bypass the functions of LolA and LolB, implying that these proteins do not perform any truly essential mechanistic steps in lipoprotein trafficking. Instead, these proteins’ key function is to prevent lethal accumulation of mislocalized lipoproteins. PMID:28416660

  10. An ABC-transporter and an outer membrane lipoprotein participate in posttranslational activation of type VI secretion in Pseudomonas aeruginosa

    Casabona, Maria G.; Silverman, Julie M.; Sall, Khady M.; Boyer, Frédéric; Couté, Yohann; Poirel, Jessica; Grunwald, Didier; Mougous, Joseph D.; Elsen, Sylvie; Attree, Ina

    2012-01-01

    Pseudomonas aeruginosa is capable of injecting protein toxins into other bacterial cells through one of its three type VI secretion systems (T6SS). The activity of this T6SS is tightly regulated on the posttranslational level by phosphorylation-dependent and -independent pathways. The phosphorylation-dependent pathway consists of a Thr kinase/phosphatase pair (PpkA/PppA) that acts on a forkhead domain-containing protein Fha1, and a periplasmic protein, TagR, that positively regulates PpkA. In the present work, we biochemically and functionally characterize three additional proteins of the phosphorylation-dependent regulatory cascade that controls T6S activation: TagT, TagS and TagQ. We show that similar to TagR, these proteins act upstream of the PpkA/PppA checkpoint and influence phosphorylation of Fha1 and export of Hcp1 and Tse1. Localization studies demonstrate that TagQ is an outer membrane lipoprotein and TagR is associated with the outer membrane. Consistent with their homology to lipoprotein outer membrane localization (Lol) components, TagT and TagS form a stable inner membrane complex with ATPase activity. However, we find that outer membrane association of T6SS lipoproteins TagQ and TssJ1, and TagR, is unaltered in a ΔtagTS background. Notably, we found that TagQ is indispensible for anchoring of TagR to the outer membrane fraction. As T6S-dependent fitness of P. aeruginosa requires TagT, S, R and Q, we conclude that these proteins likely participate in a trans-membrane signaling pathway that promotes H1-T6SS activity under optimal environmental conditions. PMID:22765374

  11. An ABC transporter and an outer membrane lipoprotein participate in posttranslational activation of type VI secretion in Pseudomonas aeruginosa.

    Casabona, Maria G; Silverman, Julie M; Sall, Khady M; Boyer, Frédéric; Couté, Yohann; Poirel, Jessica; Grunwald, Didier; Mougous, Joseph D; Elsen, Sylvie; Attree, Ina

    2013-02-01

    Pseudomonas aeruginosa is capable of injecting protein toxins into other bacterial cells through one of its three type VI secretion systems (T6SSs). The activity of this T6SS is tightly regulated on the posttranslational level by phosphorylation-dependent and -independent pathways. The phosphorylation-dependent pathway consists of a Threonine kinase/phosphatase pair (PpkA/PppA) that acts on a forkhead domain-containing protein, Fha1, and a periplasmic protein, TagR, that positively regulates PpkA. In the present work, we biochemically and functionally characterize three additional proteins of the phosphorylation-dependent regulatory cascade that controls T6S activation: TagT, TagS and TagQ. We show that similar to TagR, these proteins act upstream of the PpkA/PppA checkpoint and influence phosphorylation of Fha1 and, apparatus assembly and effector export. Localization studies demonstrate that TagQ is an outer membrane lipoprotein and TagR is associated with the outer membrane. Consistent with their homology to lipoprotein outer membrane localization (Lol) components, TagT and TagS form a stable inner membrane complex with ATPase activity. However, we find that outer membrane association of T6SS lipoproteins TagQ and TssJ1, and TagR, is unaltered in a ΔtagTS background. Notably, we found that TagQ is indispensible for anchoring of TagR to the outer membrane fraction. As T6S-dependent fitness of P. aeruginosa requires TagT, S, R and Q, we conclude that these proteins likely participate in a trans-membrane signalling pathway that promotes H1-T6SS activity under optimal environmental conditions. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  12. 46 CFR 154.170 - Outer hull steel plating.

    2010-10-01

    ... 46 Shipping 5 2010-10-01 2010-10-01 false Outer hull steel plating. 154.170 Section 154.170... STANDARDS FOR SELF-PROPELLED VESSELS CARRYING BULK LIQUEFIED GASES Design, Construction and Equipment Hull Structure § 154.170 Outer hull steel plating. (a) Except as required in paragraph (b) of this section, the...

  13. Outer space and nuclear deterrence: problems and prospects

    Gasparini Alves, P.

    1993-01-01

    The presentation deals with the role of outer-space applications and prospects for near future developments in nuclear deterrence. Outer space capabilities of United Sates, Russian Federation, Belarus, Kazakhstan, Ukraine, China, and United Kingdom as well as other states are analyzed. Conceptual problems of offensive and defensive doctrines are reviewed together with legal implications

  14. Long-Lived Glass Mirrors For Outer Space

    Bouquet, Frank L.; Maag, Carl R.; Heggen, Philip M.

    1988-01-01

    Paper summarizes available knowledge about glass mirrors for use in outer space. Strengths and weaknesses of various types of first and second reflective surfaces identified. Second-surface glass mirrors used in outer space designed to different criteria more stringent for terrestrial mirrors. Protons, electrons, cosmic rays, meteorites, and orbiting space debris affect longevities of components. Contamination also factor in space.

  15. Newborns' Face Recognition: Role of Inner and Outer Facial Features

    Turati, Chiara; Macchi Cassia, Viola; Simion, Francesca; Leo, Irene

    2006-01-01

    Existing data indicate that newborns are able to recognize individual faces, but little is known about what perceptual cues drive this ability. The current study showed that either the inner or outer features of the face can act as sufficient cues for newborns' face recognition (Experiment 1), but the outer part of the face enjoys an advantage…

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

    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. Comparative proteomic analysis of outer membrane vesicles from Shigella flexneri under different culture conditions

    Chen, Yong; Liu, Liguo; Fu, Hua; Wei, Candong, E-mail: weicando@ipbcams.ac.cn; Jin, Qi, E-mail: zdsys@vip.sina.com

    2014-10-31

    Highlights: • We utilized mTRAQ-based quantification to study protein changes in Congo red-induced OMVs. • A total of 148 proteins were identified in S. flexneri-derived OMVs. • Twenty-eight and five proteins are significantly up- and down-regulated in the CR-induced OMV, respectively. • The result implied that a special sorting mechanism of particular proteins into OMVs may exist. • Key node proteins in the protein interaction network might be important for pathogenicity. - Abstract: The production of outer membrane vesicles (OMVs) is a common and regulated process of gram-negative bacteria. Nonetheless, the processes of Shigella flexneri OMV production still remain unclear. S. flexneri is the causative agent of endemic shigellosis in developing countries. The Congo red binding of strains is associated with increased infectivity of S. flexneri. Therefore, understanding the modulation pattern of OMV protein expression induced by Congo red will help to elucidate the bacterial pathogenesis. In the present study, we investigated the proteomic composition of OMVs and the change in OMV protein expression induced by Congo red using mTRAQ-based quantitative comparative proteomics. mTRAQ labelling increased the confidence in protein identification, and 148 total proteins were identified in S. flexneri-derived OMVs. These include a variety of important virulence factors, including Ipa proteins, TolC family, murein hydrolases, and members of the serine protease autotransporters of Enterobacteriaceae (SPATEs) family. Among the identified proteins, 28 and five proteins are significantly up- and down-regulated in the Congo red-induced OMV, respectively. Additionally, by comprehensive comparison with previous studies focused on DH5a-derived OMV, we identified some key node proteins in the protein–protein interaction network that may be involved in OMV biogenesis and are common to all gram-negative bacteria.

  18. Comparative proteomic analysis of outer membrane vesicles from Shigella flexneri under different culture conditions

    Chen, Yong; Liu, Liguo; Fu, Hua; Wei, Candong; Jin, Qi

    2014-01-01

    Highlights: • We utilized mTRAQ-based quantification to study protein changes in Congo red-induced OMVs. • A total of 148 proteins were identified in S. flexneri-derived OMVs. • Twenty-eight and five proteins are significantly up- and down-regulated in the CR-induced OMV, respectively. • The result implied that a special sorting mechanism of particular proteins into OMVs may exist. • Key node proteins in the protein interaction network might be important for pathogenicity. - Abstract: The production of outer membrane vesicles (OMVs) is a common and regulated process of gram-negative bacteria. Nonetheless, the processes of Shigella flexneri OMV production still remain unclear. S. flexneri is the causative agent of endemic shigellosis in developing countries. The Congo red binding of strains is associated with increased infectivity of S. flexneri. Therefore, understanding the modulation pattern of OMV protein expression induced by Congo red will help to elucidate the bacterial pathogenesis. In the present study, we investigated the proteomic composition of OMVs and the change in OMV protein expression induced by Congo red using mTRAQ-based quantitative comparative proteomics. mTRAQ labelling increased the confidence in protein identification, and 148 total proteins were identified in S. flexneri-derived OMVs. These include a variety of important virulence factors, including Ipa proteins, TolC family, murein hydrolases, and members of the serine protease autotransporters of Enterobacteriaceae (SPATEs) family. Among the identified proteins, 28 and five proteins are significantly up- and down-regulated in the Congo red-induced OMV, respectively. Additionally, by comprehensive comparison with previous studies focused on DH5a-derived OMV, we identified some key node proteins in the protein–protein interaction network that may be involved in OMV biogenesis and are common to all gram-negative bacteria

  19. Prevention of an arms race in outer space

    Anon.

    1990-01-01

    The space age may be the to have begun in 1957, when for the first time a man-made object was lofted into orbit round the Earth. Since that date, the new problems of outer space have been discussed in the United Nations, particularly in the General Assembly, in the Committee on the Peaceful Uses of Outer Space and its subsidiary bodies, and in the Conference on Disarmament. The discussions have contributed to the conclusion of a number of international agreements concerning both military and peaceful aspects of the use of outer space. This paper reports that according to the 1967 Treaty on Principles Governing the Activities of States in the Exploration and Use of Outer Space, including the Moon and Other Celestial Bodies, known as the outer space Treaty, outer space, including the moon and other celestial bodies, is not subject to national appropriation by claim of sovereignty, by means of use or occupation, or by any other means (article II), and the parties undertake not to place in orbit around the earth any objects carrying nuclear weapons or any other kinds of weapons of ass destruction, install such weapons on celestial bodies, or station such weapons in outer space in any other manner (article IV). Detailed norms for States' actions in this environment are included in the 1979 Agreement Governing the Activities of States on the Moon and other Celestial Bodies to ensure that the Moon and other celestial bodies within the solar system, other than Earth, are used exclusively for peaceful purposes

  20. Ultrasonic examination of defects close to the outer surface

    Benoist, P.; Serre, M.; Champigny, F.

    1986-11-01

    During the examination of a pressurized water reactor vessel with an in Service Inspection Machine (MIS), various welds are scanned with immersion ultrasonic focused transducers from the inside of the vessel. Defects close to the outer surface are sometimes detected, and sizing with the successive 6 dB drop method leads to oversize some indications; this is caused by various reflections on the outer wall; the corner echo is of particular importance here. CEA and EDF have started an experimental program in order to study the response of volumetric and planar defects located near the outer surface. We present here the first results obtained with artificial defects. 2 refs

  1. Detergent-associated solution conformations of helical and beta-barrel membrane proteins.

    Mo, Yiming; Lee, Byung-Kwon; Ankner, John F; Becker, Jeffrey M; Heller, William T

    2008-10-23

    Membrane proteins present major challenges for structural biology. In particular, the production of suitable crystals for high-resolution structural determination continues to be a significant roadblock for developing an atomic-level understanding of these vital cellular systems. The use of detergents for extracting membrane proteins from the native membrane for either crystallization or reconstitution into model lipid membranes for further study is assumed to leave the protein with the proper fold with a belt of detergent encompassing the membrane-spanning segments of the structure. Small-angle X-ray scattering was used to probe the detergent-associated solution conformations of three membrane proteins, namely bacteriorhodopsin (BR), the Ste2p G-protein coupled receptor from Saccharomyces cerevisiae, and the Escherichia coli porin OmpF. The results demonstrate that, contrary to the traditional model of a detergent-associated membrane protein, the helical proteins BR and Ste2p are not in the expected, compact conformation and associated with detergent micelles, while the beta-barrel OmpF is indeed embedded in a disk-like micelle in a properly folded state. The comparison provided by the BR and Ste2p, both members of the 7TM family of helical membrane proteins, further suggests that the interhelical interactions between the transmembrane helices of the two proteins differ, such that BR, like other rhodopsins, can properly refold to crystallize, while Ste2p continues to prove resistant to crystallization from an initially detergent-associated state.

  2. Membrane Protein Properties Revealed through Data-Rich Electrostatics Calculations.

    Marcoline, Frank V; Bethel, Neville; Guerriero, Christopher J; Brodsky, Jeffrey L; Grabe, Michael

    2015-08-04

    The electrostatic properties of membrane proteins often reveal many of their key biophysical characteristics, such as ion channel selectivity and the stability of charged membrane-spanning segments. The Poisson-Boltzmann (PB) equation is the gold standard for calculating protein electrostatics, and the software APBSmem enables the solution of the PB equation in the presence of a membrane. Here, we describe significant advances to APBSmem, including full automation of system setup, per-residue energy decomposition, incorporation of PDB2PQR, calculation of membrane-induced pKa shifts, calculation of non-polar energies, and command-line scripting for large-scale calculations. We highlight these new features with calculations carried out on a number of membrane proteins, including the recently solved structure of the ion channel TRPV1 and a large survey of 1,614 membrane proteins of known structure. This survey provides a comprehensive list of residues with large electrostatic penalties for being embedded in the membrane, potentially revealing interesting functional information. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Atmospheric Mining in the Outer Solar System: Outer Planet Orbital Transfer and Lander Analyses

    Palaszewski, Bryan

    2016-01-01

    Atmospheric mining in the outer solar system has been investigated as a means of fuel production for high energy propulsion and power. Fusion fuels such as Helium 3 (3He) and deuterium can be wrested from the atmospheres of Uranus and Neptune and either returned to Earth or used in-situ for energy production. Helium 3 and deuterium were the primary gases of interest with hydrogen being the primary propellant for nuclear thermal solid core and gas core rocket-based atmospheric flight. A series of analyses were undertaken to investigate resource capturing aspects of atmospheric mining in the outer solar system. This included the gas capturing rate, storage options, and different methods of direct use of the captured gases. While capturing 3He, large amounts of hydrogen and 4He are produced. Analyses of orbital transfer vehicles (OTVs), landers, and the issues with in-situ resource utilization (ISRU) mining factories are included. Preliminary observations are presented on near-optimal selections of moon base orbital locations, OTV power levels, and OTV and lander rendezvous points. For analyses of round trip OTV flights from Uranus to Miranda or Titania, a 10- Megawatt electric (MWe) OTV power level and a 200 metricton (MT) lander payload were selected based on a relative short OTV trip time and minimization of the number of lander flights. A similar optimum power level is suggested for OTVs flying from low orbit around Neptune to Thalassa or Triton. Several moon base sites at Uranus and Neptune and the OTV requirements to support them are also addressed.

  4. Outer hair cell piezoelectricity: frequency response enhancement and resonance behavior.

    Weitzel, Erik K; Tasker, Ron; Brownell, William E

    2003-09-01

    Stretching or compressing an outer hair cell alters its membrane potential and, conversely, changing the electrical potential alters its length. This bi-directional energy conversion takes place in the cell's lateral wall and resembles the direct and converse piezoelectric effects both qualitatively and quantitatively. A piezoelectric model of the lateral wall has been developed that is based on the electrical and material parameters of the lateral wall. An equivalent circuit for the outer hair cell that includes piezoelectricity shows a greater admittance at high frequencies than one containing only membrane resistance and capacitance. The model also predicts resonance at ultrasonic frequencies that is inversely proportional to cell length. These features suggest all mammals use outer hair cell piezoelectricity to support the high-frequency receptor potentials that drive electromotility. It is also possible that members of some mammalian orders use outer hair cell piezoelectric resonance in detecting species-specific vocalizations.

  5. Inner and Outer Recursive Neural Networks for Chemoinformatics Applications.

    Urban, Gregor; Subrahmanya, Niranjan; Baldi, Pierre

    2018-02-26

    Deep learning methods applied to problems in chemoinformatics often require the use of recursive neural networks to handle data with graphical structure and variable size. We present a useful classification of recursive neural network approaches into two classes, the inner and outer approach. The inner approach uses recursion inside the underlying graph, to essentially "crawl" the edges of the graph, while the outer approach uses recursion outside the underlying graph, to aggregate information over progressively longer distances in an orthogonal direction. We illustrate the inner and outer approaches on several examples. More importantly, we provide open-source implementations [available at www.github.com/Chemoinformatics/InnerOuterRNN and cdb.ics.uci.edu ] for both approaches in Tensorflow which can be used in combination with training data to produce efficient models for predicting the physical, chemical, and biological properties of small molecules.

  6. Photoreceptor atrophy in acute zonal occult outer retinopathy

    Zibrandtsen, N.; Munch, I.C.; Klemp, K.

    2006-01-01

    Purpose: To assess retinal morphology in acute zonal occult outer retinopathy (AZOOR). Methods: Three patients with a normal ophthalmoscopic fundus appearance, a history of photopsia, and visual field loss compatible with AZOOR were examined using optical coherence tomography, automated perimetry...

  7. Proposal for the LHCb outer tracker front-end electronics

    Deppe, H; Feuerstack-Raible, M; Srowig, A; Stange, U; Hommels, B; Sluijk, T

    2001-01-01

    A market survey on available TDCs for reading out the LHCb Outer Tracker has left over only one TDC, which is not optimal for this purpose. Hence, a new readout architecture which is based on a TDC to be developed anew has been defined. This system fits optimal the requirements of the LHCb Outer Tracker and also should be much cheaper. The system and its main issues are described in this paper.

  8. Developments for the outer tracking system of the LHCb experiment

    Bachmann, S; Haas, T; Uwer, U; Walter, M; Wiedner, D

    2004-01-01

    The outer tracking system of the LHCb experiment is discussed. The outer tracking system (OT) is made of three stations and every station is made up of four detecting planes with a double layer of straw tubes. The straw tubes are mounted in detector module boxes made up of sandwich panels. The use of a counting gas with a high drift velocity is suggested to cope with high bunch crossing rate at the LHCb experiment. (Edited abstract) 3 Refs.

  9. Inner and outer cylinders of the CMS vacuum tank.

    Patrice Loïez

    2002-01-01

    The vacuum tank of the CMS magnet system consists of inner and outer stainless-steel cylinders and houses the superconducting coil. The inner cylinder contains all the barrel sub-detectors, which it supports via a system of horizontal rails. The cylinder is pictured here in the vertical position on a yellow platform mounted on the ferris-wheel support structure. This will allow it to be pivoted and inserted into the already installed outer cylinder, through which this photo was taken.

  10. Star laws: legal controls on armed conflict in outer space

    Stephens, Dale

    2016-01-01

    An undeclared military space race is unfolding yet there is no clear understanding of how international las operates in the field of armed conflict in outer space. In conjunction with McGill University Law School, Montreal, Canada, a 'Manual on international law applicable to military uses of outer space' has been drafted. This article looks at types of space weapons, previous space treaties and discusses humanitarian law.

  11. Kar5p is required for multiple functions in both inner and outer nuclear envelope fusion in Saccharomyces cerevisiae.

    Rogers, Jason V; Rose, Mark D

    2014-12-02

    During mating in the budding yeast Saccharomyces cerevisiae, two haploid nuclei fuse via two sequential membrane fusion steps. SNAREs (i.e., soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Prm3p mediate outer nuclear membrane fusion, but the inner membrane fusogen remains unknown. Kar5p is a highly conserved transmembrane protein that localizes adjacent to the spindle pole body (SPB), mediates nuclear envelope fusion, and recruits Prm3p adjacent to the SPB. To separate Kar5p's functions, we tested localization, Prm3p recruitment, and nuclear fusion efficiency in various kar5 mutants. All domains and the conserved cysteine residues were essential for nuclear fusion. Several kar5 mutant proteins localized properly but did not mediate Prm3p recruitment; other kar5 mutant proteins localized and recruited Prm3p but were nevertheless defective for nuclear fusion, demonstrating additional functions beyond Prm3p recruitment. We identified one Kar5p domain required for SPB localization, which is dependent on the half-bridge protein Mps3p. Electron microscopy revealed a kar5 mutant that arrests with expanded nuclear envelope bridges, suggesting that Kar5p is required after outer nuclear envelope fusion. Finally, a split-GFP assay demonstrated that Kar5p localizes to both the inner and outer nuclear envelope. These insights suggest a mechanism by which Kar5p mediates inner nuclear membrane fusion. Copyright © 2015 Rogers and Rose.

  12. Chlamydomonas outer arm dynein alters conformation in response to Ca2+.

    Sakato, Miho; Sakakibara, Hitoshi; King, Stephen M

    2007-09-01

    We have previously shown that Ca(2+) directly activates ATP-sensitive microtubule binding by a Chlamydomonas outer arm dynein subparticle containing the beta and gamma heavy chains (HCs). The gamma HC-associated LC4 light chain is a member of the calmodulin family and binds 1-2 Ca(2+) with K(Ca) = 3 x 10(-5) M in vitro, suggesting it may act as a Ca(2+) sensor for outer arm dynein. Here we investigate interactions between the LC4 light chain and gamma HC. Two IQ consensus motifs for binding calmodulin-like proteins are located within the stem domain of the gamma heavy chain. In vitro experiments indicate that LC4 undergoes a Ca(2+)-dependent interaction with the IQ motif domain while remaining tethered to the HC. LC4 also moves into close proximity of the intermediate chain IC1 in the presence of Ca(2+). The sedimentation profile of the gamma HC subunit changed subtly upon Ca(2+) addition, suggesting that the entire complex had become more compact, and electron microscopy of the isolated gamma subunit revealed a distinct alteration in conformation of the N-terminal stem in response to Ca(2+) addition. We propose that Ca(2+)-dependent conformational change of LC4 has a direct effect on the stem domain of the gamma HC, which eventually leads to alterations in mechanochemical interactions between microtubules and the motor domain(s) of the outer dynein arm.

  13. Influence of bacterial toxins on the GTPase activity of transducin from bovine retinal rod outer segments

    Rybin, V.O.; Gureeva, A.A.

    1986-01-01

    The action of cholera toxin, capable of ADP-ribosylation of the activator N/sub s/ protein, and pertussis toxin, capable of ADP-ribosylation of the inhibitor N/sub i/ protein of the adenylate cyclase complex, on transducin, the GTP-binding protein of the rod outer segments of the retina, was investigated. It was shown that under the action of pertussis and cholera toxins, the GTPase activity of transducin is inhibited. Pertussin toxin inhibits the GTPase of native retinal rod outer segments by 30-40%, while GTPase of homogeneous transducin produces a 70-80% inhibition. The action of toxins on transducin depends on the presence and nature of the guanylic nucleotide with which incubation is performed. On the basis of the data obtained it is suggested that pertussis toxin interacts with pretransducin and with the transducin-GDP complex, while cholera toxin ADP-ribosylates the transducin-GTP complex and does not act on transducin lacking GTP

  14. The Motion of a Single Molecule, the Lambda-Receptor, in the Bacterial Outer Membrane

    Oddershede, Lene; Dreyer, Jakob Kisbye; Grego, Sonia

    2002-01-01

    Using optical tweezers and single particle tracking, we have revealed the motion of a single protein, the lambda-receptor, in the outer membrane of living Escherichia coli bacteria. We genetically modified the lambda-receptor placing a biotin on an extracellular site of the receptor in vivo....... The efficiency of this in vivo biotinylation is very low, thus enabling the attachment of a streptavidin-coated bead binding specifically to a single biotinylated lambda-receptor. The bead was used as a handle for the optical tweezers and as a marker for the single particle tracking routine. We propose a model...

  15. Localization of cytochromes in the outer membrane of Desulfovibrio vulgaris (Hildenborough) and their role in anaerobic biocorrosion.

    Van Ommen Kloeke, F; Bryant, R D; Laishley, E J

    1995-12-01

    A protocol was developed whereby the outer and cytoplasmic membranes of the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) were isolated and partially characterized. The isolated outer membrane fractions from cultures grown under high (100 ppm) and low (5 ppm) Fe2+ conditions were compared by SDS-PAGE electrophoresis, and showed that several protein bands were derepressed under the low iron conditions, most notably at 50 kDa, and 77.5 kDa. Outer membrane isolated from low iron cultured cells was found to contain two proteins, 77.5 kDa and 62.5 kDa in size, that reacted with a heme-specific stain and were referred to as high molecular weight cytochromes. Studies conducted on the low iron isolated outer membrane by a phosphate/mild steel hydrogen evolution system showed that addition of the membrane fraction caused an immediate acceleration in H2 production. A new model for the anaerobic biocorrosion of mild steel is proposed.

  16. Molecular cloning and protein structure of a human blood group Rh polypeptide

    Cherif-Zahar, B.; Bloy, C.; Le Van Kim, C.; Blanchard, D.; Bailly, P.; Hermand, P.; Salmon, C.; Cartron, J.P.; Colin, Y.

    1990-01-01

    cDNA clones encoding a human blood group Rh polypeptide were isolated from a human bone marrow cDNA library by using a polymerase chain reaction-amplified DNA fragment encoding the known common N-terminal region of the Rh proteins. The entire primary structure of the Rh polypeptide has been deduced from the nucleotide sequence of a 1384-base-pair-long cDNA clone. Translation of the open reading frame indicates that the Rh protein is composed of 417 amino acids, including the initiator methionine, which is removed in the mature protein, lacks a cleavable N-terminal sequence, and has no consensus site for potential N-glycosylation. The predicted molecular mass of the protein is 45,500, while that estimated for the Rh protein analyzed in NaDodSO 4 /polyacrylamide gels is in the range of 30,000-32,000. These findings suggest either that the hydrophobic Rh protein behaves abnormally on NaDodSO 4 gels or that the Rh mRNA may encode a precursor protein, which is further matured by a proteolytic cleavage of the C-terminal region of the polypeptide. Hydropathy analysis and secondary structure predictions suggest the presence of 13 membrane-spanning domains, indicating that the Rh polypeptide is highly hydrophobic and deeply buried within the phospholipid bilayer. These results suggest that the expression of the Rh gene(s) might be restricted to tissues or cell lines expressing erythroid characters

  17. Software alignment of the LHCb Outer Tracker chambers

    Deissenroth, Marc

    2010-04-21

    This work presents an alignment algorithm that was developed to precisely determine the positions of the LHCb Outer Tracker detector elements. The algorithm is based on the reconstruction of tracks and exploits that misalignments of the detector change the residual between a measured hit and the reconstructed track. It considers different levels of granularities of the Outer Tracker geometry and fully accounts for correlations of all elements which are imposed by particle trajectories. In extensive tests, simulated shifts and rotations for different levels of the detector granularity have been used as input to the track reconstruction and alignment procedure. With about 260 000 tracks the misalignments are recovered with a statistical precision of O(10 - 100 {mu}m) for the translational degrees of freedom and of O(10{sup -2} - 10{sup -1} mrad) for rotations. A study has been performed to determine the impact of Outer Tracker misalignments on the performance of the track reconstruction algorithms. It shows that the achieved statistical precision does not decrease the track reconstruction performance in a significant way. During the commissioning of the LHCb detector, cosmic ray muon events have been collected. The events have been analysed and used for the first alignment of the 216 Outer Tracker modules. The module positions have been determined within {proportional_to} 90 {mu}m. The developed track based alignment algorithm has demonstrated its reliability and is one of the core algorithms which are used for the precise determination of the positions of the LHCb Outer Tracker elements. (orig.)

  18. AN OUTER ARM IN THE SECOND GALACTIC QUADRANT: STRUCTURE

    Du, Xinyu; Xu, Ye; Yang, Ji; Sun, Yan; Li, Facheng; Zhang, Shaobo; Zhou, Xin, E-mail: xydu@pmo.ac.cn, E-mail: xuye@pmo.ac.cn [Purple Mountain Observatory, Chinese Academy of Science, Nanjing 210008 (China)

    2016-05-01

    The lack of arm tracers, especially remote tracers, is one of the most difficult problems preventing us from studying the structure of the Milky Way. Fortunately, with its high-sensitivity CO survey, the Milky Way Imaging Scroll Painting (MWISP) project offers such an opportunity. Since completing about one-third of its mission, an area of l = [100, 150]°, b = [−3, 5]° has nearly been covered. The Outer arm of the Milky Way first clearly revealed its shape in the second galactic quadrant in the form of molecular gas—this is the first time that the Outer arm has been reported in such a large-scale mapping of molecular gas. Using the 115 GHz {sup 12}CO(1–0) data of MWISP at the LSR velocity ≃[−100, −60] km s{sup −1} and in the area mentioned above, we have detected 481 molecular clouds in total, and among them 332 (about 69%) are newly detected and 457 probably belong to the Outer arm. The total mass of the detected Outer arm clouds is ∼3.1 × 10{sup 6} M {sub ⊙}. Assuming that the spiral arm is a logarithmic spiral, the pitch angle is fitted as ∼13.°1. Besides combining both the CO data from MWISP and the 21 cm H i data from the Canadian Galactic Plane Survey (CGPS), the gas distribution, warp, and thickness of the Outer arm are also studied.

  19. Software alignment of the LHCb Outer Tracker chambers

    Deissenroth, Marc

    2010-01-01

    This work presents an alignment algorithm that was developed to precisely determine the positions of the LHCb Outer Tracker detector elements. The algorithm is based on the reconstruction of tracks and exploits that misalignments of the detector change the residual between a measured hit and the reconstructed track. It considers different levels of granularities of the Outer Tracker geometry and fully accounts for correlations of all elements which are imposed by particle trajectories. In extensive tests, simulated shifts and rotations for different levels of the detector granularity have been used as input to the track reconstruction and alignment procedure. With about 260 000 tracks the misalignments are recovered with a statistical precision of O(10 - 100 μm) for the translational degrees of freedom and of O(10 -2 - 10 -1 mrad) for rotations. A study has been performed to determine the impact of Outer Tracker misalignments on the performance of the track reconstruction algorithms. It shows that the achieved statistical precision does not decrease the track reconstruction performance in a significant way. During the commissioning of the LHCb detector, cosmic ray muon events have been collected. The events have been analysed and used for the first alignment of the 216 Outer Tracker modules. The module positions have been determined within ∝ 90 μm. The developed track based alignment algorithm has demonstrated its reliability and is one of the core algorithms which are used for the precise determination of the positions of the LHCb Outer Tracker elements. (orig.)

  20. Knitted outer gloves in primary hip and knee arthroplasty.

    Tanner, J; Wraighte, P; Howard, P

    2006-01-01

    A randomised trial was carried out to determine the rate of perforation to inner gloves when comparing latex with knitted gloves during hip and knee arthroplasty. Members of the surgical team were randomised to wear either two pairs of latex gloves (standard double gloving) or a knitted glove on top of a latex glove. In addition, participants completed a visual analogue assessment of their overall satisfaction with the gloves. A total of 406 inner gloves were tested for perforations over a four-month period: 23% of inner gloves were perforated when latex outer gloves were used and 6% of inner gloves were perforated when knitted outer gloves were used. In total, there were 64 perforations to the inner gloves; only one of these perforations was detected by the glove wearer. Wearing knitted outer gloves during hip and knee arthroplasty statistically significantly reduces the risk of perforation to inner latex gloves (p<0.0001).

  1. Outer Planet Missions with Electric Propulsion Systems—Part I

    Carlos Renato Huaura Solórzano

    2010-01-01

    Full Text Available For interplanetary missions, efficient electric propulsion systems can be used to increase the mass delivered to the destination. Outer planet exploration has experienced new interest with the launch of the Cassini and New Horizons Missions. At the present, new technologies are studied for better use of electric propulsion systems in missions to the outer planets. This paper presents low-thrust trajectories using the method of the transporting trajectory to Uranus, Neptune, and Pluto. They use nuclear and radio isotopic electric propulsion. These direct transfers have continuous electric propulsion of low power along the entire trajectory. The main goal of the paper is to optimize the transfers, that is, to provide maximum mass to be delivered to the outer planets.

  2. The outer magnetosphere. [composition and comparison with earth

    Schardt, A. W.; Behannon, K. W.; Lepping, R. P.; Carbary, J. F.; Eviatar, A.; Siscoe, G. L.

    1984-01-01

    Similarities between the Saturnian and terrestrial outer magnetosphere are examined. Saturn, like earth, has a fully developed magnetic tail, 80 to 100 RS in diameter. One major difference between the two outer magnetospheres is the hydrogen and nitrogen torus produced by Titan. This plasma is, in general, convected in the corotation direction at nearly the rigid corotation speed. Energies of magnetospheric particles extend to above 500 keV. In contrast, interplanetary protons and ions above 2 MeV have free access to the outer magnetosphere to distances well below the Stormer cutoff. This access presumably occurs through the magnetotail. In addition to the H+, H2+, and H3+ ions primarily of local origin, energetic He, C, N, and O ions are found with solar composition. Their flux can be substantially enhanced over that of interplanetary ions at energies of 0.2 to 0.4 MeV/nuc.

  3. Mechanism and function of the outer membrane channel TolC in multidrug resistance and physiology of enterobacteria

    Helen I. Zgurskaya

    2011-09-01

    Full Text Available TolC is an archetypal member of the Outer membrane Efflux Protein (OEP family. These proteins are involved in export of peptide and small molecule toxins across the outer membrane of Gram-negative bacteria. Genomes of some bacteria such as Pseudomonas species contain multiple copies of OEPs. In contrast, enterobacteria contain a single tolC gene, the product of which functions with multiple transporters. Inactivation of tolC has a major impact on enterobacterial physiology and virulence. Recent studies suggest that the role of TolC in physiology of enterobacteria is very broad and affects almost all aspects of cell adaptation to adverse enviroments. We review the current state of understanding TolC structure and present an integrated view of TolC function in enterobacteria. We propose that seemingly unrelated phenotypes of tolC mutants are linked together by a single most common condition – an oxidative damage to membranes.

  4. Study of Power Options for Jupiter and Outer Planet Missions

    Landis, Geoffrey A.; Fincannon, James

    2015-01-01

    Power for missions to Jupiter and beyond presents a challenging goal for photovoltaic power systems, but NASA missions including Juno and the upcoming Europa Clipper mission have shown that it is possible to operate solar arrays at Jupiter. This work analyzes photovoltaic technologies for use in Jupiter and outer planet missions, including both conventional arrays, as well as analyzing the advantages of advanced solar cells, concentrator arrays, and thin film technologies. Index Terms - space exploration, spacecraft solar arrays, solar electric propulsion, photovoltaic cells, concentrator, Fresnel lens, Jupiter missions, outer planets.

  5. Colors of Outer Solar System Objects Measured with VATT

    Romanishin, William; Tegler, S. C.; Consolmagno, G. J.

    2010-10-01

    Over the past 7 years, we have measured optical B-V and V-R colors for about 40 minor outer solar system objects using the 1.8-m Vatican Advanced Technology Telescope (VATT) located on Mt. Graham in southeast Arizona. We will present these colors and use them to update the discussion of colors of minor bodies in the outer solar system. We gratefully acknowledge funding from the NASA Planetary Astronomy Program to Northern Arizona University and the U. of Oklahoma which helped support this work.

  6. Photoprotective substance occurs primarily in outer layers of fish skin.

    Fabacher, D L; Little, E E

    1998-01-01

    Methanol extracts of dorsal skin layers, eyes, gills, and livers from ultraviolet-B (UVB) radiation-sensitive and UVB-tolerant species of freshwater fish were examined for a substance that appears to be photoprotective. Significantly larger amounts of this substance were found in extracts of outer dorsal skin layers from both UVB-sensitive and UVB-tolerant fish when compared with extracts of inner dorsal skin layers. This substance occurred in minor amounts or was not detected in eye, gill, and liver extracts. The apparent primary function of this substance in fish is to protect the cells in outer dorsal skin layers from harmful levels of UVB radiation.

  7. Stability of marginally outer trapped surfaces and symmetries

    Carrasco, Alberto; Mars, Marc, E-mail: acf@usal.e, E-mail: marc@usal.e [Facultad de Ciencias, Universidad de Salamanca, Plaza de la Merced s/n, 37008 Salamanca (Spain)

    2009-09-07

    We study the properties of stable, strictly stable and locally outermost marginally outer trapped surfaces in spacelike hypersurfaces of spacetimes possessing certain symmetries such as isometries, homotheties and conformal Killings. We first obtain results for general diffeomorphisms in terms of the so-called metric deformation tensor and then particularize to different types of symmetries. In particular, we find restrictions at the surfaces on the vector field generating the symmetry. Some consequences are discussed. As an application, we present a result on non-existence of stable marginally outer trapped surfaces in slices of FLRW.

  8. Chemical synthesis of membrane proteins by the removable backbone modification method.

    Tang, Shan; Zuo, Chao; Huang, Dong-Liang; Cai, Xiao-Ying; Zhang, Long-Hua; Tian, Chang-Lin; Zheng, Ji-Shen; Liu, Lei

    2017-12-01

    Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy. Two key steps of this protocol are: (i) installation of solubilizing Arg4-tagged RBM groups into the transmembrane peptides at any primary amino acid through Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis and (ii) native ligation of the full-length sequence, followed by removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to afford the native protein. The installation of RBM groups is achieved by using 4-methoxy-5-nitrosalicyladehyde by reduction amination to incorporate an activated O-to-N acyl transfer auxiliary. The Arg4-tag-modified membrane-spanning peptide segments behave like water-soluble peptides to facilitate their purification, ligation and mass characterization.

  9. Refolding, purification and crystallization of the FrpB outer membrane iron transporter from Neisseria meningitidis

    Saleem, Muhammad; Prince, Stephen M.; Patel, Hema; Chan, Hannah; Feavers, Ian M.; Derrick, Jeremy P.

    2012-01-01

    The refolding, purification and crystallization of FrpB from the meningitis pathogen Neisseria meningitidis is described. FrpB is an integral outer membrane protein from the human pathogen Neisseria meningitidis. It is a member of the TonB-dependent transporter family and promotes the uptake of iron across the outer membrane. There is also evidence that FrpB is an antigen and hence a potential component of a vaccine against meningococcal meningitis. FrpB incorporating a polyhistidine tag was overexpressed in Escherichia coli into inclusion bodies. The protein was then solubilized in urea, refolded and purified to homogeneity. Two separate antigenic variants of FrpB were crystallized by sitting-drop vapour diffusion. Crystals of the F5-1 variant diffracted to 2.4 Å resolution and belonged to space group C2, with unit-cell parameters a = 176.5, b = 79.4, c = 75.9 Å, β = 98.3°. Crystal-packing calculations suggested the presence of a monomer in the asymmetric unit. Crystals of the F3-3 variant also diffracted to 2.4 Å resolution and belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 85.3, b = 104.6, c = 269.1 Å. Preliminary analysis suggested the presence of an FrpB trimer in the asymmetric unit

  10. Genetic Manipulation of Outer Membrane Permeability: Generating Porous Heterogeneous Catalyst Analogs in Escherichia coli

    Patel, TN; Park, AHA; Bantat, S

    2014-12-01

    The limited permeability of the E. coli outer membrane can significantly hinder whole-cell biocatalyst performance. In this study, the SARS coronavirus small envelope protein (SCVE) was expressed in E. coli cells previously engineered for periplasmic expression of carbonic anhydrase (CA) activity. This maneuver increased small molecule uptake by the cells, resulting in increased apparent CA activity of the biocatalysts. The enhancements in activity were quantified using methods developed for traditional heterogeneous catalysis. The expression of the SCVE protein was found to significantly reduce the Thiele moduli (phi), as well as increase the effectiveness factors (eta), effective diffusivities (D-e), and permeabilities (P) of the biocatalysts. These catalytic improvements translated into superior performance of the biocatalysts for the precipitation of calcium carbonate from solution which is an attractive strategy for long-term sequestration of captured carbon dioxide. Overall, these results demonstrate that synthetic biology approaches can be used to enhance heterogeneous catalysts incorporated into microbial whole-cell scaffolds.

  11. Proteomic study via a non-gel based approach of meningococcal outer membrane vesicle vaccine obtained from strain CU385: a road map for discovering new antigens.

    Gil, Jeovanis; Betancourt, L Zaro H; Sardiñas, Gretel; Yero, Daniel; Niebla, Olivia; Delgado, Maité; García, Darien; Pajón, Rolando; Sánchez, Aniel; González, Luis J; Padrón, Gabriel; Campa, Concepción; Sotolongo, Franklin; Barberó, Ramón; Guillén, Gerardo; Herrera, Luis; Besada, Vladimir

    2009-05-01

    This work presents the results from a study of the protein composition of outer membrane vesicles from VA-MENGOC-BC (Finlay Institute, Cuba), an available vaccine against serogroup B Neisseria meningitidis. Proteins were identified by means of SCAPE, a 2DE-free method for proteome studies. More than one hundred proteins were detected by tandem liquid chromatographymass spectrometry analysis of fractions enriched in peptides devoid of histidine or arginine residues, providing a detailed description of the vaccine. A bioinformatic analysis of the identified components resulted in the identification of 31 outer membrane proteins and three conserved hypothetical proteins, allowing the cloning, expression, purification and immunological study of two of them (NMB0088 and NMB1796) as new antigens.

  12. Epoxide-mediated differential packaging of Cif and other virulence factors into outer membrane vesicles.

    Ballok, Alicia E; Filkins, Laura M; Bomberger, Jennifer M; Stanton, Bruce A; O'Toole, George A

    2014-10-01

    Pseudomonas aeruginosa produces outer membrane vesicles (OMVs) that contain a number of secreted bacterial proteins, including phospholipases, alkaline phosphatase, and the CFTR inhibitory factor (Cif). Previously, Cif, an epoxide hydrolase, was shown to be regulated at the transcriptional level by epoxides, which serve as ligands of the repressor, CifR. Here, we tested whether epoxides have an effect on Cif levels in OMVs. We showed that growth of P. aeruginosa in the presence of specific epoxides but not a hydrolysis product increased Cif packaging into OMVs in a CifR-independent fashion. The outer membrane protein, OprF, was also increased under these conditions, but alkaline phosphatase activity was not significantly altered. Additionally, we demonstrated that OMV shape and density were affected by epoxide treatment, with two distinct vesicle fractions present when cells were treated with epibromohydrin (EBH), a model epoxide. Vesicles isolated from the two density fractions exhibited different protein profiles in Western blotting and silver staining. We have shown that a variety of clinically or host-relevant treatments, including antibiotics, also alter the proteins packaged in OMVs. Proteomic analysis of purified OMVs followed by an analysis of transposon mutant OMVs yielded mutants with altered vesicle packaging. Finally, epithelial cell cytotoxicity was reduced in the vesicles formed in the presence of EBH, suggesting that this epoxide alters the function of the OMVs. Our data support a model whereby clinically or host-relevant signals mediate differential packaging of virulence factors in OMVs, which results in functional consequences for host-pathogen interactions. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  13. Outer membrane biogenesis in Escherichia coli, Neisseria meningitidis, and Helicobacter pylori: paradigm deviations in H. pylori.

    Liechti, George; Goldberg, Joanna B

    2012-01-01

    The bacterial pathogen Helicobacter pylori is capable of colonizing the gastric mucosa of the human stomach using a variety of factors associated with or secreted from its outer membrane (OM). Lipopolysaccharide (LPS) and numerous OM proteins have been shown to be involved in adhesion and immune stimulation/evasion. Many of these factors are essential for colonization and/or pathogenesis in a variety of animal models. Despite this wide array of potential targets present on the bacterial surface, the ability of H. pylori to vary its OM profile limits the effectiveness of vaccines or therapeutics that target any single one of these components. However, it has become evident that the proteins comprising the complexes that transport the majority of these molecules to the OM are highly conserved and often essential. The field of membrane biogenesis has progressed remarkably in the last few years, and the possibility now exists for targeting the mechanisms by which β-barrel proteins, lipoproteins, and LPS are transported to the OM, resulting in loss of bacterial fitness and significant altering of membrane permeability. In this review, the OM transport machinery for LPS, lipoproteins, and outer membrane proteins (OMPs) are discussed. While the principal investigations of these transport mechanisms have been conducted in Escherichia coli and Neisseria meningitidis, here these systems will be presented in the genetic context of ε proteobacteria. Bioinformatic analysis reveals that minimalist genomes, such as that of Helicobacter pylori, offer insight into the smallest number of components required for these essential pathways to function. Interestingly, in the majority of ε proteobacteria, while the inner and OM associated apparatus of LPS, lipoprotein, and OMP transport pathways appear to all be intact, most of the components associated with the periplasmic compartment are either missing or are almost unrecognizable when compared to their E. coli counterparts. Eventual

  14. Thermographic studies of outer target heat fluxes on KSTAR

    H.H. Lee

    2017-08-01

    Full Text Available A new infra-red (IR thermography system with high spatial resolution has been installed on KSTAR and is now mainly applied to measure the outer divertor heat load profile. The first measurement results of the outer divertor heat load profiles between ELMs have been applied to characterize the inter-ELMs outer divertor heat loads in KSTAR H-mode plasmas. In particular, the power decay length (λq of the divertor heat load profile has been determined by fitting the profile to a convolution of an exponential decay and a Gaussian function. The analysis on the power decay length shows a good agreement with the recent multi-machine λq scaling, which predicts λq of the inter-ELMs divertor heat load to be ∼1 mm under the standard H-mode scenario in ITER. The divertor IR thermography system has also successfully measured the strike point splitting of the outer divertor heat flux during the application of resonant magnetic perturbation (RMP fields. In addition, it has provided a clear evidence that the strike point splitting pattern depends on the RMP fields configuration.

  15. 75 FR 71734 - Outer Continental Shelf (OCS), Scientific Committee (SC)

    2010-11-24

    ... Environmental Studies Program (ESP) and environmental aspects of the offshore energy and marine minerals... oceanography, as well as studies of the social and economic impacts of OCS energy and marine minerals... DEPARTMENT OF THE INTERIOR Bureau of Ocean Energy Management, Regulation and Enforcement Outer...

  16. Detergent organisation in crystals of monomeric outer membrane phospholipase A

    Snijder, HJ; Timmins, PA; Kalk, KH; Dijkstra, BW

    The structure of the detergent in crystals of outer membrane phospholipase A (OMPLA) has been determined using neutron diffraction contrast variation. Large crystals were soaked in stabilising solutions, each containing a different H2O/D2O contrast. From the neutron diffraction at five contrasts,

  17. Outer casing of the AA antiproton production target

    CERN PhotoLab

    1979-01-01

    The first version of the antiproton production target was a tungsten rod, 11 cm long (actually a row of 11 rods, each 1 cm long) and 3 mm in diameter. The rod was embedded in graphite, pressure-seated into an outer casing made of stainless steel. The casing had fins for forced-air cooling.

  18. A progenitor of the outer membrane LamB trimer.

    Stader, J; Silhavy, T J

    1988-01-01

    During its localization to the outer membrane, LamB possesses distinctive biochemical properties as it passes through the cytoplasmic membrane. Because LamB entered this dynamic state with an attached signal sequence and leaves after cleavage, we call this export-related form of LamB the early-translocation form (et-LamB).

  19. Ethane Ices in the Outer Solar System: Spectroscopy and Chemistry

    Hudson, R. L.; Moore, M. H.; Raines, L. L.

    2009-01-01

    We report recent experiments on ethane ices made at temperatures applicable to the outer Solar System. New near- and mid-infrared data for crystalline and amorphous ethane, including new spectra for a seldom-studied solid phase that exists at 35-55 K, are presented along with radiation-chemical experiments showing the formation of more-complex hydrocarbons

  20. Wisps in the outer edge of the Keeler Gap

    Tiscareno, Matthew S.; Arnault, Ethan G.

    2015-11-01

    Superposed upon the relatively smooth outer edge of the Keeler Gap are a system of "wisps," which appear to be ring material protruding inward into the gap, usually with a sharp trailing edge and a smooth gradation back to the background edge location on the leading side (Porco et al. 2005, Science). The radial amplitude of wisps is usually 0.5 to 1 km, and their azimuthal extent is approximately a degree of longitude (~2400 km). Wisps are likely caused by an interplay between Daphnis (and perhaps other moons) and embedded moonlets within the ring, though the details remain unclear.Aside from the wisps, the Keeler Gap outer edge is the only one of the five sharp edges in the outer part of Saturn's A ring that is reasonably smooth in appearance (Tiscareno et al. 2005, DPS), with occultations indicating residuals less than 1 km upon a possibly non-zero eccentricity (R.G. French, personal communication, 2014). The other four (the inner and outer edges of the Encke Gap, the inner edge of the Keeler Gap, and the outer edge of the A ring itself) are characterized by wavy structure at moderate to high spatial frequencies, with amplitudes ranging from 2 to 30 km (Tiscareno et al. 2005, DPS).We will present a catalogue of wisp detections in Cassini images. We carry out repeated gaussian fits of the radial edge location in order to characterize edge structure and visually scan those fitted edges in order to detect wisps. With extensive coverage in longitude and in time, we will report on how wisps evolve and move, both within an orbit period and on longer timescales. We will also report on the frequency and interpretation of wisps that deviate from the standard morphology. We will discuss the implications of our results for the origin and nature of wisps, and for the larger picture of how masses interact within Saturn's rings.

  1. Novel function of Wsc proteins as a methanol-sensing machinery in the yeast Pichia pastoris.

    Ohsawa, Shin; Yurimoto, Hiroya; Sakai, Yasuyoshi

    2017-04-01

    Wsc family proteins are plasma membrane spanning sensor proteins conserved from yeasts to mammalian cells. We studied the functional roles of Wsc family proteins in the methylotrophic yeast Pichia pastoris, and found that PpWsc1 and PpWsc3 function as methanol-sensors during growth on methanol. PpWsc1 responds to a lower range of methanol concentrations than PpWsc3. PpWsc1, but not PpWsc3, also functions during high temperature stress, but PpWsc1 senses methanol as a signal that is distinct from high-temperature stress. We also found that PpRom2, which is known to function downstream of the Wsc family proteins in the cell wall integrity pathway, was also involved in sensing methanol. Based on these results, these PpWsc family proteins were demonstrated to be involved in sensing methanol and transmitting the signal via their cytoplasmic tail to the nucleus via PpRom2, which plays a critical role in regulating expression of a subset of methanol-inducible genes to coordinate well-balanced methanol metabolism. © 2017 John Wiley & Sons Ltd.

  2. Crystallization and preliminary crystallographic characterization of the iron-regulated outer membrane lipoprotein FrpD from Neisseria meningitidis

    Sviridova, E.; Bumba, Ladislav; Řezáčová, Pavlína; Procházková, Kateřina; Kavan, Daniel; Bezouška, Karel; Kutý, Michal; Šebo, Peter; Kutá-Smatanová, Ivana

    2010-01-01

    Roč. 66, - (2010), s. 1119-1123 ISSN 1744-3091 R&D Projects: GA MŠk(CZ) LC06010; GA ČR GP310/06/P150 Institutional research plan: CEZ:AV0Z50200510; CEZ:AV0Z50520514; CEZ:AV0Z60870520; CEZ:AV0Z40550506 Keywords : Fe-regulated protein D * iron-regulated proteins * outer membrane lipoproteins Subject RIV: EC - Immunology Impact factor: 0.563, year: 2010

  3. Characterization of a subunit of the outer dynein arm docking complex necessary for correct flagellar assembly in Leishmania donovani.

    Simone Harder

    Full Text Available BACKGROUND: In order to proceed through their life cycle, Leishmania parasites switch between sandflies and mammals. The flagellated promastigote cells transmitted by the insect vector are phagocytized by macrophages within the mammalian host and convert into the amastigote stage, which possesses a rudimentary flagellum only. During an earlier proteomic study of the stage differentiation of the parasite we identified a component of the outer dynein arm docking complex, a structure of the flagellar axoneme. The 70 kDa subunit of the outer dynein arm docking complex consists of three subunits altogether and is essential for the assembly of the outer dynein arm onto the doublet microtubule of the flagella. According to the nomenclature of the well-studied Chlamydomonas reinhardtii complex we named the Leishmania protein LdDC2. METHODOLOGY/PRINCIPAL FINDINGS: This study features a characterization of the protein over the life cycle of the parasite. It is synthesized exclusively in the promastigote stage and localizes to the flagellum. Gene replacement mutants of lddc2 show reduced growth rates and diminished flagellar length. Additionally, the normally spindle-shaped promastigote parasites reveal a more spherical cell shape giving them an amastigote-like appearance. The mutants lose their motility and wiggle in place. Ultrastructural analyses reveal that the outer dynein arm is missing. Furthermore, expression of the amastigote-specific A2 gene family was detected in the deletion mutants in the absence of a stage conversion stimulus. In vitro infectivity is slightly increased in the mutant cell line compared to wild-type Leishmania donovani parasites. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the correct assembly of the flagellum has a great influence on the investigated characteristics of Leishmania parasites. The lack of a single flagellar protein causes an aberrant morphology, impaired growth and altered infectiousness of the parasite.

  4. Identification and characterization of a novel porin family highlights a major difference in the outer membrane of chlamydial symbionts and pathogens.

    Karin Aistleitner

    Full Text Available The Chlamydiae constitute an evolutionary well separated group of intracellular bacteria comprising important pathogens of humans as well as symbionts of protozoa. The amoeba symbiont Protochlamydia amoebophila lacks a homologue of the most abundant outer membrane protein of the Chlamydiaceae, the major outer membrane protein MOMP, highlighting a major difference between environmental chlamydiae and their pathogenic counterparts. We recently identified a novel family of putative porins encoded in the genome of P. amoebophila by in silico analysis. Two of these Protochlamydiaouter membrane proteins, PomS (pc1489 and PomT (pc1077, are highly abundant in outer membrane preparations of this organism. Here we show that all four members of this putative porin family are toxic when expressed in the heterologous host Escherichia coli. Immunofluorescence analysis using antibodies against heterologously expressed PomT and PomS purified directly from elementary bodies, respectively, demonstrated the location of both proteins in the outer membrane of P. amoebophila. The location of the most abundant protein PomS was further confirmed by immuno-transmission electron microscopy. We could show that pomS is transcribed, and the corresponding protein is present in the outer membrane throughout the complete developmental cycle, suggesting an essential role for P. amoebophila. Lipid bilayer measurements demonstrated that PomS functions as a porin with anion-selectivity and a pore size similar to the Chlamydiaceae MOMP. Taken together, our results suggest that PomS, possibly in concert with PomT and other members of this porin family, is the functional equivalent of MOMP in P. amoebophila. This work contributes to our understanding of the adaptations of symbiotic and pathogenic chlamydiae to their different eukaryotic hosts.

  5. Components of SurA required for outer membrane biogenesis in uropathogenic Escherichia coli.

    Kristin M Watts

    2008-10-01

    Full Text Available SurA is a periplasmic peptidyl-prolyl isomerase (PPIase and chaperone of Escherichia coli and other Gram-negative bacteria. In contrast to other PPIases, SurA appears to have a distinct role in chaperoning newly synthesized porins destined for insertion into the outer membrane. Previous studies have indicated that the chaperone activity of SurA rests in its "core module" (the N- plus C-terminal domains, based on in vivo envelope phenotypes and in vitro binding and protection of non-native substrates.In this study, we determined the components of SurA required for chaperone activity using in vivo phenotypes relevant to disease causation by uropathogenic E. coli (UPEC, namely membrane resistance to permeation by antimicrobials and maturation of the type 1 pilus usher FimD. FimD is a SurA-dependent, integral outer membrane protein through which heteropolymeric type 1 pili, which confer bladder epithelial binding and invasion capacity upon uropathogenic E. coli, are assembled and extruded. Consistent with prior results, the in vivo chaperone activity of SurA in UPEC rested primarily in the core module. However, the PPIase domains I and II were not expendable for wild-type resistance to novobiocin in broth culture. Steady-state levels of FimD were substantially restored in the UPEC surA mutant complemented with the SurA N- plus C-terminal domains. The addition of PPIase domain I augmented FimD maturation into the outer membrane, consistent with a model in which domain I enhances stability of and/or substrate binding by the core module.Our results confirm the core module of E. coli SurA as a potential target for novel anti-infective development.

  6. Bloodstream-To-Eye Infections Are Facilitated by Outer Blood-Retinal Barrier Dysfunction.

    Phillip S Coburn

    Full Text Available The blood-retinal barrier (BRB functions to maintain the immune privilege of the eye, which is necessary for normal vision. The outer BRB is formed by tightly-associated retinal pigment epithelial (RPE cells which limit transport within the retinal environment, maintaining retinal function and viability. Retinal microvascular complications and RPE dysfunction resulting from diabetes and diabetic retinopathy cause permeability changes in the BRB that compromise barrier function. Diabetes is the major predisposing condition underlying endogenous bacterial endophthalmitis (EBE, a blinding intraocular infection resulting from bacterial invasion of the eye from the bloodstream. However, significant numbers of EBE cases occur in non-diabetics. In this work, we hypothesized that dysfunction of the outer BRB may be associated with EBE development. To disrupt the RPE component of the outer BRB in vivo, sodium iodate (NaIO3 was administered to C57BL/6J mice. NaIO3-treated and untreated mice were intravenously injected with 108 colony forming units (cfu of Staphylococcus aureus or Klebsiella pneumoniae. At 4 and 6 days postinfection, EBE was observed in NaIO3-treated mice after infection with K. pneumoniae and S. aureus, although the incidence was higher following S. aureus infection. Invasion of the eye was observed in control mice following S. aureus infection, but not in control mice following K. pneumoniae infection. Immunohistochemistry and FITC-dextran conjugate transmigration assays of human RPE barriers after infection with an exoprotein-deficient agr/sar mutant of S. aureus suggested that S. aureus exoproteins may be required for the loss of the tight junction protein, ZO-1, and for permeability of this in vitro barrier. Our results support the clinical findings that for both pathogens, complications which result in BRB permeability increase the likelihood of bacterial transmigration from the bloodstream into the eye. For S. aureus, however, BRB

  7. Calcium regulates ATP-sensitive microtubule binding by Chlamydomonas outer arm dynein.

    Sakato, Miho; King, Stephen M

    2003-10-31

    The Chlamydomonas outer dynein arm contains three distinct heavy chains (alpha, beta, and gamma) that exhibit different motor properties. The LC4 protein, which binds 1-2 Ca2+ with KCa = 3 x 10-5 m, is associated with the gamma heavy chain and has been proposed to act as a sensor to regulate dynein motor function in response to alterations in intraflagellar Ca2+ levels. Here we genetically dissect the outer arm to yield subparticles containing different motor unit combinations and assess the microtubule-binding properties of these complexes both prior to and following preincubation with tubulin and ATP, which was used to inhibit ATP-insensitive (structural) microtubule binding. We observed that the alpha heavy chain exhibits a dominant Ca2+-independent ATP-sensitive MT binding activity in vitro that is inhibited by attachment of tubulin to the structural microtubule-binding domain. Furthermore, we show that ATP-sensitive microtubule binding by a dynein subparticle containing only the beta and gamma heavy chains does not occur at Ca2+ concentrations below pCa 6 but is maximally activated above pCa 5. This activity was not observed in mutant dyneins containing small deletions in the microtubule-binding region of the beta heavy chain or in dyneins that lack both the alpha heavy chain and the motor domain of the beta heavy chain. These findings strongly suggest that Ca2+ binding directly to a component of the dynein complex regulates ATP-sensitive interactions between the beta heavy chain and microtubules and lead to a model for how individual motor units are controlled within the outer dynein arm.

  8. Antibiotic Trapping by Plasmid-Encoded CMY-2 beta-Lactamase Combined with Reduced Outer Membrane Permeability as a Mechanism of Carbapenem Resistance in Escherichia coli

    Goessens, W.H.F.; van der Bij, A.K.; van Boxtel, R.; Pitout, J.D.D.; van Ulsen, J.P.; Melles, D.C.; Tommassen, J.

    2013-01-01

    A liver transplant patient was admitted with cholangitis, for which meropenem therapy was started. Initial cultures showed a carbapenem-susceptible (CS) Escherichia coli strain, but during admission, a carbapenem-resistant (CR) E. coli strain was isolated. Analysis of the outer membrane protein

  9. Discovery of Salmonella Virulence Factors Translocated via Outer Membrane Vesicles to Murine Macrophages.

    Yoon, Hyunjin; Ansong, Charles; Adkins, Joshua N.; Heffron, Fred

    2011-06-01

    We have previously shown that the regulators SpvR, FruR, IHF, PhoP/PhoQ, SsrA/SsrB, SlyA, Hnr, RpoE, SmpB, CsrA, RpoS, Crp, OmpR/EnvZ, and Hfq are essential for Salmonella Typhimurium virulence in mice. Here we use quantitative LC-MS-based proteomics profiling of in-frame deletion mutants of these 14 regulators to identify proteins that are coordinately regulated by these virulence regulators and are thus presumably novel factors contributing to Salmonella pathogenesis. Putative candidate proteins from proteomics analysis were determined, which exhibited similar abundance profiles to those of Salmonella pathogenicity island (SPI)-2 type III secretion system (TTSS) proteins. A subset of 5 proteins including STM0082, STM1548, PdgL, STM1633, and STM3595 was selected for further analysis. All 5 proteins were expressed inside macrophage cells and STM0082 (SrfN) was secreted into host cytoplasm. Furthermore, deletion of STM0082 attenuated virulence in mice when administered intraperitoneally as determined by competitive index. srfN transcription was positively regulated by SsrAB, however, secretion was independent of SPI-2 TTSS as well as SPI-1 TTSS and flagella. Proteins including PagK and STM2585A, which are positively regulated by PhoP/PhoQ, have sec signal peptides as predicted for SrfN and were secreted into macrophage cytoplasm regardless of SPI-2 TTSS. Isolation of outer membrane vesicles (OMVs) revealed the presence of SrfN, PagK, and STM2585A inside vesicle compartments. This result is the first case showing delivery of virulence effectors via OMVs in S. Typhimurium. Moreover, Hfq regulation of SrfN translation suggests that small non-coding RNAs may be responsible for regulating effector protein expression.

  10. Focus on the Outer Membrane Factor OprM, the Forgotten Player from Efflux Pumps Assemblies

    Gilles Phan

    2015-11-01

    Full Text Available Antibiotics have been used extensively during several decades and we are now facing the emergence of multidrug resistant strains. It has become a major public concern, urging the need to discover new strategies to combat them. Among the different ways used by bacteria to resist antibiotics, the active efflux is one of the main mechanisms. In Gram-negative bacteria the efflux pumps are comprised of three components forming a long edifice crossing the complete cell wall from the inside to the outside of the cell. Blocking these pumps would permit the restoration of the effectiveness of the current antibiotherapy which is why it is important to increase our knowledge on the different proteins involved in these complexes. A tremendous number of experiments have been performed on the inner membrane protein AcrB from Escherichia coli and, to a lesser extent, the protein partners forming the AcrAB-TolC pump, but less information is available concerning the efflux pumps from other virulent Gram-negative bacteria. The present review will focus on the OprM outer membrane protein from the MexAB-OprM pump of Pseudomonas aeruginosa, highlighting similarities and differences compare to the archetypal AcrAB-TolC in terms of structure, function, and assembly properties.

  11. Detailed Performance of the Outer Tracker at LHCb

    Tuning, N

    2014-01-01

    The LHCb Outer Tracker is a gaseous detector covering an area of 5x6m2 with 12 double layers of straw tubes. Based on data of the first LHC running period from 2010 to 2012, the performance in terms of the single hit resolution and efficiency are presented. Details on the ionization length and subtle effects regarding signal reflections and the subsequent time-walk correction are given. The efficiency to detect a hit in the central half of the straw is estimated to be 99.2%, and the position resolution is determined to be approximately 200 um, depending on the detailed implementation of the internal alignment of individual detector modules. The Outer Tracker received a dose in the hottest region corresponding to 0.12 C/cm, and no signs of gain deterioration or other ageing effects are observed.

  12. The carbon budget in the outer solar nebula

    Simonelli, D.P.; Pollack, J.B.; Mckay, C.P.; Reynolds, R.T.; Summers, A.L.

    1989-01-01

    The compositional contrast between the giant-planet satellites and the significantly rockier Pluto/Charon system is indicative of different formation mechanisms; cosmic abundance calculations, in conjunction with an assumption of the Pluto/Charon system's direct formation from solar nebula condensates, strongly suggest that most of the carbon in the outer solar nebula was in CO form, in keeping with both the inheritance from the dense molecular clouds in the interstellar medium, and/or the Lewis and Prinn (1980) kinetic-inhibition model of solar nebula chemistry. Laboratory studies of carbonaceous chondrites and Comet Halley flyby studies suggest that condensed organic material, rather than elemental carbon, is the most likely candidate for the small percentage of the carbon-bearing solid in the outer solar nebula. 71 refs

  13. LO2/LH2 propulsion for outer planet orbiter spacecraft

    Garrison, P. W.; Sigurdson, K. B.

    1983-01-01

    Galileo class orbiter missions (750-1500 kg) to the outer planets require a large postinjection delta-V for improved propulsion performance. The present investigation shows that a pump-fed low thrust LO2/LH2 propulsion system can provide a significantly larger net on-orbit mass for a given delta-V than a state-of-the-art earth storable, N2O4/monomethylhydrazine pressure-fed propulsion system. A description is given of a conceptual design for a LO2/LH2 pump-fed propulsion system developed for a Galileo class mission to the outer planets. Attention is given to spacecraft configuration, details regarding the propulsion system, the thermal control of the cryogenic propellants, and aspects of mission performance.

  14. Defining the limits of outer space for regulatory purposes

    Bittencourt Neto, Olavo de Oliviera

    2015-01-01

    With different countries ascribing to different theories of air space and outer space law, Dr. Bittencourt Neto proposes in this Brief a reassessment of the international law related to the extension of state territories vertically. Taking into consideration the vast number of proposals offered by scholars and diplomatic delegations on this subject matter, as well as the principles of comparative law, a compromise to allow for peaceful development is the only way forward. The author argues for setting the delimitation of the frontier between air space and outer space at 100 km above mean sea level through an international treaty. This would also regulate passage rights for space objects during launchings and reentries, as long as those space activities are peaceful, conducted in accordance with international Law and respecting the sovereign interests of the territorial State. Continuing expansion of the commercial space industry and conflicting national laws require a stable and fair legal framework best ...

  15. Hubble 2020: Outer Planet Atmospheres Legacy (OPAL) Program

    Simon, Amy

    2017-08-01

    Long time base observations of the outer planets are critical in understanding the atmospheric dynamics and evolution of the gas giants. We propose yearly monitoring of each giant planet for the remainder of Hubble's lifetime to provide a lasting legacy of increasingly valuable data for time-domain studies. The Hubble Space Telescope is a unique asset to planetary science, allowing high spatial resolution data with absolute photometric knowledge. For the outer planets, gas/ice giant planets Jupiter, Saturn, Uranus and Neptune, many phenomena happen on timescales of years to decades, and the data we propose are beyond the scope of a typical GO program. Hubble is the only platform that can provide high spatial resolution global studies of cloud coloration, activity, and motion on a consistent time basis to help constrain the underlying mechanics.

  16. Low velocity encounters of minor bodies with the outer planets

    Carusi, A.; Perozzi, E.; Valsecchi, G.B.

    1983-01-01

    Previous studies of close encounters of minor bodies with Jupiter have shown that the perturbations are stronger either if the encounter is very deep or if the velocity of the minor body relative to the planet is low. In the present research the author investigates the effects of low velocity encounters between fictitious minor bodies and the four outer planets. Two possible outcomes of this type of encounter are the temporary satellite capture of the minor body by the planet, and the exchange of perihelion with aphelion of the minor body orbit. Different occurrence rates of these processes are found for different planets, and the implications for the orbital evolution of minor bodies in the outer Solar System are discussed. (Auth.)

  17. Truss topology optimization with discrete design variables by outer approximation

    Stolpe, Mathias

    2015-01-01

    Several variants of an outer approximation method are proposed to solve truss topology optimization problems with discrete design variables to proven global optimality. The objective is to minimize the volume of the structure while satisfying constraints on the global stiffness of the structure...... for classical outer approximation approaches applied to optimal design problems. A set of two- and three-dimensional benchmark problems are solved and the numerical results suggest that the proposed approaches are competitive with other special-purpose global optimization methods for the considered class...... under the applied loads. We extend the natural problem formulation by adding redundant force variables and force equilibrium constraints. This guarantees that the designs suggested by the relaxed master problems are capable of carrying the applied loads, a property which is generally not satisfied...

  18. Rotational instability in the outer region of protoplanetary disks

    Ono, Tomohiro [Department of Astronomy, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502 (Japan); Nomura, Hideko; Takeuchi, Taku, E-mail: ono.t@kusastro.kyoto-u.ac.jp [Department of Earth and Planetary Sciences, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo 152-8551 (Japan)

    2014-05-20

    We analytically calculate the marginally stable surface density profile for the rotational instability of protoplanetary disks. The derived profile can be utilized for considering the region in a rotating disk where radial pressure gradient force is comparable to the gravitational force, such as an inner edge, steep gaps or bumps, and an outer region of the disk. In this paper, we particularly focus on the rotational instability in the outer region of disks. We find that a protoplanetary disk with a surface density profile of similarity solution becomes rotationally unstable at a certain radius, depending on its temperature profile and a mass of the central star. If the temperature is relatively low and the mass of the central star is high, disks have rotationally stable similarity profiles. Otherwise, deviation from the similarity profiles of surface density could be observable, using facilities with high sensitivity, such as ALMA.

  19. Rotational instability in the outer region of protoplanetary disks

    Ono, Tomohiro; Nomura, Hideko; Takeuchi, Taku

    2014-01-01

    We analytically calculate the marginally stable surface density profile for the rotational instability of protoplanetary disks. The derived profile can be utilized for considering the region in a rotating disk where radial pressure gradient force is comparable to the gravitational force, such as an inner edge, steep gaps or bumps, and an outer region of the disk. In this paper, we particularly focus on the rotational instability in the outer region of disks. We find that a protoplanetary disk with a surface density profile of similarity solution becomes rotationally unstable at a certain radius, depending on its temperature profile and a mass of the central star. If the temperature is relatively low and the mass of the central star is high, disks have rotationally stable similarity profiles. Otherwise, deviation from the similarity profiles of surface density could be observable, using facilities with high sensitivity, such as ALMA.

  20. Progressive outer retinal necrosis-like retinitis in immunocompetent hosts.

    Chawla, Rohan; Tripathy, Koushik; Gogia, Varun; Venkatesh, Pradeep

    2016-08-10

    We describe two young immunocompetent women presenting with bilateral retinitis with outer retinal necrosis involving posterior pole with centrifugal spread and multifocal lesions simulating progressive outer retinal necrosis (PORN) like retinitis. Serology was negative for HIV and CD4 counts were normal; however, both women were on oral steroids at presentation for suspected autoimmune chorioretinitis. The retinitis in both eyes responded well to oral valaciclovir therapy. However, the eye with the more fulminant involvement developed retinal detachment with a loss of vision. Retinal atrophy was seen in the less involved eye with preservation of vision. Through these cases, we aim to describe a unique evolution of PORN-like retinitis in immunocompetent women, which was probably aggravated by a short-term immunosuppression secondary to oral steroids. 2016 BMJ Publishing Group Ltd.

  1. Possible origin of Saturn's newly discovered outer ring

    Moehlmann, D.

    1986-01-01

    Within a planetogonic model the self-gravitationally caused formation of pre-planetary and pre-satellite rings from an earlier thin disk is reported. The theoretically derived orbital radii of these rings are compared with the orbital levels in the planetary system and the satellite systems of Jupiter, Saturn and Uranus. From this comparison it is concluded that at the radial position of Saturn's newly discovered outer ring an early pre-satellite ring of more or less evolved satellites could have existed. These satellites should have been disturbed in their evolution by the gravitation of the neighbouring massive satellite Titan. The comparison also may indicate similarities between the asteroidal belt and the newly discovered outer ring of Saturn

  2. TFG facilitates outer coat disassembly on COPII transport carriers to promote tethering and fusion with ER-Golgi intermediate compartments.

    Hanna, Michael G; Block, Samuel; Frankel, E B; Hou, Feng; Johnson, Adam; Yuan, Lin; Knight, Gavin; Moresco, James J; Yates, John R; Ashton, Randolph; Schekman, Randy; Tong, Yufeng; Audhya, Anjon

    2017-09-12

    The conserved coat protein complex II (COPII) mediates the initial steps of secretory protein trafficking by assembling onto subdomains of the endoplasmic reticulum (ER) in two layers to generate cargo-laden transport carriers that ultimately fuse with an adjacent ER-Golgi intermediate compartment (ERGIC). Here, we demonstrate that Trk-fused gene (TFG) binds directly to the inner layer of the COPII coat. Specifically, the TFG C terminus interacts with Sec23 through a shared interface with the outer COPII coat and the cargo receptor Tango1/cTAGE5. Our findings indicate that TFG binding to Sec23 outcompetes these other associations in a concentration-dependent manner and ultimately promotes outer coat dissociation. Additionally, we demonstrate that TFG tethers vesicles harboring the inner COPII coat, which contributes to their clustering between the ER and ERGIC in cells. Together, our studies define a mechanism by which COPII transport carriers are retained locally at the ER/ERGIC interface after outer coat disassembly, which is a prerequisite for fusion with ERGIC membranes.

  3. Imaging Shock Fronts in the Outer Ejecta of Eta Carinae

    Smith, Nathan

    2017-08-01

    Although Eta Car has been imaged many times with HST to monitor the central star and the bright Homunculus Nebula, we propose the first WFC3 imaging of Eta Car to study the more extended Outer Ejecta from previous eruptions. WFC3 has two key filters that have not been used before to image Eta Car, which will provide critical physical information about its eruptive history: (1) F280N with WFC3/UVIS will produce the first Mg II 2800 image of Eta Car, the sharpest image of its complex Outer Ejecta, and will unambiguously trace shock fronts, and (2) F126N with WFC3/IR will sample [Fe II] 12567 arising in the densest post-shock gas. Eta Car is surrounded by a bright, soft X-ray shell seen in Chandra images, which arises from the fastest 1840s ejecta overtaking slower older material. Our recent proper motion measurements show that the outer knots were ejected in two outbursts several hundred years before the 1840s eruption, and spectroscopy of light echoes has recently revealed extremely fast ejecta during the 1840s that indicate an explosive event. Were those previous eruptions explosive as well? If so, were they as energetic, did they also have such fast ejecta, and did they have the same geometry? The structure and excitation of the Outer Ejecta hold unique clues for reconstructing Eta Car's violent mass loss history. The locations of shock fronts in circumstellar material provide critical information, because they identify past discontinuities in the mass loss. This is one of the only ways to investigate the long term (i.e. centuries) evolution and duty cycle of eruptive mass loss in the most massive stars.

  4. Photoreceptor atrophy in acute zonal occult outer retinopathy

    Zibrandtsen, N.; Munch, I.C.; Klemp, K.

    2008-01-01

    appearance were examined using optical coherence tomography (OCT), automated perimetry and electroretinography (ERG). RESULTS: Both patients demonstrated photoreceptor atrophy corresponding to partial or complete scotomata with reduced or extinct electroretinographic responses. Attenuation or complete loss...... of all the segments composing the photoreceptor layer was found by OCT. Full-field ERG revealed affection of the 30 Hz flicker responses and subnormal photopic responses in both patients and subnormal scotopic responses in case 1. Multifocal electroretinography (mERG) revealed localized outer retinal...

  5. Photoreceptor atrophy in acute zonal occult outer retinopathy

    Zibrandtsen, N.; Munch, I.C.; Klemp, K.

    2008-01-01

    examined using optical coherence tomography (OCT), automated perimetry and electroretinography (ERG). Both patients demonstrated photoreceptor atrophy corresponding to partial or complete scotomata with reduced or extinct electroretinographic responses. Attenuation or complete loss of all the segments...... composing the photoreceptor layer was found by OCT. Full-field ERG revealed affection of the 30 Hz flicker responses and subnormal photopic responses in both patients and subnormal scotopic responses in case 1. Multifocal electroretinography (mERG) revealed localized outer retinal dysfunction. The field...

  6. Progressive outer retinal necrosis and immunosuppressive therapy in myasthenia gravis.

    Coisy, Solène; Ebran, Jean-Marc; Milea, Dan

    2014-01-01

    Progressive outer retinal necrosis (PORN) is a rare but devastating infectious retinitis associated with varicella zoster virus (VZV) and responsible for severe visual loss. A 59-year-old man treated for generalized myasthenia with oral azathioprine and prednisone presented with severe unilateral necrotizing retinitis. Polymerase chain reaction of the aqueous and vitreous humors was diagnostic for VZV PORN. VZV PORN is a severe potential ocular complication of immunosuppression, prompting urgent diagnosis and appropriate treatment.

  7. Progressive Outer Retinal Necrosis and Immunosuppressive Therapy in Myasthenia Gravis

    Solène Coisy

    2014-04-01

    Full Text Available Introduction: Progressive outer retinal necrosis (PORN is a rare but devastating infectious retinitis associated with varicella zoster virus (VZV and responsible for severe visual loss. Case Report: A 59-year-old man treated for generalized myasthenia with oral azathioprine and prednisone presented with severe unilateral necrotizing retinitis. Polymerase chain reaction of the aqueous and vitreous humors was diagnostic for VZV PORN. Conclusion: VZV PORN is a severe potential ocular complication of immunosuppression, prompting urgent diagnosis and appropriate treatment.

  8. Total water content thresholds for shallow landslides, Outer Western Carpathians

    Bil, M.; Andrašík, R.; Zahradníček, Pavel; Kubeček, J.; Sedonik, J.; Štěpánek, Petr

    2016-01-01

    Roč. 13, č. 2 (2016), s. 337-347 ISSN 1612-510X R&D Projects: GA ČR GA13-19831S; GA MŠk(CZ) EE2.3.20.0248 Institutional support: RVO:67179843 Keywords : data quality -control * debris flows * rainfall thresholds * equivalent * depth * failures * example * europe * model * Landslides * Threshold * Snowmelt * Time series * Antecedent rainfall * Outer Western Carpathians Subject RIV: EH - Ecology, Behaviour Impact factor: 3.657, year: 2016

  9. Multimission nuclear electric propulsion system for outer planet exploration missions

    Mondt, J.F.

    1981-01-01

    A 100-kW reactor power system with a specific mass of 15 to 30 kg/kW/sub e/ and an electric thrust system with a specific mass of 5 to 10 kg/kW/sub e/ can be combined into a nuclear electric propulsion system. The system can be used for outer planet missions as well as earth orbital transfer vehicle missions. 5 refs

  10. Infectious polymorphic toxins delivered by outer membrane exchange discriminate kin in myxobacteria.

    Vassallo, Christopher N; Cao, Pengbo; Conklin, Austin; Finkelstein, Hayley; Hayes, Christopher S; Wall, Daniel

    2017-08-18

    Myxobacteria are known for complex social behaviors including outer membrane exchange (OME), in which cells exchange large amounts of outer membrane lipids and proteins upon contact. The TraA cell surface receptor selects OME partners based on a variable domain. However, traA polymorphism alone is not sufficient to precisely discriminate kin. Here, we report a novel family of OME-delivered toxins that promote kin discrimination of OME partners. These SitA lipoprotein toxins are polymorphic and widespread in myxobacteria. Each sitA is associated with a cognate sitI immunity gene, and in some cases a sitB accessory gene. Remarkably, we show that SitA is transferred serially between target cells, allowing the toxins to move cell-to-cell like an infectious agent. Consequently, SitA toxins define strong identity barriers between strains and likely contribute to population structure, maintenance of cooperation, and strain diversification. Moreover, these results highlight the diversity of systems evolved to deliver toxins between bacteria.

  11. Living among giants exploring and settling the outer solar system

    Carroll, Michael

    2015-01-01

    The outer Solar System is rich in resources and may be the best region in which to search for life beyond Earth. In fact, it may ultimately be the best place for Earthlings to set up permanent abodes. This book surveys the feasibility of that prospect, covering the fascinating history of exploration that kicks off our adventure into the outer Solar System.   Although other books provide surveys of the outer planets, Carroll approaches it from the perspective of potential future human exploration, exploitation and settlement, using insights from today’s leading scientists in the field. These experts take us to targets such as the moons Titan, Triton, Enceladus, Iapetus and Europa, and within the atmospheres of the gas and ice giants. In these pages you will experience the thrill of discovery awaiting those who journey through the giant worlds and their moons.   All the latest research is included, as are numerous illustrations, among them original paintings by the author, a renowned prize-winning space art...

  12. Neogene sedimentation on the outer continental margin, southern Bering Sea

    Vallier, T.L.; Underwood, M.B.; Gardner, J.V.; Barron, J.A.

    1980-01-01

    Neogene sedimentary rocks and sediments from sites on the outer continental margin in the southern Bering Sea and on the Alaska Peninsula are dominated by volcanic components that probably were eroded from an emergent Aleutian Ridge. A mainland continental source is subordinate. Most sediment in the marine environment was transported to the depositional sites by longshore currents, debris flows, and turbidity currents during times when sea level was near the outermost continental shelf. Fluctuations of sea level are ascribed both to worldwide glacio-eustatic effects and to regional vertical tectonics. Large drainage systems, such as the Yukon and Kuskokwim Rivers, had little direct influence on sedimentation along the continental slope and Unmak Plateau in the southern Bering Sea. Sediments from those drainage systems probably were transported to the floor of the Aleutian Basin, to the numerous shelf basins that underlie the outer continental shelf, and to the Arctic Ocean after passing through the Bering Strait. Environments of deposition at the sites along the outer continental margin have not changed significantly since the middle Miocene. The site on the Alaska Peninsula, however, is now emergent following shallow-marine and transitional sedimentation during the Neogene. ?? 1980.

  13. Aging and Phase Stability of Waste Package Outer Barrier

    Tammy S. Edgecumble Summers

    2001-08-23

    This Analysis Model Report (AMR) was prepared in accordance with the Work Direction and Planning Document, ''Aging and Phase Stability of Waste Package Outer Barrier'' (CRWMS M&O 1999a). ICN 01 of this AMR was developed following guidelines provided in TWP-MGR-MD-000004 REV 01, ''Technical Work Plan for: Integrated Management of Technical Product Input Department'' (BSC 2001, Addendum B). It takes into consideration the Enhanced Design Alternative II (EDA II), which has been selected as the preferred design for the Engineered Barrier System (EBS) by the License Application Design Selection (LADS) program team (CRWMS M&O 1999b). The salient features of the EDA II design for this model are a waste package (WP) consisting of an outer barrier of Alloy 22 and an inner barrier of Type 316L stainless steel. This report provides information on the phase stability of Alloy 22l, the current waste-package-outer-barrier (WPOB) material. These phase stability studies are currently divided into three general areas: (1) Long-range order reactions; (2) Intermetallic and carbide precipitation in the base metal; and (3) Intermetallic and carbide precipitation in welded samples.

  14. NIF Double Shell outer/inner shell collision experiments

    Merritt, E. C.; Loomis, E. N.; Wilson, D. C.; Cardenas, T.; Montgomery, D. S.; Daughton, W. S.; Dodd, E. S.; Desjardins, T.; Renner, D. B.; Palaniyappan, S.; Batha, S. H.; Khan, S. F.; Smalyuk, V.; Ping, Y.; Amendt, P.; Schoff, M.; Hoppe, M.

    2017-10-01

    Double shell capsules are a potential low convergence path to substantial alpha-heating and ignition on NIF, since they are predicted to ignite and burn at relatively low temperatures via volume ignition. Current LANL NIF double shell designs consist of a low-Z ablator, low-density foam cushion, and high-Z inner shell with liquid DT fill. Central to the Double Shell concept is kinetic energy transfer from the outer to inner shell via collision. The collision determines maximum energy available for compression and implosion shape of the fuel. We present results of a NIF shape-transfer study: two experiments comparing shape and trajectory of the outer and inner shells at post-collision times. An outer-shell-only target shot measured the no-impact shell conditions, while an `imaging' double shell shot measured shell conditions with impact. The `imaging' target uses a low-Z inner shell and is designed to perform in similar collision physics space to a high-Z double shell but can be radiographed at 16keV, near the viable 2DConA BL energy limit. Work conducted under the auspices of the U.S. DOE by LANL under contract DE-AC52-06NA25396.

  15. Distribution of flexural deflection in the worldwide outer rise area

    Lin, Zi-Jun; Lin, Jing-Yi; Lin, Yi-Chin; Chin, Shao-Jinn; Chen, Yen-Fu

    2015-04-01

    The outer rise on the fringe of a subduction system is caused by an accreted load on the flexed oceanic lithosphere. The magnitude of the deflection is usually linked to the stress state beard by the oceanic plate. In a coupled subduction zone, the stress is abundantly accumulated across the plate boundary which should affect the flexural properties of the subducted plate. Thus, the variation of the outer rise in shape may reflect the seismogenic characteristics of the subduction system. In this study, we intent to find the correlation between the flexure deflection (Wb) of the outer rise and the subduction zone properties by comparing several slab parameters and the Wb distribution. The estimation of Wb is performed based on the available bathymetry data and the statistic analysis of earthquakes is from the global ISC earthquake catalog for the period of 1900-2015. Our result shows a progressive change of Wb in space, suggesting a robust calculation. The average Wb of worldwise subduction system spreads from 348 to 682 m. No visible distinction in the ranging of Wb was observed for different subduction zones. However, in a weak coupling subduction system, the standard variation of Wb has generally larger value. Relatively large Wb generally occurs in the center of the trench system, whereas small Wb for the two ends of trench. The comparison of Wb and several slab parameters shows that the Wb may be correlated with the maximal magnitude and the number of earthquakes. Otherwise, no clear relationship with other parameters can be obtained.

  16. Students’ misconceptions about Newton's second law in outer space

    Temiz, B K; Yavuz, A

    2014-01-01

    Students’ misconceptions about Newton's second law in frictionless outer space were investigated. The research was formed according to an epistemic game theoretical framework. The term ‘epistemic’ refers to students’ participation in problem-solving activities as a means of constructing new knowledge. The term ‘game’ refers to a coherent activity that consists of moves and rules. A set of questions in which students are asked to solve two similar Newton's second law problems, one of which is on the Earth and the other in outer space, was administered to 116 undergraduate students. The findings indicate that there is a significant difference between students’ epistemic game preferences and race-type (outer space or frictional surface) question. So students who used Newton's second law on the ground did not apply this law and used primitive reasoning when it came to space. Among these students, voluntary interviews were conducted with 18 students. Analysis of interview transcripts showed that: (1) the term ‘space’ causes spontaneity among students that prevents the use of the law; (2) students hesitate to apply Newton's second law in space due to the lack of a condition—the friction; (3) students feel that Newton's second law is not valid in space for a variety of reasons, but mostly for the fact that the body in space is not in contact with a surface. (paper)

  17. Structural Basis for a Ribofuranosyl Binding Protein: Insights into the Furanose Specific Transport

    Bagaria, A.; Swaminathan, S.; Kumaran, D.; Burley, S. K.

    2011-04-01

    The ATP-binding cassette transporters (ABC-transporters) are members of one of the largest protein superfamilies, with representatives in all extant phyla. These integral membrane proteins utilize the energy of ATP hydrolysis to carry out certain biological processes, including translocation of various substrates across membranes and non-transport related processes such as translation of RNA and DNA repair. Typically, such transport systems in bacteria consist of an ATP binding component, a transmembrane permease, and a periplasmic receptor or binding protein. Soluble proteins found in the periplasm of gram-negative bacteria serve as the primary receptors for transport of many compounds, such as sugars, small peptides, and some ions. Ligand binding activates these periplasmic components, permitting recognition by the membrane spanning domain, which supports for transport and, in some cases, chemotaxis. Transport and chemotaxis processes appear to be independent of one another, and a few mutants of bifunctional periplasmic components reveal the absence of one or the other function. Previously published high-resolution X-ray structures of various periplasmic ligand binding proteins include Arabinose binding protein (ABP), Allose binding protein (ALBP), Glucose-galactose binding protein (GBP) and Ribose binding protein (RBP). Each of these proteins consists of two structurally similar domains connected by a three-stranded hinge region, with ligand buried between the domains. Upon ligand binding and release, various conformational changes have been observed. For RBP, open (apo) and closed (ligand bound) conformations have been reported and so for MBP. The closed/active form of the protein interacts with the integral membrane component of the system in both transport and chemotaxis. Herein, we report 1.9{angstrom} resolution X-ray structure of the R{sub f}BP periplasmic component of an ABC-type sugar transport system from Hahella chejuensis (UniProt Id Q2S7D2) bound to

  18. The carbon budget in the outer solar nebula.

    Simonelli, D P; Pollack, J B; McKay, C P; Reynolds, R T; Summers, A L

    1989-01-01

    Detailed models of the internal structures of Pluto and Charon, assuming rock and water ice as the only constituents, indicate that the mean silicate mass fraction of this two-body system is on the order of 0.7; thus the Pluto/Charon system is significantly "rockier" than the satellites of the giant planets (silicate mass fraction approximately 0.55). This compositional contrast reflects different formation mechanisms: it is likely that Pluto and Charon formed directly from the solar nebula, while the circumplanetary nebulae that produced the giant planet satellites were derived from envelopes that surrounded the forming giant planets (envelopes in which icy planetesimals dissolved more readily than rocky planetesimals). Simple cosmic abundance calculations, and the assumption that the Pluto/Charon system formed directly from solar nebula condensates, strongly suggest that the majority of the carbon in the outer solar nebula was in the form of carbon monoxide; these results are consistent with (1) inheritance from the dense molecular clouds in the interstellar medium (where CH4/CO nebula chemistry. Theoretical predictions of the C/H enhancements in the atmospheres of the giant planets, when compared to the actual observed enhancements, suggest that 10%, or slightly more, of the carbon in the outer solar nebula was in the form of condensed materials (although the amount of condensed C may have dropped slightly with increasing heliocentric distance). Strict compositional limits computed for the Pluto/Charon system using the densities of CH4 and CO ices indicate that these pure ices are at best minor components in the interiors of these bodies, and imply that CH4 and CO ices were not the dominant C-bearing solids in the outer nebula. Clathrate-hydrates could not have appropriated enough CH4 or CO to be the major form of condensed carbon, although such clathrates may be necessary to explain the presence of methane on Pluto after its formation from a CO-rich nebula

  19. Water and Volatiles in the Outer Solar System

    Grasset, O.; Castillo-Rogez, J.; Guillot, T.; Fletcher, L. N.; Tosi, F.

    2017-10-01

    Space exploration and ground-based observations have provided outstanding evidence of the diversity and the complexity of the outer solar system. This work presents our current understanding of the nature and distribution of water and water-rich materials from the water snow line to the Kuiper Belt. This synthesis is timely, since a thorough exploration of at least one object in each region of the outer solar system has now been achieved. Next steps, starting with the Juno mission now in orbit around Jupiter, will be more focused on understanding the processes at work than on describing the general characteristics of each giant planet systems. This review is organized in three parts. First, the nature and the distribution of water and volatiles in giant and intermediary planets are described from their inner core to their outer envelopes. A special focus is given to Jupiter and Saturn, which are much better understood than the two ice giants (Uranus and Neptune) thanks to the Galileo and Cassini missions. Second, the icy moons will be discussed. Space missions and ground-based observations have revealed the variety of icy surfaces in the outer system. While Europa, Enceladus, and maybe Titan present past or even active tectonic and volcanic activities, many other moons have been dead worlds for more than 3 billion years. Ice compositions found at these bodies are also complex and it is now commonly admitted that icy surfaces are never composed of pure ices. A detailed review of the distribution of non-ice materials on the surfaces and in the tenuous atmospheres of the moons is proposed, followed by a more focused discussion on the nature and the characteristics of the liquid layers trapped below the cold icy crusts that have been suggested in the icy Galilean moons, and in Enceladus, Dione, and Titan at Saturn. Finally, the recent observations collected by Dawn at Ceres and New Horizons at Pluto, as well as the state of knowledge of other transneptunian objects

  20. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes.

    Bruno Hernáez

    2016-04-01

    Full Text Available African swine fever virus (ASFV is a nucleocytoplasmic large DNA virus (NCLDV that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs.

  1. Destabilization of the Outer and Inner Mitochondrial Membranes by Core and Linker Histones

    Cascone, Annunziata; Bruelle, Celine; Lindholm, Dan; Bernardi, Paolo; Eriksson, Ove

    2012-01-01

    Background Extensive DNA damage leads to apoptosis. Histones play a central role in DNA damage sensing and may mediate signals of genotoxic damage to cytosolic effectors including mitochondria. Methodology/Principal Findings We have investigated the effects of histones on mitochondrial function and membrane integrity. We demonstrate that both linker histone H1 and core histones H2A, H2B, H3, and H4 bind strongly to isolated mitochondria. All histones caused a rapid and massive release of the pro-apoptotic intermembrane space proteins cytochrome c and Smac/Diablo, indicating that they permeabilize the outer mitochondrial membrane. In addition, linker histone H1, but not core histones, permeabilized the inner membrane with a collapse of the membrane potential, release of pyridine nucleotides, and mitochondrial fragmentation. Conclusions We conclude that histones destabilize the mitochondrial membranes, a mechanism that may convey genotoxic signals to mitochondria and promote apoptosis following DNA damage. PMID:22523586

  2. Relevance of lysine snorkeling in the outer transmembrane domain of small viral potassium ion channels.

    Gebhardt, Manuela; Henkes, Leonhard M; Tayefeh, Sascha; Hertel, Brigitte; Greiner, Timo; Van Etten, James L; Baumeister, Dirk; Cosentino, Cristian; Moroni, Anna; Kast, Stefan M; Thiel, Gerhard

    2012-07-17

    Transmembrane domains (TMDs) are often flanked by Lys or Arg because they keep their aliphatic parts in the bilayer and their charged groups in the polar interface. Here we examine the relevance of this so-called "snorkeling" of a cationic amino acid, which is conserved in the outer TMD of small viral K(+) channels. Experimentally, snorkeling activity is not mandatory for Kcv(PBCV-1) because K29 can be replaced by most of the natural amino acids without any corruption of function. Two similar channels, Kcv(ATCV-1) and Kcv(MT325), lack a cytosolic N-terminus, and neutralization of their equivalent cationic amino acids inhibits their function. To understand the variable importance of the cationic amino acids, we reanalyzed molecular dynamics simulations of Kcv(PBCV-1) and N-terminally truncated mutants; the truncated mutants mimic Kcv(ATCV-1) and Kcv(MT325). Structures were analyzed with respect to membrane positioning in relation to the orientation of K29. The results indicate that the architecture of the protein (including the selectivity filter) is only weakly dependent on TMD length and protonation of K29. The penetration depth of Lys in a given protonation state is independent of the TMD architecture, which leads to a distortion of shorter proteins. The data imply that snorkeling can be important for K(+) channels; however, its significance depends on the architecture of the entire TMD. The observation that the most severe N-terminal truncation causes the outer TMD to move toward the cytosolic side suggests that snorkeling becomes more relevant if TMDs are not stabilized in the membrane by other domains.

  3. Grain Size Data from the NOAA Outer Continental Shelf Environmental Assessment Program (OCSEAP)

    National Oceanic and Atmospheric Administration, Department of Commerce — This data set contains grain size data from samples acquired under the NOAA Outer Continental Shelf Environmental Assessment Program (OCSEAP) from the Outer...

  4. In vivo evidence of TonB shuttling between the cytoplasmic and outer membrane in Escherichia coli.

    Larsen, Ray A; Letain, Tracy E; Postle, Kathleen

    2003-07-01

    Gram-negative bacteria are able to convert potential energy inherent in the proton gradient of the cytoplasmic membrane into active nutrient transport across the outer membrane. The transduction of energy is mediated by TonB protein. Previous studies suggest a model in which TonB makes sequential and cyclic contact with proteins in each membrane, a process called shuttling. A key feature of shuttling is that the amino-terminal signal anchor must quit its association with the cytoplasmic membrane, and TonB becomes associated solely with the outer membrane. However, the initial studies did not exclude the possibility that TonB was artifactually pulled from the cytoplasmic membrane by the fractionation process. To resolve this ambiguity, we devised a method to test whether the extreme TonB amino-terminus, located in the cytoplasm, ever became accessible to the cys-specific, cytoplasmic membrane-impermeant molecule, Oregon Green(R) 488 maleimide (OGM) in vivo. A full-length TonB and a truncated TonB were modified to carry a sole cysteine at position 3. Both full-length TonB and truncated TonB (consisting of the amino-terminal two-thirds) achieved identical conformations in the cytoplasmic membrane, as determined by their abilities to cross-link to the cytoplasmic membrane protein ExbB and their abilities to respond conformationally to the presence or absence of proton motive force. Full-length TonB could be amino-terminally labelled in vivo, suggesting that it was periplasmically exposed. In contrast, truncated TonB, which did not associate with the outer membrane, was not specifically labelled in vivo. The truncated TonB also acted as a control for leakage of OGM across the cytoplasmic membrane. Further, the extent of labelling for full-length TonB correlated roughly with the proportion of TonB found at the outer membrane. These findings suggest that TonB does indeed disengage from the cytoplasmic membrane during energy transduction and shuttle to the outer membrane.

  5. Legal Implications of Military Uses of Outer Space

    Catena, Johanna

    2002-01-01

    Acquisition of Space Weapons, the Legal, Political and Military Impact for International Peace and At the dawn of a new century an immediate danger is upon us: The weaponization of outer space, including potential cost implications upon the prospect of ushering an era of peace and prosperity. But, can such statements be explained as pure sentimentality for hopes of a new era? Or is the danger misplaced that the threat to peace and security is an ever more ominous? By militarising outer space one could monitor crisis areas that could become a potential threat and this would in turn build confidence and security amongst nations. However the Outer Space Treaty prohibits placing in orbit nuclear and other weapons of mass destruction. This does not include other military systems. Many countries feel the prohibition should be extended in the Treaty. Other military systems may involve anti-satellite weapons, (ASATS), emitting or simply placing technologies in space using laser and /or particle beams from space to intercept presently specific military targets such as ballistic missiles and hostile satellites, but in the future this may extend to destroying a target on earth. Military presence in space however, is not founded on weapons alone, but also through military surveillance systems and seen by some countries as an effective measure in verification on arms control. It is also seen as intensifying an arms race. At the forefront of the debate for space weapons is the possibility of countries deploying a National Missile Defence system. How does one reconcile such a system with present treaties? There has always been a direct relationship between weapons and space exploration, particularly if traced through the history of the late nineteenth century to the era of the space race. Konstantin Tsiolkovsky, (1857 - 1935), was one of the founders to astronautics. Robert Goddard, (1882-1945) an Englishman, developed Tsiolkovskys' work further. He built the first liquid

  6. 33 CFR 165.1402 - Apra Outer Harbor, Guam-regulated navigation area.

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Apra Outer Harbor, Guam-regulated....1402 Apra Outer Harbor, Guam—regulated navigation area. (a) The following is a regulated navigation area—The waters of the Pacific Ocean and Apra Outer Harbor enclosed by a line beginning at latitude 13...

  7. 78 FR 29091 - Safety Zone; Outer Banks Bluegrass Festival; Shallowbag Bay, Manteo, NC

    2013-05-17

    ...-AA00 Safety Zone; Outer Banks Bluegrass Festival; Shallowbag Bay, Manteo, NC AGENCY: Coast Guard, DHS... the Outer Banks Bluegrass Festival. This action is necessary to protect the life and property of the... vessels from a portion of Shallowbag Bay River during the Outer Banks Bluegrass Festival Fireworks display...

  8. Colours of the Outer Solar System Origins Survey: An Update

    Schwamb, Megan E.; Fraser, Wesley C.; Pike, Rosemary E.; Bannister, Michele T.; Marsset, Michaël; Kavelaars, J. J.; Benecchi, Susan; Delsanti, Audrey C.; Lehner, Matthew J.; Wang, Shiang-Yu; Thirouin, Audrey; Nesvorný, David

    2018-01-01

    The vast majority of the known dwarf-planet sized bodies are bright enough to be studied through optical and infrared spectroscopy. As a result, we have an understanding of the surface properties for the largest Kuiper belt objects (KBOs) which retain their primordial inventory of volatile ices. For the typically smaller > 22 mag KBO, we must rely instead on what colors reveal by proxy; yet this picture remains incomplete. Most KBO physical property studies examine the hodgepodge set of objects discovered by various surveys with different and varying detection biases that make it difficult if not impossible to reliably estimate the sizes of the different surface color groupings (compositional classes) residing in the modern-day Kuiper belt.The Colours of the Outer Solar System Origins Survey (Col-OSSOS) probes the surface properties within the Kuiper belt primarily through near simultaneous g,r and J colors with the Gemini North Telescope and u-band with Canada-France-Hawaii Telescope. The project aims to target ~100 KBOs brighter than 23.6 r‧ mag found by the Outer Solar System Origins Survey (OSSOS), a survey with a well-measured detection efficiency. Thus, Col-OSSOS provides the first brightness-complete, compositional-dynamical map of the Outer Solar System, probing in a new light the radial color distribution in the primordial planetesimal disk from which KBOs originated. We will provide an update on the current status of the program highlighting results from the first two years of the survey; including size estimates of the two color KBO subgroups (the red and neutral surfaces) within the dynamically excited Kuiper belt and implications for the early planetesimal disk composition based on neutral-colored binaries found in the cold classical Kuiper belt.

  9. Early photoreceptor outer segment loss and retinoschisis in Cohen syndrome.

    Uyhazi, Katherine E; Binenbaum, Gil; Carducci, Nicholas; Zackai, Elaine H; Aleman, Tomas S

    2018-06-01

    To describe early structural and functional retinal changes in a patient with Cohen syndrome. A 13-month-old Caucasian girl of Irish and Spanish ancestry was noted to have micrognathia and laryngomalacia at birth, which prompted a genetic evaluation that revealed biallelic deletions in COH1 (VPS13B) (a maternally inherited 60-kb deletion involving exons 26-32 and a paternally inherited 3.5-kb deletion within exon 17) consistent with Cohen syndrome. She underwent a complete ophthalmic examination, full-field flash electroretinography and retinal imaging with spectral domain optical coherence tomography. Central vision was central, steady, and maintained. There was bilateral myopic astigmatic refractive error. Fundus exam was notable for dark foveolar pigmentation, but no obvious abnormalities of either eye. Spectral domain optical coherence tomography cross sections through the fovea revealed a normal appearing photoreceptor outer nuclear layer but loss of the interdigitation signal between the photoreceptor outer segments and the apical retinal pigment epithelium. Retinoschisis involving the inner nuclear layer of both eyes and possible ganglion cell layer thinning were also noted. There was a detectable electroretinogram with similarly reduced amplitudes of rod- (white, 0.01 cd.s.m -2 ) and cone-mediated (3 cd.s.m -2 , 30 Hz) responses. Photoreceptor outer segment abnormalities and retinoschisis may represent the earliest structural retinal change detected by spectral domain optical coherence tomography in patients with Cohen syndrome, suggesting a complex pathophysiology with primary involvement of the photoreceptor cilium and disorganization of the structural integrity of the inner retina.

  10. General Corrosion and Localized Corrosion of Waste Package Outer Barrier

    K.G. Mon

    2004-10-01

    The waste package design for the License Application is a double-wall waste package underneath a protective drip shield (BSC 2004 [DIRS 168489]; BSC 2004 [DIRS 169480]). The purpose and scope of this model report is to document models for general and localized corrosion of the waste package outer barrier (WPOB) to be used in evaluating waste package performance. The WPOB is constructed of Alloy 22 (UNS N06022), a highly corrosion-resistant nickel-based alloy. The inner vessel of the waste package is constructed of Stainless Steel Type 316 (UNS S31600). Before it fails, the Alloy 22 WPOB protects the Stainless Steel Type 316 inner vessel from exposure to the external environment and any significant degradation. The Stainless Steel Type 316 inner vessel provides structural stability to the thinner Alloy 22 WPOB. Although the waste package inner vessel would also provide some performance for waste containment and potentially decrease the rate of radionuclide transport after WPOB breach before it fails, the potential performance of the inner vessel is far less than that of the more corrosion-resistant Alloy 22 WPOB. For this reason, the corrosion performance of the waste package inner vessel is conservatively ignored in this report and the total system performance assessment for the license application (TSPA-LA). Treatment of seismic and igneous events and their consequences on waste package outer barrier performance are not specifically discussed in this report, although the general and localized corrosion models developed in this report are suitable for use in these scenarios. The localized corrosion processes considered in this report are pitting corrosion and crevice corrosion. Stress corrosion cracking is discussed in ''Stress Corrosion Cracking of the Drip Shield, the Waste Package Outer Barrier, and the Stainless Steel Structural Material'' (BSC 2004 [DIRS 169985]).

  11. Outer Sphere Adsorption of Pb(II)EDTA on Goethite

    Bargar, John R

    1999-07-16

    FTIR and EXAFS spectroscopic measurements were performed on Pb(II)EDTA adsorbed on goethite as functions of pH (4-6), Pb(II)EDTA concentration (0.11 {micro}M - 72 {micro}M), and ionic strength (16 {micro}M - 0.5M). FTIR measurements show no evidence for carboxylate-Fe(III) bonding or protonation of EDTA at Pb:EDTA = 1:1. Both FTIR and EXAFS measurements suggest that EDTA acts as a hexadentate ligand, with all four of its carboxylate and both amine groups bonded to Pb(II). No evidence was observed for inner-sphere Pb(II)-goethite bonding at Pb:EDTA = 1:1. Hence, the adsorbed complexes should have composition Pb(II)EDTA{sup 2{minus}}. Since substantial uptake of PbEDTA(II){sup 2{minus}} occurred in the samples, we infer that Pb(II)EDTA{sup 2{minus}} adsorbed as outer-sphere complexes and/or as complexes that lose part of their solvation shells and hydrogen bond directly to goethite surface sites. We propose the term ''hydration-sphere'' for the latter type of complexes because they should occupy space in the primary hydration spheres of goethite surface functional groups, and to distinguish this mode of sorption from common structural definitions of inner- and outer-sphere complexes. The similarity of Pb(II) uptake isotherms to those of other divalent metal ions complexed by EDTA suggests that they too adsorb by these mechanisms. The lack of evidence for inner-sphere EDTA-Fe(III) bonding suggests that previously proposed metal-ligand - promoted dissolution mechanisms should be modified, specifically to account for the presence of outer-sphere precursor species.

  12. Analysis of the outer membrane proteome and secretome of Bacteroides fragilis reveals a multiplicity of secretion mechanisms.

    Marlena M Wilson

    Full Text Available Bacteroides fragilis is a widely distributed member of the human gut microbiome and an opportunistic pathogen. Cell surface molecules produced by this organism likely play important roles in colonization, communication with other microbes, and pathogenicity, but the protein composition of the outer membrane (OM and the mechanisms used to transport polypeptides into the extracellular space are poorly characterized. Here we used LC-MS/MS to analyze the OM proteome and secretome of B. fragilis NCTC 9343 grown under laboratory conditions. Of the 229 OM proteins that we identified, 108 are predicted to be lipoproteins, and 61 are predicted to be TonB-dependent transporters. Based on their proximity to genes encoding TonB-dependent transporters, many of the lipoprotein genes likely encode proteins involved in nutrient or small molecule uptake. Interestingly, protease accessibility and biotinylation experiments indicated that an unusually large fraction of the lipoproteins are cell-surface exposed. We also identified three proteins that are members of a novel family of autotransporters, multiple potential type I protein secretion systems, and proteins that appear to be components of a type VI secretion apparatus. The secretome consisted of lipoproteins and other proteins that might be substrates of the putative type I or type VI secretion systems. Our proteomic studies show that B. fragilis differs considerably from well-studied Gram-negative bacteria such as Escherichia coli in both the spectrum of OM proteins that it produces and the range of secretion strategies that it utilizes.

  13. Partitioning the proteome: phase separation for targeted analysis of membrane proteins in human post-mortem brain.

    Jane A English

    Full Text Available Neuroproteomics is a powerful platform for targeted and hypothesis driven research, providing comprehensive insights into cellular and sub-cellular disease states, Gene × Environmental effects, and cellular response to medication effects in human, animal, and cell culture models. Analysis of sub-proteomes is becoming increasingly important in clinical proteomics, enriching for otherwise undetectable proteins that are possible markers for disease. Membrane proteins are one such sub-proteome class that merit in-depth targeted analysis, particularly in psychiatric disorders. As membrane proteins are notoriously difficult to analyse using traditional proteomics methods, we evaluate a paradigm to enrich for and study membrane proteins from human post-mortem brain tissue. This is the first study to extensively characterise the integral trans-membrane spanning proteins present in human brain. Using Triton X-114 phase separation and LC-MS/MS analysis, we enriched for and identified 494 membrane proteins, with 194 trans-membrane helices present, ranging from 1 to 21 helices per protein. Isolated proteins included glutamate receptors, G proteins, voltage gated and calcium channels, synaptic proteins, and myelin proteins, all of which warrant quantitative proteomic investigation in psychiatric and neurological disorders. Overall, our sub-proteome analysis reduced sample complexity and enriched for integral membrane proteins by 2.3 fold, thus allowing for more manageable, reproducible, and targeted proteomics in case vs. control biomarker studies. This study provides a valuable reference for future neuroproteomic investigations of membrane proteins, and validates the use Triton X-114 detergent phase extraction on human post mortem brain.

  14. Frames and outer frames for Hilbert C^*-modules

    Arambašić, Ljiljana; Bakić, Damir

    2015-01-01

    The goal of the present paper is to extend the theory of frames for countably generated Hilbert $C^*$-modules over arbitrary $C^*$-algebras. In investigating the non-unital case we introduce the concept of outer frame as a sequence in the multiplier module $M(X)$ that has the standard frame property when applied to elements of the ambient module $X$. Given a Hilbert $\\A$-module $X$, we prove that there is a bijective correspondence of the set of all adjointable surjections from the generalize...

  15. Dynamically hot Super-Earths from outer giant planet scattering

    Huang, Chelsea X.; Petrovich, Cristobal; Deibert, Emily

    2016-01-01

    The hundreds of multiple planetary systems discovered by the \\textit{Kepler} mission are typically observed to reside in close-in ($\\lesssim0.5$ AU), low-eccentricity, and low-inclination orbits. We run N-body experiments to study the effect that unstable outer ($\\gtrsim1$ AU) giant planets, whose end orbital configurations resemble those in the Radial Velocity population, have on these close-in multiple super-Earth systems. Our experiments show that the giant planets greatly reduce the multi...

  16. Design of a Bearingless Outer Rotor Induction Motor

    Yuxin Sun

    2017-05-01

    Full Text Available A bearingless induction (BI motor with an outer rotor for flywheel energy storage systems is proposed due to the perceived advantages of simple rotor structure, non-contact support and high speed operation. Firstly, the configuration and operation principle of the proposed motor are described. Then several leading dimensional parameters are optimally calculated for achieving the maximum average values and the minimum ripples of torque output and suspension force. Finally, by using the finite element method, the characteristics and performance of the proposed machine are analyzed and verified.

  17. Outer brain barriers in rat and human development

    Brøchner, Christian B; Holst, Camilla Bjørnbak; Møllgård, Kjeld

    2015-01-01

    Complex barriers at the brain's surface, particularly in development, are poorly defined. In the adult, arachnoid blood-cerebrospinal fluid (CSF) barrier separates the fenestrated dural vessels from the CSF by means of a cell layer joined by tight junctions. Outer CSF-brain barrier provides...... diffusion restriction between brain and subarachnoid CSF through an initial radial glial end feet layer covered with a pial surface layer. To further characterize these interfaces we examined embryonic rat brains from E10 to P0 and forebrains from human embryos and fetuses (6-21st weeks post...

  18. Aircraft crash upon outer containment of nuclear power plant

    Abbas, H.; Paul, D.K.; Godbole, P.N.; Nayak, G.C.

    1996-01-01

    In this paper, analysis of an aircraft crash upon an outer containment of a nuclear power plant is presented. The effect of target yielding is considered simultaneously by calculating the reaction time in a time marching scheme. The concrete model employed is capable of predicting the cracking and yielding. The response for different cracking strains and different locations of aircraft strike for different aircraft has been studied. Critical location of aircraft strike for the containment has been investigated. The analytical procedure and the material model used are found to be capable of representing the aircraft impact response of the containment structure. (orig.)

  19. Progressive outer retinal necrosis after rituximab and cyclophosphamide therapy

    Mohit Dogra

    2018-01-01

    Full Text Available We report a case of progressive outer retinal necrosis (PORN in a patient of microscopic polyangitis (MPA, being treated with immunosuppressive drugs such as cyclophosphamide and rituximab. Her aqueous tap was positive for Varicella Zoster virus and she was treated with oral and intravitreal antivirals, along with discontinuation of one of the immunosuppressive agents, i.e. rituximab, which might have led to reactivation of the virus causing necrotizing retinitis lesions. Rituximab and cyclophosphamide are extremely potent drugs, which are necessary to manage immunological disorders such as MPA. However, they may predispose the patient to serious complications like viral infections, including PORN.

  20. Progressive outer retinal necrosis after rituximab and cyclophosphamide therapy.

    Dogra, Mohit; Bajgai, Priya; Kumar, Ashok; Sharma, Aman

    2018-04-01

    We report a case of progressive outer retinal necrosis (PORN) in a patient of microscopic polyangitis (MPA), being treated with immunosuppressive drugs such as cyclophosphamide and rituximab. Her aqueous tap was positive for Varicella Zoster virus and she was treated with oral and intravitreal antivirals, along with discontinuation of one of the immunosuppressive agents, i.e. rituximab, which might have led to reactivation of the virus causing necrotizing retinitis lesions. Rituximab and cyclophosphamide are extremely potent drugs, which are necessary to manage immunological disorders such as MPA. However, they may predispose the patient to serious complications like viral infections, including PORN.

  1. Uranus, Neptune, Pluto, and the outer solar system

    Elkins-Tanton, Linda T

    2010-01-01

    Unlike all the planets closer to the Sun, known since antiquity, the farthest reaches are the discoveries of the modern world. Uranus was discovered in 1781, Neptune in 1846, Pluto in 1930, the Kuiper belt group of objects in 1992, and though the Oort cloud has been theorized since 1950, its first member was found in 2004. The discovery of the outer planets made such an impression on the minds of mankind that they were immortalized in the names of the newly discovered elements: uranium, neptunium, and plutonium, an astonishingly deadly constituent of atomic bombs. Uranus, Neptune, Pluto, and t

  2. The straw tube technology for the LHCb outer tracking system

    Bachmann, S; Bagaturia, I; Deppe, H; Eisele, F; Haas, T; Hajduk, L; Langenegger, U; Michalowski, J; Nawrot, A; Polok, G; Pellegrino, A; Schuijlenburg, H; Schwierz, R; Sluijk, T; Spelt, J

    2004-01-01

    For the outer tracking system of the LHCb spectrometer 53.760 straws of 2.5 m length will be used. They are arranged in detector modules of 5 m length and 0.34 m width. The envisaged spatial resolution over the entire active area is 200$mu$m resulting in stringent requirements on the accuracy for the module construction. In this paper we discuss the optimisation of the straws, design and construction of detector modules. The long term operation properties of straws in two different counting g...

  3. Immunogenicity of Nontypeable Haemophilus influenzae Outer Membrane Vesicles and Protective Ability in the Chinchilla Model of Otitis Media.

    Winter, Linda E; Barenkamp, Stephen J

    2017-10-01

    Outer membrane vesicles (OMVs) produced by Gram-negative bacteria are enriched in several outer membrane components, including major and minor outer membrane proteins and lipooligosaccharide. We assessed the functional activity of nontypeable Haemophilus influenzae (NTHi) OMV-specific antisera and the protective ability of NTHi OMVs as vaccine antigens in the chinchilla otitis media model. OMVs were purified from three HMW1/HMW2-expressing NTHi strains, two of which were also engineered to overexpress Hia proteins. OMV-specific antisera raised in guinea pigs were assessed for their ability to mediate killing of representative NTHi in an opsonophagocytic assay. The three OMV-specific antisera mediated killing of 18 of 65, 24 of 65, and 30 of 65 unrelated HMW1/HMW2-expressing NTHi strains. Overall, they mediated killing of 39 of 65 HMW1/HMW2-expressing strains. The two Hia-expressing OMV-specific antisera mediated killing of 17 of 25 and 14 of 25 unrelated Hia-expressing NTHi strains. Overall, they mediated killing of 20 of 25 Hia-expressing strains. OMVs from prototype NTHi strain 12 were used to immunize chinchillas and the course of middle ear infection was monitored following intrabullar challenge with the homologous strain. All control animals developed culture-positive otitis media, as did two of three HMW1/HMW2-immunized animals. All OMV-immunized animals, with or without supplemental HMW1/HMW2 immunization, were completely protected against otitis media. NTHi OMVs are the first immunogens examined in this model that provided complete protection with sterile immunity after NTHi strain 12 challenge. These data suggest that NTHi OMVs hold significant potential as components of protective NTHi vaccines, possibly in combination with HMW1/HMW2 proteins. Copyright © 2017 American Society for Microbiology.

  4. The Effects of Earth's Outer Core's Viscosity on Geodynamo Models

    Dong, C.; Jiao, L.; Zhang, H.

    2017-12-01

    Geodynamo process is controlled by mathematic equations and input parameters. To study effects of parameters on geodynamo system, MoSST model has been used to simulate geodynamo outputs under different outer core's viscosity ν. With spanning ν for nearly three orders when other parameters fixed, we studied the variation of each physical field and its typical length scale. We find that variation of ν affects the velocity field intensely. The magnetic field almost decreases monotonically with increasing of ν, while the variation is no larger than 30%. The temperature perturbation increases monotonically with ν, but by a very small magnitude (6%). The averaged velocity field (u) of the liquid core increases with ν as a simple fitted scaling relation: u∝ν0.49. The phenomenon that u increases with ν is essentially that increasing of ν breaks the Taylor-Proudman constraint and drops the critical Rayleigh number, and thus u increases under the same thermal driving force. Forces balance is analyzed and balance mode shifts with variation of ν. When compared with former studies of scaling laws, this study supports the conclusion that in a certain parameter range, the magnetic field strength doesn't vary much with the viscosity, but opposes to the assumption that the velocity field has nothing to do with the outer core viscosity.

  5. Safety Framework for Nuclear Power Source Applications in Outer Space

    2009-01-01

    Nuclear power sources (NPS) for use in outer space have been developed and used in space applications where unique mission requirements and constraints on electrical power and thermal management precluded the use of non-nuclear power sources. Such missions have included interplanetary missions to the outer limits of the Solar System, for which solar panels were not suitable as a source of electrical power because of the long duration of these missions at great distances from the Sun. According to current knowledge and capabilities, space NPS are the only viable energy option to power some space missions and significantly enhance others. Several ongoing and foreseeable missions would not be possible without the use of space NPS. Past, present and foreseeable space NPS applications include radioisotope power systems (for example, radioisotope thermoelectric generators and radioisotope heater units) and nuclear reactor systems for power and propulsion. The presence of radioactive materials or nuclear fuels in space NPS and their consequent potential for harm to people and the environment in Earth's biosphere due to an accident require that safety should always be an inherent part of the design and application of space NPS. NPS applications in outer space have unique safety considerations compared with terrestrial applications. Unlike many terrestrial nuclear applications, space applications tend to be used infrequently and their requirements can vary significantly depending upon the specific mission. Mission launch and outer space operational requirements impose size, mass and other space environment limitations not present for many terrestrial nuclear facilities. For some applications, space NPS must operate autonomously at great distances from Earth in harsh environments. Potential accident conditions resulting from launch failures and inadvertent re-entry could expose NPS to extreme physical conditions. These and other unique safety considerations for the use of

  6. Low-Outgassing Photogrammetry Targets for Use in Outer Space

    Gross, Jason N.; Sampler, Henry; Reed, Benjamin B.

    2011-01-01

    A short document discusses an investigation of materials for photogrammetry targets for highly sensitive optical scientific instruments to be operated in outer space and in an outer-space-environment- simulating thermal vacuum chamber on Earth. A key consideration in the selection of photogrammetry-target materials for vacuum environments is the need to prevent contamination that could degrade the optical responses of the instruments. Therefore, in addition to the high levels and uniformity of reflectivity required of photogrammetry-target materials suitable for use in air, the materials sought must exhibit minimal outgassing. Commercially available photogrammetry targets were found to outgas excessively under the thermal and vacuum conditions of interest; this finding prompted the investigators to consider optically equivalent or superior, lower-outgassing alternative target materials. The document lists several materials found to satisfy the requirements, but does not state explicitly whether the materials can be used individually or must be combined in the proper sequence into layered target structures. The materials in question are an aluminized polyimide tape, an acrylic pressure- sensitive adhesive, a 500-A-thick layer of vapor-deposited aluminum, and spherical barium titanate glass beads having various diameters from 20 to 63 microns..

  7. Dynamical limits on dark mass in the outer solar system

    Hogg, D.W.; Quinlan, G.D.; Tremaine, S.

    1991-01-01

    Simplified model solar systems with known observational errors are considered in conducting a dynamical search for dark mass and its minimum detectable amount, and in determining the significance of observed anomalies. The numerical analysis of the dynamical influence of dark mass on the orbits of outer planets and comets is presented in detail. Most conclusions presented are based on observations of the four giant planets where the observational errors in latitude and longitude are independent Gaussian variables with a standard deviation. Neptune's long orbital period cannot be predicted by modern ephemerides, and no evidence of dark mass is found in considering this planet. Studying the improvement in fit when observations are fitted to models that consider dark mass is found to be an efficient way to detect dark mass. Planet X must have a mass of more than about 10 times the minimum detectable mass to locate the hypothetical planet. It is suggested that the IRAS survey would have already located the Planet X if it is so massive and close that it dynamically influences the outer planets. Orbital residuals from comets are found to be more effective than those from planets in detecting the Kuiper belt. 35 refs

  8. Fields and plasmas in the outer solar system. [Review

    Smith, E J [Jet Propulsion Lab., Pasadena, CA (USA); Wolfe, J H [National Aeronautics and Space Administration, Moffett Field, CA (USA). Ames Research Center

    1979-04-01

    The most significant information about fields and plasmas in the outer solar system, based on observations by Pioneer 10 and 11 investigations, is reviewed. The characteristic evolution of solar wind streams beyond 1 AU has been observed. The region within which the velocity increases continuously near 1 AU is replaced at larger distances by a thick interaction region with abrupt jumps in the solar wind speed at the leading and trailing edges. These abrupt increases, accompanied by corresponding jumps in the field magnitude and in the solar wind density and temperature, consist typically of a forward and a reverse shock. The existance of two distinct corotating regions, separated by sharp boundaries, is a characteristic feature of the interplanetary medium in the outer solar system. Within the interaction regions, compression effects are dominant and the field strength, plasma density, plasma temperature and the level of fluctuations are enhanced. Within the intervening quiet regions, rarefaction effects dominante and the field magnitude, solar wind density and fluctuation level are very low. These changes in the structure of interplanetary space have significant consequences for the many energetic particles propagating through the medium.

  9. The CMS Outer Tracker for HL-LHC

    Dierlamm, Alexander Hermann

    2018-01-01

    The LHC is planning an upgrade program, which will bring the luminosity to about $5-7\\times10^{34}$~cm$^{-2}$s$^{-1}$ in 2026, with a goal of an integrated luminosity of 3000 fb$^{-1}$ by the end of 2037. This High Luminosity LHC scenario, HL-LHC, will require a preparation program of the LHC detectors known as Phase-2 Upgrade. The current CMS Tracker is already running beyond design specifications and will not be able to cope with the HL-LHC radiation conditions. CMS will need a completely new Tracker in order to fully exploit the highly demanding operating conditions and the delivered luminosity. The new Outer Tracker system is designed to provide robust tracking as well as Level-1 trigger capabilities using closely spaced modules composed of silicon macro-pixel and/or strip sensors. Research and development activities are ongoing to explore options and develop module components and designs for the HL-LHC environment. The design choices for the CMS Outer Tracker Upgrade are discussed along with some highlig...

  10. Outer boundary as arrested history in general relativity

    Lau, Stephen R

    2002-01-01

    We present explicit outer boundary conditions for the canonical variables of general relativity. The conditions are associated with the causal evolution of a finite Cauchy domain, a so-called quasilocal boost, and they suggest a consistent scheme for modelling such an evolution numerically. The scheme involves a continuous boost in the spacetime orthogonal complement 'orthogonal' T p (B) of the tangent space T p (B) belonging to each point p on the system boundary B. We show how the boost rate may be computed numerically via equations similar to those appearing in canonical investigations of black-hole thermodynamics (although here holding at an outer two-surface rather than the bifurcate two-surface of a Killing horizon). We demonstrate the numerical scheme on a model example, the quasilocal boost of a spherical three-ball in Minkowski spacetime. Developing our general formalism with recent hyperbolic formulations of the Einstein equations in mind, we use Anderson and York's 'Einstein-Christoffel' hyperbolic system as the evolution equations for our numerical simulation of the model

  11. Outer boundary as arrested history in general relativity

    Lau, Stephen R.

    2002-06-01

    We present explicit outer boundary conditions for the canonical variables of general relativity. The conditions are associated with the causal evolution of a finite Cauchy domain, a so-called quasilocal boost, and they suggest a consistent scheme for modelling such an evolution numerically. The scheme involves a continuous boost in the spacetime orthogonal complement ⊥Tp(B) of the tangent space Tp(B) belonging to each point p on the system boundary B. We show how the boost rate may be computed numerically via equations similar to those appearing in canonical investigations of black-hole thermodynamics (although here holding at an outer two-surface rather than the bifurcate two-surface of a Killing horizon). We demonstrate the numerical scheme on a model example, the quasilocal boost of a spherical three-ball in Minkowski spacetime. Developing our general formalism with recent hyperbolic formulations of the Einstein equations in mind, we use Anderson and York's 'Einstein-Christoffel' hyperbolic system as the evolution equations for our numerical simulation of the model.

  12. Outer boundary as arrested history in general relativity

    Lau, S

    2002-01-01

    We present explicit outer boundary conditions for the canonical variables of general relativity. The conditions are associated with the causal evolution of a finite Cauchy domain, a so-called quasilocal boost, and they suggest a consistent scheme for modelling such an evolution numerically. The scheme involves a continuous boost in the spacetime orthogonal complement 'orthogonal' T sub p (B) of the tangent space T sub p (B) belonging to each point p on the system boundary B. We show how the boost rate may be computed numerically via equations similar to those appearing in canonical investigations of black-hole thermodynamics (although here holding at an outer two-surface rather than the bifurcate two-surface of a Killing horizon). We demonstrate the numerical scheme on a model example, the quasilocal boost of a spherical three-ball in Minkowski spacetime. Developing our general formalism with recent hyperbolic formulations of the Einstein equations in mind, we use Anderson and York's 'Einstein-Christoffel' hy...

  13. Solar wind velocity and temperature in the outer heliosphere

    Gazis, P. R.; Barnes, A.; Mihalov, J. D.; Lazarus, A. J.

    1994-01-01

    At the end of 1992, the Pioneer 10, Pioneer 11, and Voyager 2 spacecraft were at heliocentric distances of 56.0, 37.3, and 39.0 AU and heliographic latitudes of 3.3 deg N, 17.4 deg N, and 8.6 deg S, respectively. Pioneer 11 and Voyager 2 are at similar celestial longitudes, while Pioneer 10 is on the opposite side of the Sun. All three spacecraft have working plasma analyzers, so intercomparison of data from these spacecraft provides important information about the global character of the solar wind in the outer heliosphere. The averaged solar wind speed continued to exhibit its well-known variation with solar cycle: Even at heliocentric distances greater than 50 AU, the average speed is highest during the declining phase of the solar cycle and lowest near solar minimum. There was a strong latitudinal gradient in solar wind speed between 3 deg and 17 deg N during the last solar minimum, but this gradient has since disappeared. The solar wind temperature declined with increasing heliocentric distance out to a heliocentric distance of at least 20 AU; this decline appeared to continue at larger heliocentric distances, but temperatures in the outer heliosphere were suprisingly high. While Pioneer 10 and Voyager 2 observed comparable solar wind temperatures, the temperature at Pioneer 11 was significantly higher, which suggests the existence of a large-scale variation of temperature with heliographic longitude. There was also some suggestion that solar wind temperatures were higher near solar minimum.

  14. Upgrades of the CMS Outer Tracker for HL-LHC

    AUTHOR|(CDS)2067159

    2016-01-01

    The LHC machine is planning an upgrade program which will smoothly bring the luminosity to about 5×1034cm$^{−2}$s$^{−1}$ around 2028, to possibly reach an integrated luminosity of 3000 fb$^{−1}$ in the following decade. This High Luminosity LHC scenario, HL-LHC, will require a preparation program of the LHC detectors known as Phase-2 upgrade. The current CMS Outer Tracker, already running close to its design limits, will not be able to survive HL-LHC radiation conditions and CMS will need a completely new device, in order to fully exploit the highly demanding operating conditions and the delivered luminosity. The new Tracker should have also L1 trigger capabilities. To achieve such goals, R&D; activities are ongoing to explore options and develop solutions that would allow including tracking information at Level-1. The design choices for the CMS Outer Tracker upgrades are discussed along with some highlights of the R&D; activities.

  15. Functional Architecture of the Outer Arm Dynein Conformational Switch*

    King, Stephen M.; Patel-King, Ramila S.

    2012-01-01

    Dynein light chain 1 (LC1/DNAL1) is one of the most highly conserved components of ciliary axonemal outer arm dyneins, and it associates with both a heavy chain motor unit and tubulin located within the A-tubule of the axonemal outer doublet microtubules. In a variety of model systems, lack of LC1 or expression of mutant forms leads to profound defects in ciliary motility, including the failure of the hydrodynamic coupling needed for ciliary metachronal synchrony, random stalling during the power/recovery stroke transition, an aberrant response to imposed viscous load, and in some cases partial failure of motor assembly. These phenotypes have led to the proposal that LC1 acts as part of a mechanical switch to control motor function in response to alterations in axonemal curvature. Here we have used NMR chemical shift mapping to define the regions perturbed by a series of mutations in the C-terminal domain that yield a range of phenotypic effects on motility. In addition, we have identified the subdomain of LC1 involved in binding microtubules and characterized the consequences of an Asn → Ser alteration within the terminal leucine-rich repeat that in humans causes primary ciliary dyskinesia. Together, these data define a series of functional subdomains within LC1 and allow us to propose a structural model for the organization of the dynein heavy chain-LC1-microtubule ternary complex that is required for the coordinated activity of dynein motors in cilia. PMID:22157010

  16. Photoionization of the outer electrons in noble gas endohedral atoms

    Amusia, M. Ya.; Baltenkov, A. S.; Chernysheva, L. V.

    2008-01-01

    We suggest a prominent modification of the outer shell photoionization cross section in noble gas (NG) endohedral atoms NG-C n under the action of the electron shell of fullerene C n . This shell leads to two important effects: a strong enhancement of the cross section due to fullerene shell polarization under the action of the incoming electromagnetic wave and to prominent oscillation of this cross section due to the reflection of a photoelectron from the NG by the fullerene shell. Both factors lead to powerful maxima in the outer shell ionization cross sections of NG-C n , which we call giant endohedral resonances. The oscillator strength reaches a very large value in the atomic scale, 25. We consider atoms of all noble gases except He. The polarization of the fullerene shell is expressed in terms of the total photoabsorption cross section of the fullerene. The photoelectron reflection is taken into account in the framework of the so-called bubble potential, which is a spherical δ-type potential. It is assumed in the derivations that the NG is centrally located in the fullerene. It is also assumed, in accordance with the existing experimental data, that the fullerene radius R C is much larger than the atomic radius r A and the thickness Δ C of the fullerene shell. As was demonstrated recently, these assumptions allow us to represent the NG-C n photoionization cross section as a product of the NG cross section and two well-defined calculated factors

  17. Modeling of the outer electron belt during magnetic storms

    Desorgher, L.; Buehler, P.; Zehnder, A.; Daly, E.; Adams, L.

    1999-01-01

    The flux dropout of relativistic electrons in the earth's outer radiation belt, during the main phase of the 26 March 1995 magnetic storm is examined. Outer belt measurements by the Radiation Environment Monitor, REM aboard the STRV-1b satellite are presented to characterize this dropout. In order to simulate the dynamics of the electron belt during the storm main phase a particle tracing code was developed which allows to trace the trajectories of equatorially mirroring electrons in a dynamic magnetospheric electromagnetic field. Two simulations were performed in a non-stationary magnetic field, one taking only the induced electric field into account (fully adiabatic motion), and one with an additional non-stationary convection electric field. The simulations show, that adiabatic deceleration can produce the observed count rate decrease and also the observed inward motion of the count rate peak. The convection electric field causes diffusion, which can take particles from low L values out to the magnetopause and contribute to an additional loss of particles, which is suggested by the observations

  18. Solar system astrophysics planetary atmospheres and the outer solar system

    Milone, Eugene F

    2014-01-01

    The second edition of Solar System Astrophysics: Planetary Atmospheres and the Outer Solar System provides a timely update of our knowledge of planetary atmospheres and the bodies of the outer solar system and their analogs in other planetary systems. This volume begins with an expanded treatment of the physics, chemistry, and meteorology of the atmospheres of the Earth, Venus, and Mars, moving on to their magnetospheres and then to a full discussion of the gas and ice giants and their properties. From here, attention switches to the small bodies of the solar system, beginning with the natural satellites. Then comets, meteors, meteorites, and asteroids are discussed in order, and the volume concludes with the origin and evolution of our solar system. Finally, a fully revised section on extrasolar planetary systems puts the development of our system in a wider and increasingly well understood galactic context. All of the material is presented within a framework of historical importance. This book and its sist...

  19. Hybrid rocket propulsion systems for outer planet exploration missions

    Jens, Elizabeth T.; Cantwell, Brian J.; Hubbard, G. Scott

    2016-11-01

    Outer planet exploration missions require significant propulsive capability, particularly to achieve orbit insertion. Missions to explore the moons of outer planets place even more demanding requirements on propulsion systems, since they involve multiple large ΔV maneuvers. Hybrid rockets present a favorable alternative to conventional propulsion systems for many of these missions. They typically enjoy higher specific impulse than solids, can be throttled, stopped/restarted, and have more flexibility in their packaging configuration. Hybrids are more compact and easier to throttle than liquids and have similar performance levels. In order to investigate the suitability of these propulsion systems for exploration missions, this paper presents novel hybrid motor designs for two interplanetary missions. Hybrid propulsion systems for missions to Europa and Uranus are presented and compared to conventional in-space propulsion systems. The hybrid motor design for each of these missions is optimized across a range of parameters, including propellant selection, O/F ratio, nozzle area ratio, and chamber pressure. Details of the design process are described in order to provide guidance for researchers wishing to evaluate hybrid rocket motor designs for other missions and applications.

  20. Secretory IgA is Concentrated in the Outer Layer of Colonic Mucus along with Gut Bacteria

    Eric W. Rogier

    2014-04-01

    Full Text Available Antibodies of the secretory IgA (SIgA class comprise the first line of antigen-specific immune defense, preventing access of commensal and pathogenic microorganisms and their secreted products into the body proper. In addition to preventing infection, SIgA shapes the composition of the gut microbiome. SIgA is transported across intestinal epithelial cells into gut secretions by the polymeric immunoglobulin receptor (pIgR. The epithelial surface is protected by a thick network of mucus, which is composed of a dense, sterile inner layer and a loose outer layer that is colonized by commensal bacteria. Immunofluorescence microscopy of mouse and human colon tissues demonstrated that the SIgA co-localizes with gut bacteria in the outer mucus layer. Using mice genetically deficient for pIgR and/or mucin-2 (Muc2, the major glycoprotein of intestinal mucus, we found that Muc2 but not SIgA was necessary for excluding gut bacteria from the inner mucus layer in the colon. Our findings support a model whereby SIgA is anchored in the outer layer of colonic mucus through combined interactions with mucin proteins and gut bacteria, thus providing immune protection against pathogens while maintaining a mutually beneficial relationship with commensals.

  1. Outer Membrane Proteome of Veillonella parvula: A Diderm Firmicute of the Human Microbiome

    Daniel I. Poppleton

    2017-06-01

    Full Text Available Veillonella parvula is a biofilm-forming commensal found in the lungs, vagina, mouth, and gastro-intestinal tract of humans, yet it may develop into an opportunistic pathogen. Furthermore, the presence of Veillonella has been associated with the development of a healthy immune system in infants. Veillonella belongs to the Negativicutes, a diverse clade of bacteria that represent an evolutionary enigma: they phylogenetically belong to Gram-positive (monoderm Firmicutes yet maintain an outer membrane (OM with lipopolysaccharide similar to classic Gram-negative (diderm bacteria. The OMs of Negativicutes have unique characteristics including the replacement of Braun's lipoprotein by OmpM for tethering the OM to the peptidoglycan. Through phylogenomic analysis, we have recently provided bioinformatic annotation of the Negativicutes diderm cell envelope. We showed that it is a unique type of envelope that was present in the ancestor of present-day Firmicutes and lost multiple times independently in this phylum, giving rise to the monoderm architecture; however, little experimental data is presently available for any Negativicutes cell envelope. Here, we performed the first experimental proteomic characterization of the cell envelope of a diderm Firmicute, producing an OM proteome of V. parvula. We initially conducted a thorough bioinformatics analysis of all 1,844 predicted proteins from V. parvula DSM 2008's genome using 12 different localization prediction programs. These results were complemented by protein extraction with surface exposed (SE protein tags and by subcellular fractionation, both of which were analyzed by liquid chromatography tandem mass spectrometry. The merging of proteomics and bioinformatics results allowed identification of 78 OM proteins. These include a number of receptors for TonB-dependent transport, the main component of the BAM system for OM protein biogenesis (BamA, the Lpt system component LptD, which is responsible for

  2. Autofluorescence from the outer retina and subretinal space: hypothesis and review.

    Spaide, Richard

    2008-01-01

    To review the pathophysiologic principles underlying increased autofluorescence from the outer retina and subretinal space using selected diseases as examples. The ocular imaging information and histopathologic features, when known, were integrated for diseases causing increased autofluorescence from the outer retina and subretinal space. Inferences were taken from this information and used to create a classification scheme. These diseases are principally those that cause separation of the outer retina from the retinal pigment epithelium, thereby preventing proper phagocytosis of photoreceptor outer segments. The separation can arise from increased exudation into the subretinal space or inadequate removal of fluid from the subretinal space. Lack of normal outer segment processing initially leads to increased accumulation of outer segments on the outer retina and subretinal space. Over time, this material is visible as an increasingly thick coating on the outer retina, is yellow, and is autofluorescent. Over time, atrophy develops with thinning of the deposited material and decreasing autofluorescence. The accumulated material is ultimately capable of inducing damage to the retinal pigment epithelium. Diseases causing accumulation of the material include central serous chorioretinopathy, vitelliform macular dystrophy, acute exudative polymorphous vitelliform maculopathy, choroidal tumors, and vitreomacular traction syndrome. The physical separation of the retinal outer segments from the retinal pigment epithelium hinders proper phagocytosis of the outer segments. Accumulation of the shed but not phagocytized outer segments plays a role in disease manifestations for a number of macular diseases.

  3. Turbine exhaust diffuser with region of reduced flow area and outer boundary gas flow

    Orosa, John

    2014-03-11

    An exhaust diffuser system and method for a turbine engine. The outer boundary may include a region in which the outer boundary extends radially inwardly toward the hub structure and may direct at least a portion of an exhaust flow in the diffuser toward the hub structure. At least one gas jet is provided including a jet exit located on the outer boundary. The jet exit may discharge a flow of gas downstream substantially parallel to an inner surface of the outer boundary to direct a portion of the exhaust flow in the diffuser toward the outer boundary to effect a radially outward flow of at least a portion of the exhaust gas flow toward the outer boundary to balance an aerodynamic load between the outer and inner boundaries.

  4. Atmospheric Mining in the Outer Solar System: Outer Planet In-Space Bases and Moon Bases for Resource Processing

    Palaszewski, Bryan

    2017-01-01

    Atmospheric mining in the outer solar system has been investigated as a means of fuel production for high energy propulsion and power. Fusion fuels such as Helium 3 (3He) and deuterium can be wrested from the atmospheres of Uranus and Neptune and either returned to Earth or used in-situ for energy production. Helium 3 and deuterium were the primary gases of interest with hydrogen being the primary propellant for nuclear thermal solid core and gas core rocket-based atmospheric flight. A series of analyses were undertaken to investigate resource capturing aspects of atmospheric mining in the outer solar system. This included the gas capturing rate, storage options, and different methods of direct use of the captured gases. While capturing 3He, large amounts of hydrogen and 4He are produced. The propulsion and transportation requirements for all of the major moons of Uranus and Neptune are presented. Analyses of orbital transfer vehicles (OTVs), landers, factories, and the issues with in-situ resource utilization (ISRU) low gravity processing factories are included. Preliminary observations are presented on near-optimal selections of moon base orbital locations, OTV power levels, and OTV and lander rendezvous points. Several artificial gravity in-space base designs and orbital sites at Uranus and Neptune and the OTV requirements to support them are also addressed.

  5. Methane Group Ions in Saturn’s Outer Magnetosphere?

    Sittler, E. C.; Hartle, R. E.; Cooper, J. F.; Johnson, R. E.; Smith, H.; Shappirio, M.; Reisenfeld, D. B.

    2009-12-01

    Yelle et al. [2008] have estimated from Cassini Ion Neutral Mass Spectrometer (INMS) measurements that methane is escaping from Titan’s upper atmosphere at the rate of 2.5-3.0×109 mol/cm2/s and in order to explain this loss rate Strobel [2008] has proposed a hydrodynamic escape model to explain such high loss rates. This translates to loss of 2.8×1027 methane mol/s. The consequence of this work is the formation of a methane torus around Saturn which will dissociate to CH3 and other fragments of methane. The CH3 will then become ionized to form CH3+ with pickup energies ≈ keV after which it can be detected by the Cassini Plasma Spectrometer (CAPS) Ion Mass Spectrometer (IMS). Up till now the ion composition within Saturn’s outer magnetosphere in the vicinity of Titan’s orbit have yielded negative results with water group ions W+ dominating. The water group ions probably result from the emission of fast neutrals from the Enceladus torus via charge exchange reactions but still gravitationally bound to Saturn [see Johnson et al., 2005 and Sittler et al. 2006] and then become ionized in the outer magnetosphere as ~≈keV pickup ions. The CAPS IMS produces two ion composition data products, one called Straight Through (ST) and the other Linear Electric Field (LEF). The first has a higher sensitivity, while the latter has a greater discrimination in time-of-flight (TOF). For ST data O+ and CH4+ have similar TOF with the primary discriminator being the O- fragment which appears at a different TOF than for mass 16 ions. One can also look for other discriminators called ghost peaks. In case of LEF W+ ions produce TOF peak close to that for atomic O+ and the methane will produce TOF close to that for atomic C+ which has a significantly different(shorter) TOF than O+. We will be reporting on our continual search for methane ions within Saturn’s outer magnetosphere. References: 1. Yelle, R. V., J. Cui and I.C.F. Müller-Wodarg, JGR, 2008. 2. Strobel, D. F., Icarus

  6. Metabolic remodeling precedes mitochondrial outer membrane permeabilization in human glioma xenograft cells.

    Ponnala, Shivani; Chetty, Chandramu; Veeravalli, Krishna Kumar; Dinh, Dzung H; Klopfenstein, Jeffrey D; Rao, Jasti S

    2012-02-01

    Glioma cancer cells adapt to changing microenvironment and shift from mitochondrial oxidative phosphorylation to aerobic glycolysis for their metabolic needs irrespective of oxygen availability. In the present study, we show that silencing MMP-9 in combination with uPAR/cathepsin B switch the glycolytic metabolism of glioma cells to oxidative phosphorylation (OXPHOS) and generate reactive oxygen species (ROS) to predispose glioma cells to mitochondrial outer membrane permeabilization. shRNA for MMP-9 and uPAR (pMU) as well as shRNA for MMP-9 and cathepsin B (pMC) activated complexes of mitochondria involved in OXPHOS and inhibited glycolytic hexokinase expression. The decreased interaction of hexokinase 2 with mitochondria in the treated cells indicated the inhibition of glycolysis activation. Overexpression of Akt reversed the pMU- and pMC-mediated OXPHOS to glycolysis switch. The OXPHOS un-coupler oligomycin A altered the expression levels of the Bcl-2 family of proteins; treatment with pMU or pMC reversed this effect and induced mitochondrial outer membrane permeabilization. In addition, our results show changes in mitochondrial pore transition to release cytochrome c due to changes in the VDAC-Bcl-XL and BAX-BAK interaction with pMU and pMC treatments. Taken together, our results suggest that pMU and pMC treatments switch glioma cells from the glycolytic to the OXPHOS pathway through an inhibitory effect on Akt, ROS induction and an increase of cytosolic cytochrome c accumulation. These results demonstrate the potential of pMU and pMC as therapeutic candidates for the treatment of glioma.

  7. Molecular recognition in myxobacterial outer membrane exchange: functional, social and evolutionary implications.

    Wall, Daniel

    2014-01-01

    Through cooperative interactions, bacteria can build multicellular communities. To ensure that productive interactions occur, bacteria must recognize their neighbours and respond accordingly. Molecular recognition between cells is thus a fundamental behaviour, and in bacteria important discoveries have been made. This MicroReview focuses on a recently described recognition system in myxobacteria that is governed by a polymorphic cell surface receptor called TraA. TraA regulates outer membrane exchange (OME), whereby myxobacterial cells transiently fuse their OMs to efficiently transfer proteins and lipids between cells. Unlike other transport systems, OME is rather indiscriminate in what OM goods are transferred. In contrast, the recognition of partnering cells is discriminatory and only occurs between cells that bear identical or closely related TraA proteins. Therefore TraA functions in kin recognition and, in turn, OME helps regulate social interactions between myxobacteria. Here, I discuss and speculate on the social and evolutionary implications of OME and suggest it helps to guide their transition from free-living cells into coherent and functional populations. © 2013 John Wiley & Sons Ltd.

  8. Getting to the Outer Leaflet: Physiology of Phosphatidylserine Exposure at the Plasma Membrane.

    Bevers, Edouard M; Williamson, Patrick L

    2016-04-01

    Phosphatidylserine (PS) is a major component of membrane bilayers whose change in distribution between inner and outer leaflets is an important physiological signal. Normally, members of the type IV P-type ATPases spend metabolic energy to create an asymmetric distribution of phospholipids between the two leaflets, with PS confined to the cytoplasmic membrane leaflet. On occasion, membrane enzymes, known as scramblases, are activated to facilitate transbilayer migration of lipids, including PS. Recently, two proteins required for such randomization have been identified: TMEM16F, a scramblase regulated by elevated intracellular Ca(2+), and XKR8, a caspase-sensitive protein required for PS exposure in apoptotic cells. Once exposed at the cell surface, PS regulates biochemical reactions involved in blood coagulation, and bone mineralization, and also regulates a variety of cell-cell interactions. Exposed on the surface of apoptotic cells, PS controls their recognition and engulfment by other cells. This process is exploited by parasites to invade their host, and in specialized form is used to maintain photoreceptors in the eye and modify synaptic connections in the brain. This review discusses what is known about the mechanism of PS exposure at the surface of the plasma membrane of cells, how actors in the extracellular milieu sense surface exposed PS, and how this recognition is translated to downstream consequences of PS exposure. Copyright © 2016 the American Physiological Society.

  9. Fluid flow near the surface of earth's outer core

    Bloxham, Jeremy; Jackson, Andrew

    1991-01-01

    This review examines the recent attempts at extracting information on the pattern of fluid flow near the surface of the outer core from the geomagnetic secular variation. Maps of the fluid flow at the core surface are important as they may provide some insight into the process of the geodynamo and may place useful constraints on geodynamo models. In contrast to the case of mantle convection, only very small lateral variations in core density are necessary to drive the flow; these density variations are, by several orders of magnitude, too small to be imaged seismically; therefore, the geomagnetic secular variation is utilized to infer the flow. As substantial differences exist between maps developed by different researchers, the possible underlying reasons for these differences are examined with particular attention given to the inherent problems of nonuniqueness.

  10. Acute Zonal Occult Outer Retinopathy with Atypical Findings

    Dimitrios Karagiannis

    2014-01-01

    Full Text Available Background. To report a case of acute zonal occult outer retinopathy (AZOOR with atypical electrophysiology findings. Case Presentation. A 23-year-old-female presented with visual acuity deterioration in her right eye accompanied by photopsia bilaterally. Corrected distance visual acuity at presentation was 20/50 in the right eye and 20/20 in the left eye. Fundus examination was unremarkable. Visual field (VF testing revealed a large scotoma. Pattern and full-field electroretinograms (PERG and ERG revealed macular involvement associated with generalized retinal dysfunction. Electrooculogram (EOG light rise and the Arden ratio were within normal limits bilaterally. The patient was diagnosed with AZOOR due to clinical findings, visual field defect, and ERG findings. Conclusion. This is a case of AZOOR with characteristic VF defects and clinical symptoms presenting with atypical EOG findings.

  11. Progressive outer retinal necrosis presenting as cherry red spot.

    Yiu, Glenn; Young, Lucy H

    2012-10-01

    To report a case of progressive outer retinal necrosis (PORN) presenting as a cherry red spot. Case report. A 53-year-old woman with recently diagnosed HIV and varicella-zoster virus (VZV) aseptic meningitis developed rapid sequential vision loss in both eyes over 2 months. Her exam showed a "cherry red spot" in both maculae with peripheral atrophy and pigmentary changes, consistent with PORN. Due to her late presentation and the rapid progression of her condition, she quickly developed end-stage vision loss in both eyes. PORN should be considered within the differential diagnosis of a "cherry red spot." Immune-deficient patients with a history of herpetic infection who present with visual loss warrant prompt ophthalmological evaluation.

  12. Progressive outer retinal necrosis in immunocompromised kidney allograft recipient.

    Turno-Kręcicka, A; Boratyńska, M; Tomczyk-Socha, M; Mazanowska, O

    2015-06-01

    Ocular complications in patients who underwent renal transplantation are attributed to side effects of the immunosuppressive regimen. Progressive outer retinal necrosis (PORN) syndrome is a clinical variant of necrotizing herpetic retinopathy and it occurs almost exclusively in patients with acquired immunodeficiency syndrome. We present a case of a human immunodeficiency virus-negative patient who underwent renal transplant and, after a few years, developed bilateral PORN associated with viral infections. Varicella zoster virus (VZV) and BK virus were identified by polymerase chain reaction from the vitreous fluid. It is unclear which of the viruses identified had the dominant role in the pathogenesis of PORN and other organ damage, or whether their actions were synergistic. Adequate antiviral immune surveillance, as well as pre-transplant vaccination against VZV, may reduce the incidence of VZV infection and its complications. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Progressive outer retinal necrosis: manifestation of human immunodeficiency virus infection.

    Lo, Phey Feng; Lim, Rongxuan; Antonakis, Serafeim N; Almeida, Goncalo C

    2015-05-06

    We present the case of a 54-year-old man who developed progressive outer retinal necrosis (PORN) as an initial manifestation of HIV infection without any significant risk factors for infection with HIV. PORN is usually found as a manifestation of known AIDS late in the disease. Our patient presented with transient visual loss followed by decrease in visual acuity and facial rash. Subsequent investigation revealed anterior chamber tap positive for varicella zoster virus (VZV), as well as HIV positivity, with an initial CD4 count of 48 cells/µL. Systemic and intravitreal antivirals against VZV, and highly active antiretroviral therapy against HIV were started, which halted further progression of retinal necrosis. This case highlights the importance of suspecting PORN where there is a rapidly progressive retinitis, and also testing the patient for HIV, so appropriate treatment can be started. 2015 BMJ Publishing Group Ltd.

  14. On the theory of helium diffusion in stellar outer layers

    Ponce D, S.; Verga, A.D.

    1986-01-01

    We discuss the approximations usually made in the different approaches to diffusion in stellar outer layers. We analyze the hypotheses of binary diffusion and diffusion over a non altered background both analytically and numerically. Numerical calculations are applied to central stars of planetary nebulae in which a depletion of helium is observed. We find that in this case helium diffusion may be considered as a binary process but cannot be decoupled from the structure computation. We present an alternative method for studying diffusion and apply it to the central stars. We thus solve a stationary hydrodynamic model for a completely ionized H-He plasma, which takes into account consistently the behavior of all the species. We find equilibrium abundance distributions very different from those obtained according to the trace element approaches while helium and electron densities increase with depth in the atmosphere, protons tend to decrease. However, preliminary studies of the stability show that these are not the actual distributions. (author)

  15. Testing of a femtosecond pulse laser in outer space

    Lee, Joohyung; Lee, Keunwoo; Jang, Yoon-Soo; Jang, Heesuk; Han, Seongheum; Lee, Sang-Hyun; Kang, Kyung-In; Lim, Chul-Woo; Kim, Young-Jin; Kim, Seung-Woo

    2014-01-01

    We report a test operation of an Er-doped fibre femtosecond laser which was conducted for the first time in outer space. The fibre-based ultrashort pulse laser payload was designed to meet space-use requirements, undergone through ground qualification tests and finally launched into a low-earth orbit early in 2013. Test results obtained during a one-year mission lifetime confirmed stable mode-locking all the way through although the radiation induced attenuation (RIA) in the Er-doped gain fibre caused an 8.6% reduction in the output power. This successful test operation would help facilitate diverse scientific and technological applications of femtosecond lasers in space and earth atmosphere in the near future. PMID:24875665

  16. Microbial Morphology and Motility as Biosignatures for Outer Planet Missions

    Nadeau, Jay; Lindensmith, Chris; Deming, Jody W.; Fernandez, Vicente I.; Stocker, Roman

    2016-10-01

    Meaningful motion is an unambiguous biosignature, but because life in the Solar System is most likely to be microbial, the question is whether such motion may be detected effectively on the micrometer scale. Recent results on microbial motility in various Earth environments have provided insight into the physics and biology that determine whether and how microorganisms as small as bacteria and archaea swim, under which conditions, and at which speeds. These discoveries have not yet been reviewed in an astrobiological context. This paper discusses these findings in the context of Earth analog environments and environments expected to be encountered in the outer Solar System, particularly the jovian and saturnian moons. We also review the imaging technologies capable of recording motility of submicrometer-sized organisms and discuss how an instrument would interface with several types of sample-collection strategies.

  17. The CMS Outer HCAL SiPM Upgrade

    CMS Collaboration

    2014-01-01

    The CMS Outer Hadron Calorimeter (HO) is the first large scale hadron collider detector to use SiPMs. By late January 2014 the installation of 1656 of 2376 channels was completed. The HO readout system provides for active temperature stabilisation of the SiPMs to less than 0.1$^\\circ$C using Peltier coolers, temperature measurement, and software feedback. Each channel has independently controlled bias voltage with a resolution of 25mV. Each SiPM is read out by 40MHz QIE ADCs. We report on the system design, schedule and progress. The next phase for the detector is commissioning during 2014 before the 2015 LHC run. We report on the status of commissioning and plans for operation. We discuss the calibration strategy with local cosmic ray runs using the HO's self trigger ability.

  18. The CMS Outer HCAL SiPM Upgrade

    Lobanov, Artur

    2015-01-01

    The CMS Outer Hadron Calorimeter (HO) is the first large scale hadron collider detector to use SiPMs. By late January 2014 the installation of 1656 of 2376 channels was completed. The HO readout system provides for active temperature stabilization of the SiPMs to less than 0.1$^\\circ$C using Peltier coolers, temperature measurement, and software feedback. Each channel has independently controlled bias voltage with a resolution of 25~mV. Each SiPM is read out by 40~MHz QIE ADCs. We report on the system design, schedule and progress. The next phase for the detector is commissioning during 2014 before the 2015 LHC run. We report on the status of commissioning and plans for operation. We discuss the calibration strategy with local cosmic ray runs using the HO's self trigger ability. We discuss the plans for a global CMS operations run in November 2014.

  19. Biogenesis and function of Porphyromonas gingivalis outer membrane vesicles

    Xie, H

    2015-01-01

    Porphyromonas gingivalis is one of the keystone pathogens associated with chronic periodontitis. All P. gingivalis strains examined thus far produce outer membrane vesicles. Recent studies have found that vesicles possess some well-known virulence factors of P. gingivalis such as adhesins, toxins and proteolytic enzymes. Carrying most of the characteristic features of their parent P. gingivalis cells, vesicles communicate with host cells and other members of microbial biofilms, resulting in the transmission of virulence factors into these host cells and the formation of pathogenic bacteria-dominated microbial communities. An in-depth understanding of both the nature and role of vesicles in the pathogenicity of P. gingivalis is both important and timely, particularly when speaking of periodontitis and its related systemic effects. PMID:26343879

  20. COOL DUST IN THE OUTER RING OF NGC 1291

    Hinz, J. L.; Engelbracht, C. W.; Skibba, R.; Montiel, E.; Crocker, A.; Calzetti, D.; Donovan Meyer, J.; Sandstrom, K.; Walter, F.; Groves, B.; Meidt, S. E.; Johnson, B. D.; Hunt, L.; Aniano, G.; Draine, B.; Murphy, E. J.; Armus, L.; Dale, D. A.; Galametz, M.; Kennicutt, R. C.

    2012-01-01

    We examine Herschel Space Observatory images of one nearby prototypical outer ring galaxy, NGC 1291, and show that the ring becomes more prominent at wavelengths longer than 160 μm. The mass of cool dust in the ring dominates the total dust mass of the galaxy, accounting for at least 70% of it. The temperature of the emitting dust in the ring (T = 19.5 ± 0.3 K) is cooler than that of the inner galaxy (T = 25.7 ± 0.7 K). We discuss several explanations for the difference in dust temperature, including age and density differences in the stellar populations of the ring versus the bulge.

  1. Large-scale density structures in the outer heliosphere

    Belcher, J. W.; Lazarus, A. J.; Mcnutt, R. L., Jr.; Gordon, G. S., Jr.

    1993-01-01

    The Plasma Science experiment on the Voyager 2 spacecraft has measured the solar wind density from 1 to 38 AU. Over this distance, the solar wind density decreases as the inverse square of the heliocentric distance. However, there are large variations in this density at a given radius. Such changes in density are the dominant cause of changes in the solar wind ram pressure in the outer heliosphere and can cause large perturbations in the location of the termination shock of the solar wind. Following a simple model suggested by Suess, we study the non-equilibrium, dynamic location of the termination shock as it responds to these pressure changes. The results of this study suggest that the termination shock is rarely if ever at its equilibrium distance and may depart from that distance by as much as 50 AU at times.

  2. Tick in the outer ear canal: Two case reports

    Özalkan Özkan

    2013-01-01

    Full Text Available Serious systemic diseases such as Crimean Congo Hemorrhagic Fever, Lyme Disease, tularemia, typhus and Q fever can be transmitted by ticks. An 8-year-old boy and a 34-year-old woman were brought to our clinic with ear pains. No symptoms such as fever, headache or lethargy to suggest CCHF were present. The patients both lived in rural areas and were engaged in farming. Full blood count, liver and kidney function tests and serum electrolytes were within normal limits at laboratory tests. Prothrombin time, partial thromboplastin time and INR were normal. The tick was grasped with alligator forceps and removed from the outer ear in one piece with the help of 0-degree rigid otoendoscopy.

  3. Potential alternative energy technologies on the Outer Continental Shelf.

    Elcock, D.; Environmental Assessment

    2007-04-20

    This technical memorandum (TM) describes the technology requirements for three alternative energy technologies for which pilot and/or commercial projects on the U.S. Outer Continental Shelf (OCS) are likely to be proposed within the next five to seven years. For each of the alternative technologies--wind, wave, and ocean current--the TM first presents an overview. After each technology-specific overview, it describes the technology requirements for four development phases: site monitoring and testing, construction, operation, and decommissioning. For each phase, the report covers the following topics (where data are available): facility description, electricity generated, ocean area (surface and bottom) occupied, resource requirements, emissions and noise sources, hazardous materials stored or used, transportation requirements, and accident potential. Where appropriate, the TM distinguishes between pilot-scale (or demonstration-scale) facilities and commercial-scale facilities.

  4. Using Stem Cells to Model Diseases of the Outer Retina

    Camille Yvon

    2015-01-01

    Full Text Available Retinal degeneration arises from the loss of photoreceptors or retinal pigment epithelium (RPE. It is one of the leading causes of irreversible blindness worldwide with limited effective treatment options. Generation of induced pluripotent stem cell (IPSC-derived retinal cells and tissues from individuals with retinal degeneration is a rapidly evolving technology that holds a great potential for its use in disease modelling. IPSCs provide an ideal platform to investigate normal and pathological retinogenesis, but also deliver a valuable source of retinal cell types for drug screening and cell therapy. In this review, we will provide some examples of the ways in which IPSCs have been used to model diseases of the outer retina including retinitis pigmentosa (RP, Usher syndrome (USH, Leber congenital amaurosis (LCA, gyrate atrophy (GA, juvenile neuronal ceroid lipofuscinosis (NCL, Best vitelliform macular dystrophy (BVMD and age related macular degeneration (AMD.

  5. Solar system astrophysics planetary atmospheres and the outer solar system

    Milone, Eugene F

    2008-01-01

    Solar System Astrophysics opens with coverage of the atmospheres, ionospheres and magnetospheres of the Earth, Venus and Mars and the magnetosphere of Mercury. The book then provides an introduction to meteorology and treating the physics and chemistry of these areas in considerable detail. What follows are the structure, composition, particle environments, satellites, and rings of Jupiter, Saturn, Uranus and Neptune, making abundant use of results from space probes. Solar System Astrophysics follows the history, orbits, structure, origin and demise of comets and the physics of meteors and provides a thorough treatment of meteorites, the asteroids and, in the outer solar system, the Kuiper Belt objects. The methods and results of extrasolar planet searches, the distinctions between stars, brown dwarfs, and planets, and the origins of planetary systems are examined. Historical introductions precede the development and discussion in most chapters. A series of challenges, useful as homework assignments or as foc...

  6. The CMS Outer Tracker Upgrade for the High Luminosity LHC

    Luetic, Jelena

    2017-01-01

    The era of the High Luminosity Large Hadron Collider will pose unprecedented challenges for detector design and operation. The planned luminosity of the upgraded machine is $5$x$10^{34} $ cm$^{-2}$s$^{-1}$, reaching an integrated luminosity of more than 3000 fb$^{-1}$ by the end of 2037. The CMS Tracker detector will have to be replaced in order to fully exploit the delivered luminosity and cope with the demanding operating conditions. The new detector will provide robust tracking as well as input for the first level trigger. This report is focusing on the replacement of the CMS Outer Tracker system, describing the new layout and technological choices together with some highlights of research and development activities.

  7. Forbush decreases and particle acceleration in the outer heliosphere

    Van Allen, J.A.; Mihalov, J.D.

    1990-01-01

    Major solar flare activity in 1989 has provided examples of the local acceleration of protons at 28 AU (Pioneer 11) and of the propagation of Forbush decreases in galactic cosmic ray intensity to a heliocentric radial distance of 47 AU (Pioneer 10). The combination of these and previous data at lesser distances shows (a) that Forbush decreases propagate with essentially constant magnitude to (at least) 47 AU and with similar magnitude at widely different ecliptic longitudes and (b) that the times for recovery from such decreases become progressively greater as the radial distance increases, being of the order of months in the outer heliosphere. A phenomenological scheme for (b) is proposed and fresh support is given to the hypothesis that the solar cycle modulation of the galactic cosmic ray intensity is attributable primarily to overlapping Forbush decreases which are more frequent and of greater magnitude near times of maximum solar activity than at times of lesser activity

  8. A thermal study of pipes with outer transverse fins

    S. Gil

    2016-10-01

    Full Text Available This paper provides results of thermal investigations on pipes with outer transverse fins produced by placing a strip, being a form of helical spring which functions as a radiator, on the basis pipe. The investigations were carried out at the facility that enables measurements with respect to both natural and forced convection. Performance of the investigated pipes was assessed in relation to a non-finned pipe and a pipe welded with the use of Metal Active Gas (MAG technology. The experiments have shown that the finned pipe welding technology does not markedly affect their thermal efficiency, which has been confirmed by performed model calculations, while the welding technology has a crucial impact on their operating performance.

  9. Using Stem Cells to Model Diseases of the Outer Retina.

    Yvon, Camille; Ramsden, Conor M; Lane, Amelia; Powner, Michael B; da Cruz, Lyndon; Coffey, Peter J; Carr, Amanda-Jayne F

    2015-01-01

    Retinal degeneration arises from the loss of photoreceptors or retinal pigment epithelium (RPE). It is one of the leading causes of irreversible blindness worldwide with limited effective treatment options. Generation of induced pluripotent stem cell (IPSC)-derived retinal cells and tissues from individuals with retinal degeneration is a rapidly evolving technology that holds a great potential for its use in disease modelling. IPSCs provide an ideal platform to investigate normal and pathological retinogenesis, but also deliver a valuable source of retinal cell types for drug screening and cell therapy. In this review, we will provide some examples of the ways in which IPSCs have been used to model diseases of the outer retina including retinitis pigmentosa (RP), Usher syndrome (USH), Leber congenital amaurosis (LCA), gyrate atrophy (GA), juvenile neuronal ceroid lipofuscinosis (NCL), Best vitelliform macular dystrophy (BVMD) and age related macular degeneration (AMD).

  10. Knockout of an outer membrane protein operon of anaplasma marginale by transposon mutagenesis

    Large amounts of data generated by genomics, transcriptomics and proteomics technologies have increased our understanding of the biology of Anaplasma marginale. However, these data have also led to new assumptions that require testing, ideally through classic genetic mutation. One example is the def...

  11. The Neisseria meningitidis Outer Membrane Lipoprotein FrpD Binds the RTX Protein FrpC

    Procházková, Kateřina; Osička, Radim; Linhartová, Irena; Halada, Petr; Šulc, Miroslav; Šebo, Peter

    2005-01-01

    Roč. 280, č. 5 (2005), s. 3251-3258 ISSN 0021-9258 R&D Projects: GA ČR GA310/02/1448 Grant - others:Howard Hughes Medical Institute International Research Scholarship Award 55000334 Institutional research plan: CEZ:AV0Z50200510 Keywords : neisseria meningitidis * FrpD * FrpC Subject RIV: EE - Microbiology, Virology Impact factor: 5.854, year: 2005

  12. Magnetohydrodynamic Convection in the Outer Core and its Geodynamic Consequences

    Kuang, Weijia; Chao, Benjamin F.; Fang, Ming

    2004-01-01

    The Earth's fluid outer core is in vigorous convection through much of the Earth's history. In addition to generating and maintaining Earth s time-varying magnetic field (geodynamo), the core convection also generates mass redistribution in the core and a dynamical pressure field on the core-mantle boundary (CMB). All these shall result in various core-mantle interactions, and contribute to surface geodynamic observables. For example, electromagnetic core-mantle coupling arises from finite electrically conducting lower mantle; gravitational interaction occurs between the cores and the heterogeneous mantle; mechanical coupling may also occur when the CMB topography is aspherical. Besides changing the mantle rotation via the coupling torques, the mass-redistribution in the core shall produce a spatial-temporal gravity anomaly. Numerical modeling of the core dynamical processes contributes in several geophysical disciplines. It helps explain the physical causes of surface geodynamic observables via space geodetic techniques and other means, e.g. Earth's rotation variation on decadal time scales, and secular time-variable gravity. Conversely, identification of the sources of the observables can provide additional insights on the dynamics of the fluid core, leading to better constraints on the physics in the numerical modeling. In the past few years, our core dynamics modeling efforts, with respect to our MoSST model, have made significant progress in understanding individual geophysical consequences. However, integrated studies are desirable, not only because of more mature numerical core dynamics models, but also because of inter-correlation among the geophysical phenomena, e.g. mass redistribution in the outer core produces not only time-variable gravity, but also gravitational core-mantle coupling and thus the Earth's rotation variation. They are expected to further facilitate multidisciplinary studies of core dynamics and interactions of the core with other

  13. High-energy outer radiation belt dynamic modeling

    Chiu, Y.T.; Nightingale, R.W.; Rinaldi, M.A.

    1989-01-01

    Specification of the average high-energy radiation belt environment in terms of phenomenological montages of satellite measurements has been available for some time. However, for many reasons both scientific and applicational (including concerns for a better understanding of the high-energy radiatino background in space), it is desirable to model the dynamic response of the high-energy radiation belts to sources, to losses, and to geomagnetic activity. Indeed, in the outer electron belt, this is the only mode of modeling that can handle the large intensity fluctuations. Anticipating the dynamic modeling objective of the upcoming Combined Release and Radiation Effects Satellite (CRRES) program, we have undertaken to initiate the study of the various essential elements in constructing a dynamic radiation belt model based on interpretation of satellite data according to simultaneous radial and pitch-angle diffusion theory. In order to prepare for the dynamic radiation belt modeling based on a large data set spanning a relatively large segment of L-values, such as required for CRRES, it is important to study a number of test cases with data of similar characteristics but more restricted in space-time coverage. In this way, models of increasing comprehensiveness can be built up from the experience of elucidating the dynamics of more restrictive data sets. The principal objectives of this paper are to discuss issues concerning dynamic modeling in general and to summarize in particular the good results of an initial attempt at constructing the dynamics of the outer electron radiation belt based on a moderately active data period from Lockheed's SC-3 instrument flown on board the SCATHA (P78-2) spacecraft. Further, we shall discuss the issues brought out and lessons learned in this test case

  14. Normal Mode Derived Models of the Physical Properties of Earth's Outer Core

    Irving, J. C. E.; Cottaar, S.; Lekic, V.; Wu, W.

    2017-12-01

    Earth's outer core, the largest reservoir of metal in our planet, is comprised of an iron alloy of an uncertain composition. Its dynamical behaviour is responsible for the generation of Earth's magnetic field, with convection driven both by thermal and chemical buoyancy fluxes. Existing models of the seismic velocity and density of the outer core exhibit some variation, and there are only a small number of models which aim to represent the outer core's density.It is therefore important that we develop a better understanding of the physical properties of the outer core. Though most of the outer core is likely to be well mixed, it is possible that the uppermost outer core is stably stratified: it may be enriched in light elements released during the growth of the solid, iron enriched, inner core; by elements dissolved from the mantle into the outer core; or by exsolution of compounds previously dissolved in the liquid metal which will eventually be swept into the mantle. The stratified layer may host MAC or Rossby waves and it could impede communication between the chemically differentiated mantle and outer core, including screening out some of the geodynamo's signal. We use normal mode center frequencies to estimate the physical properties of the outer core in a Bayesian framework. We estimate the mineral physical parameters needed to best produce velocity and density models of the outer core which are consistent with the normal mode observations. We require that our models satisfy realistic physical constraints. We create models of the outer core with and without a distinct uppermost layer and assess the importance of this region.Our normal mode-derived models are compared with observations of body waves which travel through the outer core. In particular, we consider SmKS waves which are especially sensitive to the uppermost outer core and are therefore an important way to understand the robustness of our models.

  15. Overexpression of MicA induces production of OmpC-enriched outer membrane vesicles that protect against Salmonella challenge.

    Choi, Hyun-Il; Kim, Moonjeong; Jeon, Jinseong; Han, Jin Kwan; Kim, Kwang-Sun

    2017-08-26

    Outer membrane vesicles (OMVs) derived from bacteria are promising candidates for subunit vaccines. Stresses that modulate the composition of outer membrane proteins (OMPs) are important for OMV synthesis. Small RNAs (sRNAs) expressed in response to stress regulate OMPs, although the mechanism underlying sRNA-mediated OMV biogenesis and its utility for developing vaccine platforms remains to be elucidated. Here, we characterized the role of a sRNA, MicA, which regulates OmpA, a major OMP involved in both production of OMVs and reactive immunity against Salmonella challenge. A Salmonella strain overexpressing MicA generated more OMVs than a control strain. In addition, OmpC was the major component of MicA-derived OMV proteins. MicA-derived OMVs induced Th1- and Th17-type immune responses in vitro and reduced Salmonella-mediated lethality in a mouse model. Thus, OmpA-regulatory sRNA-derived OMVs may facilitate production of Salmonella-protective vaccines. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Employing Escherichia coli-derived outer membrane vesicles as an antigen delivery platform elicits protective immunity against Acinetobacter baumannii infection

    Huang, Weiwei; Wang, Shijie; Yao, Yufeng; Xia, Ye; Yang, Xu; Li, Kui; Sun, Pengyan; Liu, Cunbao; Sun, Wenjia; Bai, Hongmei; Chu, Xiaojie; Li, Yang; Ma, Yanbing

    2016-11-01

    Outer membrane vesicles (OMVs) have proven to be highly immunogenic and induced an immune response against bacterial infection in human clinics and animal models. We sought to investigate whether engineered OMVs can be a feasible antigen-delivery platform for efficiently inducing specific antibody responses. In this study, Omp22 (an outer membrane protein of A. baumannii) was displayed on E. coli DH5α-derived OMVs (Omp22-OMVs) using recombinant gene technology. The morphological features of Omp22-OMVs were similar to those of wild-type OMVs (wtOMVs). Immunization with Omp22-OMVs induced high titers of Omp22-specific antibodies. In a murine sepsis model, Omp22-OMV immunization significantly protected mice from lethal challenge with a clinically isolated A. baumannii strain, which was evidenced by the increased survival rate of the mice, the reduced bacterial burdens in the lung, spleen, liver, kidney, and blood, and the suppressed serum levels of inflammatory cytokines. In vitro opsonophagocytosis assays showed that antiserum collected from Omp22-OMV-immunized mice had bactericidal activity against clinical isolates, which was partly specific antibody-dependent. These results strongly indicated that engineered OMVs could display a whole heterologous protein (~22 kDa) on the surface and effectively induce specific antibody responses, and thus OMVs have the potential to be a feasible vaccine platform.

  17. Microsphere erosion in outer hydrogel membranes creating macroscopic porosity to counter biofouling-induced sensor degradation.

    Vaddiraju, S; Wang, Y; Qiang, L; Burgess, D J; Papadimitrakopoulos, F

    2012-10-16

    Biofouling and tissue inflammation present major challenges toward the realization of long-term implantable glucose sensors. Following sensor implantation, proteins and cells adsorb on sensor surfaces to not only inhibit glucose flux but also signal a cascade of inflammatory events that eventually lead to permeability-reducing fibrotic encapsulation. The use of drug-eluting hydrogels as outer sensor coatings has shown considerable promise to mitigate these problems via the localized delivery of tissue response modifiers to suppress inflammation and fibrosis, along with reducing protein and cell absorption. Biodegradable poly (lactic-co-glycolic) acid (PLGA) microspheres, encapsulated within a poly (vinyl alcohol) (PVA) hydrogel matrix, present a model coating where the localized delivery of the potent anti-inflammatory drug dexamethasone has been shown to suppress inflammation over a period of 1-3 months. Here, it is shown that the degradation of the PLGA microspheres provides an auxiliary venue to offset the negative effects of protein adsorption. This was realized by: (1) the creation of fresh porosity within the PVA hydrogel following microsphere degradation (which is sustained until the complete microsphere degradation) and (2) rigidification of the PVA hydrogel to prevent its complete collapse onto the newly created void space. Incubation of the coated sensors in phosphate buffered saline (PBS) led to a monotonic increase in glucose permeability (50%), with a corresponding enhancement in sensor sensitivity over a 1 month period. Incubation in serum resulted in biofouling and consequent clogging of the hydrogel microporosity. This, however, was partially offset by the generated macroscopic porosity following microsphere degradation. As a result of this, a 2-fold recovery in sensor sensitivity for devices with microsphere/hydrogel composite coatings was observed as opposed to similar devices with blank hydrogel coatings. These findings suggest that the use of

  18. Mechanism of protein import across the chloroplast envelope.

    Chen, K; Chen, X; Schnell, D J

    2000-01-01

    The development and maintenance of chloroplasts relies on the contribution of protein subunits from both plastid and nuclear genomes. Most chloroplast proteins are encoded by nuclear genes and are post-translationally imported into the organelle across the double membrane of the chloroplast envelope. Protein import into the chloroplast consists of two essential elements: the specific recognition of the targeting signals (transit sequences) of cytoplasmic preproteins by receptors at the outer envelope membrane and the subsequent translocation of preproteins simultaneously across the double membrane of the envelope. These processes are mediated via the co-ordinate action of protein translocon complexes in the outer (Toc apparatus) and inner (Tic apparatus) envelope membranes.

  19. Legal Provisions Applicable to the Definition of Outer Space

    Thorin, T.

    2002-01-01

    Whether it be the adjective "spatial" or the definition "space", these two terms have, in many respects, a non-identifiable dimension, which serves as a reference point for all players in this field, without being concerned with the exact area of application. This is evident from the vast diversity of corporate names, acronyms, logos and other designations that we often use. Among some of the most worldwide common include: NASA, ISS, ESA, and so on. Without of course forgetting , a field which concerns all legal experts and should not be overlooked is "space law". Thus, it is apparent that although the "space" community (i.e. influential and space- minded governments and relevant international authorities) has been involved in this field over the last few decades, no specific and universally-accepted definition has been adopted to date. Apart from certain demands made or unilateral positions taken by a given state particularly concerned by the matter, it is important to underline that the international community has refrained from making legislation in this area, apart from some rather limited or symbolic provisions introduced. This vagueness, in legal terms, should clearly be taken as the assertion of nationalistic demands, but also shows divergence or even antagonism between states fuelled by hypothetical profits, as was the case when attempts were made to establish maritime boundaries. We can thus by now summarise this issue by asking the following question: "Where does outer space begin?" We shall begin by looking at the sketchy legal references that we have at our disposal, which as lawyers we must use to attempt to find a solution to practical commercial or scientific contingencies which we are increasingly confronted with. Such references include the Treaty on Principles Governing the Activities of States in the Exploration and Use of Outer Space, Including the Moon and Other Celestial Bodies of 10th October 1967, constituting the fundamental space charter

  20. Pressure Gradients in the Inner Surf and Outer Swash Zone

    Kidwell, A.; Puleo, J. A.; Torres-Freyermuth, A.

    2010-12-01

    The swash zone is a highly dynamic region of the beach profile. Although there has been significant progression in understanding the complex hydrodynamics of the swash zone, an improvement in the understanding of the sediment transport mechanisms deserves further investigation. Prior studies have demonstrated that the existing formulations derived from the energetics-type formulation do not accurately and consistently predict sediment transport. Thus, measurements and numerical modeling can contribute in the improvement of the current predictive capability of sediment transport. A potential enhancement to nearshore sediment transport is the horizontal pressure gradient. However, measuring the dynamic pressure gradient in nearshore flows is a difficult task. For instance, standard pressure sensors are generally ill-suited for this type of measurement in shallow swash flows due to the obstructing size of the sensor and the potential for flow interference. With improved measurement apparati and techniques, it is possible to obtain measurements of the horizontal pressure gradient. Our current research includes laboratory and numerical model investigation of the horizontal pressure gradient in the inner surf and outer swash zone. An inexpensive differential pressure gauge is employed allowing for a pressure port on the order of 2 mm diameter. Four pressure sensor pairs are installed 1 cm above the bed with a cross-shore spacing of 8 cm. The sensors are deployed just outside of and at various locations within the outer swash zone to determine spatio-temporal pressure variations. The measurement of total pressure coupled with the corresponding free surface measurements from co-located capacitance wave gauges yields time series of the hydrostatic and dynamic pressure and pressure gradients. A VOF-type RANS model is employed in this investigation. Firstly, the numerical model is validated with swash measurements. Then, model simulations will be performed in order to