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Sample records for cell wall glycoproteins

  1. Cell wall proteome of Clostridium thermocellum and detection of glycoproteins.

    PubMed

    Yu, Tingting; Xu, Xinping; Peng, Yanfeng; Luo, Yuanming; Yang, Keqian

    2012-06-20

    Clostridium thermocellum, a thermophilic anaerobe, has the unusual capacity to convert cellulosic biomass into ethanol and hydrogen. In this work, the cell wall proteome of C. thermocellum was investigated. The proteins in the cell wall fraction of C. thermocellum prepared by the boiling SDS method were released by mutanolysin digestion and resolved on two-dimensional (2D) gel. One hundred and thirty-two proteins were identified by mass spectrometry, among which the extracellular solute-binding protein (CbpB/cthe_1020), enolase, glyceraldehyde-3-phosphate dehydrogenase and translation elongation factor EF-Tu were detected as highly abundant proteins. Besides the known surface localized proteins, including FtsZ, MinD, GroEL, DnaK, many enzymes involved in bioenergetics, such as alcohol dehydrogenases and hydrogenases were also detected. By glycan stain and MS analysis of glycopeptides, we identified CbpB as a glycoprotein, which is the second glycoprotein from C. thermocellum characterized. The fact that CbpB was highly abundant in the cell wall region and glycosylated, reflects its importance in substrate assimilation. Our results indicate cell wall proteins constitute a significant portion of cellular proteins and may play important physiological roles (i.e. bioenergetics) in this bacterium. The insights described are relevant for the development of C. thermocellum as a biofuel producer. PMID:22494898

  2. Cell wall O-glycoproteins and N-glycoproteins: aspects of biosynthesis and function.

    PubMed

    Nguema-Ona, Eric; Vicré-Gibouin, Maïté; Gotté, Maxime; Plancot, Barbara; Lerouge, Patrice; Bardor, Muriel; Driouich, Azeddine

    2014-01-01

    Cell wall O-glycoproteins and N-glycoproteins are two types of glycomolecules whose glycans are structurally complex. They are both assembled and modified within the endomembrane system, i.e., the endoplasmic reticulum (ER) and the Golgi apparatus, before their transport to their final locations within or outside the cell. In contrast to extensins (EXTs), the O-glycan chains of arabinogalactan proteins (AGPs) are highly heterogeneous consisting mostly of (i) a short oligo-arabinoside chain of three to four residues, and (ii) a larger β-1,3-linked galactan backbone with β-1,6-linked side chains containing galactose, arabinose and, often, fucose, rhamnose, or glucuronic acid. The fine structure of arabinogalactan chains varies between, and within plant species, and is important for the functional activities of the glycoproteins. With regards to N-glycans, ER-synthesizing events are highly conserved in all eukaryotes studied so far since they are essential for efficient protein folding. In contrast, evolutionary adaptation of N-glycan processing in the Golgi apparatus has given rise to a variety of organism-specific complex structures. Therefore, plant complex-type N-glycans contain specific glyco-epitopes such as core β,2-xylose, core α1,3-fucose residues, and Lewis(a) substitutions on the terminal position of the antenna. Like O-glycans, N-glycans of proteins are essential for their stability and function. Mutants affected in the glycan metabolic pathways have provided valuable information on the role of N-/O-glycoproteins in the control of growth, morphogenesis and adaptation to biotic and abiotic stresses. With regards to O-glycoproteins, only EXTs and AGPs are considered herein. The biosynthesis of these glycoproteins and functional aspects are presented and discussed in this review. PMID:25324850

  3. Cell wall O-glycoproteins and N-glycoproteins: aspects of biosynthesis and function

    PubMed Central

    Nguema-Ona, Eric; Vicré-Gibouin, Maïté; Gotté, Maxime; Plancot, Barbara; Lerouge, Patrice; Bardor, Muriel; Driouich, Azeddine

    2014-01-01

    Cell wall O-glycoproteins and N-glycoproteins are two types of glycomolecules whose glycans are structurally complex. They are both assembled and modified within the endomembrane system, i.e., the endoplasmic reticulum (ER) and the Golgi apparatus, before their transport to their final locations within or outside the cell. In contrast to extensins (EXTs), the O-glycan chains of arabinogalactan proteins (AGPs) are highly heterogeneous consisting mostly of (i) a short oligo-arabinoside chain of three to four residues, and (ii) a larger β-1,3-linked galactan backbone with β-1,6-linked side chains containing galactose, arabinose and, often, fucose, rhamnose, or glucuronic acid. The fine structure of arabinogalactan chains varies between, and within plant species, and is important for the functional activities of the glycoproteins. With regards to N-glycans, ER-synthesizing events are highly conserved in all eukaryotes studied so far since they are essential for efficient protein folding. In contrast, evolutionary adaptation of N-glycan processing in the Golgi apparatus has given rise to a variety of organism-specific complex structures. Therefore, plant complex-type N-glycans contain specific glyco-epitopes such as core β,2-xylose, core α1,3-fucose residues, and Lewisa substitutions on the terminal position of the antenna. Like O-glycans, N-glycans of proteins are essential for their stability and function. Mutants affected in the glycan metabolic pathways have provided valuable information on the role of N-/O-glycoproteins in the control of growth, morphogenesis and adaptation to biotic and abiotic stresses. With regards to O-glycoproteins, only EXTs and AGPs are considered herein. The biosynthesis of these glycoproteins and functional aspects are presented and discussed in this review. PMID:25324850

  4. The three-dimensional structure of the cell wall glycoprotein of Chlorogonium elongatum.

    PubMed

    Shaw, P J; Hills, G J

    1984-06-01

    The green alga Chlorogonium elongatum, a member of the Volvocales, possesses a crystalline cell wall composed of hydroxyproline-rich glycoprotein similar to the primary cell wall glycoproteins of higher plants. Electron microscopy and computer image processing have been used to determine the crystal structure of the Chlorogonium cell wall in three dimensions to a resolution of 2.0 nm. The structure is composed of heterologous dimers. Each subunit of the dimer comprises a long, thin spacer domain and a large globular domain, which is the site of the intra- and inter-dimer interactions. There are also sites of intersubunit interactions at the opposite ends of the rod domains. We suggest that the rods are composed predominantly of glycosylated polyproline helix, as has been suggested for higher plant cell wall glycoproteins and has been shown for the cell wall glycoprotein of Chlamydomonas reinhardtii, which is closely related to Chlorogonium. PMID:6490737

  5. [Hydroxyproline: Rich glycoproteins of the plant and cell wall

    SciTech Connect

    Varner, J.E.

    1993-01-01

    Since xylem tissue includes the main cell types which are lignified, we are interested in gene expression of glycine-rich proteins and proline-rich proteins, and other proteins which are involved in secondary cell wall thickening during xylogenesis. Since the main feature of xylogenesis is the deposition of additional wall components, study of the mechanism of xylogenesis will greatly advance our knowledge of the synthesis and assembly of wall macromolecules. We are using the in vitro xylogenesis system from isolated Zinnia mesophyll cells to isolate genes which are specifically expressed during xylogenesis. We have used subtractive hybridization methods to isolate a number of cDNA clones for differentially regulated genes from the cells after hormonal induction. So far, we have partially characterized 18 different cDNA clones from 239 positive clones. These differentially regulated genes can be divided into three sets according to the characteristics of gene expression in the induction medium and the control medium. The first set is induced in both the induction medium and the control medium without hormones. The second set is induced mainly in the induction medium and in the control medium with the addition of NAA alone. Two of thesegenes are exclusively induced by auxin. The third set of genes is induced mainly in the induction medium. Since these genes are not induced by either auxin or cytokinin alone, they may be directly involved in the process of xylogenesis. Our experiments on the localization of H[sub 2]O[sub 2] production reinforce the earlier ideas of others that H[sub 2]O[sub 2] is involved in normal lignification.

  6. The cell-wall glycoproteins of the green alga Scenedesmus obliquus. The predominant cell-wall polypeptide of Scenedesmus obliquus is related to the cell-wall glycoprotein gp3 of Chlamydomonas reinhardtii.

    PubMed

    Voigt, Jürgen; Stolarczyk, Adam; Zych, Maria; Malec, Przemysław; Burczyk, Jan

    2014-02-01

    The green alga Scenedesmus obliquus contains a multilayered cell wall, ultrastructurally similar to that of Chlamydomonas reinhardtii, although its proportion of hydroxyproline is considerably lower. Therefore, we have investigated the polypeptide composition of the insoluble and the chaotrope-soluble wall fractions of S. obliquus. The polypeptide pattern of the chaotrope-soluble wall fraction was strongly modified by chemical deglycosylation with anhydrous hydrogen fluoride (HF) in pyridine indicating that most of these polypeptides are glycosylated. Polypeptide constituents of the chaotrope-soluble cell-wall fraction with apparent molecular masses of 240, 270, 265, and 135 kDa cross-reacted with a polyclonal antibody raised against the 100 kDa deglycosylation product of the C. reinhardtii cell-wall glycoprotein GP3B. Chemical deglycosylation of the chaotrope-soluble wall fraction resulted in a 135 kDa major polypeptide and a 106 kDa minor component reacting with the same antibody. This antibody recognized specific peptide epitopes of GP3B. When the insoluble wall fraction of S. obliquus was treated with anhydrous HF/pyridine, three polypeptides with apparent molecular masses of 144, 135, and 65 kDa were solubilized, which also occured in the deglycosylated chaotrope-soluble wall fraction. These findings indicate that theses glycoproteins are cross-linked to the insoluble wall fraction via HF-sensitive bonds. PMID:24388513

  7. High-resolution electron microscopy of glycoproteins: the crystalline cell wall of Lobomonas.

    PubMed

    Roberts, K; Shaw, P J; Hills, G J

    1981-10-01

    Lobomonas piriformis is a member of an order of green algae (Volvocales) that have crystalline glycoprotein cell walls. As part of a program of investigation of these glycoproteins and their architecture we have studied the cell wall of Lobomonas by a variety of chemical, electron-microscopical and image-analysis techniques. Lobomonas and Vitreochlamys incisa show a very similar structure in their cell walls and represent I of the 4 classes into which all the structures of the wall of these algae that we have so far examined fall. The 2 classes that we have previously studied in detail, represented by Chlamydomonas reinhardii and chlorogonium elongatum, have a crystalline component of the wall that is a more or less smooth continuous surface overlying an amorphous inner wall layer. Although Lobomonas also has this 2-layer structure, the crystalline layer consists of distinct plates, each of which is built around a single, very coherent crystal lattice. The polar nature of the architecture of the cell wall is shown by sectioning and by examination of the cell-wall surface by metal-shadowing of carbon replicas, both of intact cells and of isolated cell-wall plates. There are great similarities in chemical composition between the glycoproteins of the cell wall of C. reinhardii and those of Lobomonas. Both has a large content of hydroxyproline in their amino acid composition and a sugar/hydroxyproline ratio of about 6.0, and both contain sugar sulphates. Lobomonas however has a large glucose content, whereas Chlamydamonas has almost none. Electron micrographs of walls stained with methylamine tungstate and shadowed specimens show that the Lobomonas crystal structure is entirely different from that of C. Reinhardii, and that there is a distinctly different structure in the centre of the plates from that at their edges, although the transition between the 2 areas occurs with no distortion of the crystal lattice. Computer image analysis has been used to calculate

  8. Identification of a highly conserved hydroxyproline-rich glycoprotein in the cell walls of Chlamydomonas reinhardtii and two other Volvocales.

    PubMed

    Adair, W S; Appel, H

    1989-10-01

    The unicellular alga Chlamydomonas reinhardtii Dang, has a cell wall made entirely from hydroxyproline-rich glycoproteins (HRGPs). We recently employed a quantiative in vitro reconstitution system (Adair et al. 1987, J. Cell Biol. 105, 2373-2382) to assign outer-wall HRGPs of C. reinhardtii to specific sublayers, and describe the major interactions responsible for their assembly. Some of these interactions appear to involve relatively conserved HRGP domains, as evidenced by interspecific cell-wall reconstitution between C. reinhardtii and two multicellular Volvocales (Volvoxcarteri lyengar and Gonium pectorale Müller). In the present report we provide biochemical and immunological evidence that the outer cell-walls of V. carteri and G. pectorale both contain prominent HRGPs closely related to C. reinhardtii GP2. Identification of conserved GP2 homologues indicates a molecular basis for interspecific reconstitution and provides a useful avenue for characterization of HRGP domains mediating cell-wall formation in these algae. PMID:24201668

  9. Cell wall glycoproteins at interaction sites between parasitic giant dodder (Cuscuta reflexa) and its host Pelargonium zonale.

    PubMed

    Striberny, Bernd; Krause, Kirsten

    2015-01-01

    The process of host plant penetration by parasitic dodder (genus Cuscuta) is accompanied by molecular and structural changes at the host/parasite interface. Recently, changes in pectin methyl esterification levels in the host cell walls abutting parasitic cells in established infection sites were reported. In addition to that, we show here that the composition of cell wall glycoproteins in Cuscuta-infected Pelargonium zonale undergoes substantial changes. While several arabinogalactan protein epitopes exhibit decreased abundances in the vicinity of the Cuscuta reflexa haustorium, extensins tend to increase in the infected areas. PMID:26367804

  10. Cell wall glycoproteins at interaction sites between parasitic giant dodder (Cuscuta reflexa) and its host Pelargonium zonale

    PubMed Central

    Striberny, Bernd; Krause, Kirsten

    2015-01-01

    The process of host plant penetration by parasitic dodder (genus Cuscuta) is accompanied by molecular and structural changes at the host/parasite interface. Recently, changes in pectin methyl esterification levels in the host cell walls abutting parasitic cells in established infection sites were reported. In addition to that, we show here that the composition of cell wall glycoproteins in Cuscuta-infected Pelargonium zonale undergoes substantial changes. While several arabinogalactan protein epitopes exhibit decreased abundances in the vicinity of the Cuscuta reflexa haustorium, extensins tend to increase in the infected areas. PMID:26367804

  11. Mass spectrometry-based proteomics of fungal wall glycoproteins.

    PubMed

    Yin, Qing Yuan; de Groot, Piet W J; de Koster, Chris G; Klis, Frans M

    2008-01-01

    The manifold functions of fungal wall glycoproteins include maintenance of cell wall integrity, homotypic and heterotypic adhesion, biofilm formation, acquisition of iron and sterols, protein degradation and coping with oxidative stress. Transcriptome studies indicate that the expression levels of most cell wall glycoproteins can vary widely and are tightly controlled. However, owing to their complex and variable glycosylation, fungal wall glycoproteins are difficult to analyze using traditional proteomics approaches. Recent advances in mass spectrometry-based proteomics have enabled rapid and sensitive identification and quantitation of fungal wall glycoproteins; this will be particularly useful for studying the dynamics of the subproteome of fungal wall glycoproteins, and for the development of novel vaccines and diagnostic tools. PMID:18096391

  12. (Hydroxyproline-rich glycoprotein of the plant cell wall): Report on work from June 1987 to June 1988

    SciTech Connect

    Not Available

    1988-01-01

    In soybean seed costs the accumulation of the hydroxproline-rich glycoprotein extensin is regulated in a developmental and tissue-specific manner. The time course of appearance of extensin during seed development was studied by Western blot analysis and by immunogold-silver localization. Using these techniques extensin was first detected at 16 to 18 d after anthesis, increasing during development to high levels at 24 d after anthesis. Immunogold-silver localization of extensin in the seed coat showed marked depostion of the glycoprotein in the walls of palisade epidermal cells and hourglass cells. The immunolocalization of extensin in developing soybean seeds was also made by a new technique - tissue printing on nitrocellulose paper. This technique shows that extensin is primarily localized in the seed coal, hilum, and vascular elements of the seed.

  13. The first biantennary bacterial secondary cell wall polymer and its influence on S-layer glycoprotein assembly.

    PubMed Central

    Steindl, Christian; Schäffer, Christina; Wugeditsch, Thomas; Graninger, Michael; Matecko, Irena; Müller, Norbert; Messner, Paul

    2002-01-01

    The cell surface of Aneurinibacillus thermoaerophilus DSM 10155 is covered with a square surface (S)-layer glycoprotein lattice. This S-layer glycoprotein, which was extracted with aqueous buffers after a freeze-thaw cycle of the bacterial cells, is the only completely water-soluble S-layer glycoprotein to be reported to date. The purified S-layer glycoprotein preparation had an overall carbohydrate content of 19%. Detailed chemical investigations indicated that the S-layer O-glycans of previously established structure accounted for 13% of total glycosylation. The remainder could be attributed to a peptidoglycan-associated secondary cell wall polymer. Structure analysis was performed using purified secondary cell wall polymer-peptidoglycan complexes. NMR spectroscopy revealed the first biantennary secondary cell wall polymer from the domain Bacteria, with the structure alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->4)-[alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)]-beta-L-Man p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->O)-PO(2)(-)-O-PO(2)(-)-(O-->6)-MurNAc- (where MurNAc is N -acetylmuramic acid). The neutral polysaccharide is linked via a pyrophosphate bond to the C-6 atom of every fourth N -acetylmuramic acid residue, in average, of the A1gamma-type peptidoglycan. In vivo, the biantennary polymer anchored the S-layer glycoprotein very effectively to the cell wall, probably due to the doubling of motifs for a proposed lectin-like binding between the polymer and the N-terminus of the S-layer protein. When the cellular support was removed during S-layer glycoprotein isolation, the co-purified polymer mediated the solubility of the S

  14. Carbohydrate-Dependent Binding of Langerin to SodC, a Cell Wall Glycoprotein of Mycobacterium leprae

    PubMed Central

    Kim, Hee Jin; Brennan, Patrick J.; Heaslip, Darragh; Udey, Mark C.; Modlin, Robert L.

    2014-01-01

    Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin-dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by two-dimensional (2D) Western blotting, and by surface plasmon resonance demonstrated that r-langerin possessed carbohydrate-dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose-capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin-reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate-dependent affinity of rSodC (equilibrium dissociation constant [KD] = 0.862 μM) that was 20-fold greater than for M. leprae ManLAM (KD = 18.69 μM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells. PMID:25422308

  15. Carbohydrate-dependent binding of langerin to SodC, a cell wall glycoprotein of Mycobacterium leprae.

    PubMed

    Kim, Hee Jin; Brennan, Patrick J; Heaslip, Darragh; Udey, Mark C; Modlin, Robert L; Belisle, John T

    2015-02-01

    Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin-dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by two-dimensional (2D) Western blotting, and by surface plasmon resonance demonstrated that r-langerin possessed carbohydrate-dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose-capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin-reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate-dependent affinity of rSodC (equilibrium dissociation constant [KD] = 0.862 μM) that was 20-fold greater than for M. leprae ManLAM (KD = 18.69 μM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells. PMID:25422308

  16. Mitochondrial Function in Cell Wall Glycoprotein Synthesis in Saccharomyces cerevisiae NCYC 625 (Wild Type) and [rho0] Mutants

    PubMed Central

    Iung, Annie Rakotoarivony; Coulon, Joël; Kiss, Ferenc; Ekome, Jacques Ngondi; Vallner, Judit; Bonaly, Roger

    1999-01-01

    We studied phosphopeptidomannans (PPMs) of two Saccharomyces cerevisiae NCYC 625 strains (S. diastaticus): a wild type strain grown aerobically, anaerobically, and in the presence of antimycin and a [rho0] mutant grown aerobically and anaerobically. The aerobic wild-type cultures were highly flocculent, but all others were weakly flocculent. Ligands implicated in flocculation of mutants or antimycin-treated cells were not aggregated as much by concanavalin A as were those of the wild type. The [rho0] mutants and antimycin-treated cells differ from the wild type in PPM composition and invertase, acid phosphatase, and glucoamylase activities. PPMs extracted from different cells differ in the protein but not in the glycosidic moiety. The PPMs were less stable in mitochondrion-deficient cells than in wild-type cells grown aerobically, and this difference may be attributable to defective mitochondrial function during cell wall synthesis. The reduced flocculation of cells grown in the presence of antimycin, under anaerobiosis, or carrying a [rho0] mutation may be the consequence of alterations of PPM structures which are the ligands of lectins, both involved in this cell-cell recognition phenomenon. These respiratory chain alterations also affect peripheral, biologically active glycoproteins such as extracellular enzymes and peripheral PPMs. PMID:10583995

  17. [Hydroxyproline: Rich glycoproteins of the plant and cell wall]. Annual technical progress report, 1993

    SciTech Connect

    Varner, J.E.

    1993-06-01

    Since xylem tissue includes the main cell types which are lignified, we are interested in gene expression of glycine-rich proteins and proline-rich proteins, and other proteins which are involved in secondary cell wall thickening during xylogenesis. Since the main feature of xylogenesis is the deposition of additional wall components, study of the mechanism of xylogenesis will greatly advance our knowledge of the synthesis and assembly of wall macromolecules. We are using the in vitro xylogenesis system from isolated Zinnia mesophyll cells to isolate genes which are specifically expressed during xylogenesis. We have used subtractive hybridization methods to isolate a number of cDNA clones for differentially regulated genes from the cells after hormonal induction. So far, we have partially characterized 18 different cDNA clones from 239 positive clones. These differentially regulated genes can be divided into three sets according to the characteristics of gene expression in the induction medium and the control medium. The first set is induced in both the induction medium and the control medium without hormones. The second set is induced mainly in the induction medium and in the control medium with the addition of NAA alone. Two of thesegenes are exclusively induced by auxin. The third set of genes is induced mainly in the induction medium. Since these genes are not induced by either auxin or cytokinin alone, they may be directly involved in the process of xylogenesis. Our experiments on the localization of H{sub 2}O{sub 2} production reinforce the earlier ideas of others that H{sub 2}O{sub 2} is involved in normal lignification.

  18. Tunicamycins: translocase-I inhibitors that target bacterial cell wall and mammalian N-glycoproteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tunicamycins, streptovirudins, and corynetoxins are natural products that target the biosynthesis of bacterial peptidoglycan and eukaryotic N-glycoproteins. The mechanism of action is known, with the tunicamycin-Mg**2+ complex established as a transition state analog for hexosamine-1-phosphate:pren...

  19. Unique N-Glycan Moieties of the 66-kDa Cell Wall Glycoprotein from the Red Microalga Porphyridium sp.

    PubMed Central

    Levy-Ontman, Oshrat; Arad, Shoshana (Malis); Harvey, David J.; Parsons, Thomas B.; Fairbanks, Antony; Tekoah, Yoram

    2011-01-01

    We report here the structural determination of the N-linked glycans in the 66-kDa glycoprotein, part of the unique sulfated complex cell wall polysaccharide of the red microalga Porphyridium sp. Structures were elucidated by a combination of normal phase/reverse phase HPLC, positive ion MALDI-TOF MS, negative ion electrospray ionization, and MS/MS. The sugar moieties of the glycoprotein consisted of at least four fractions of N-linked glycans, each composed of the same four monosaccharides, GlcNAc, Man, 6-O-MeMan, and Xyl, with compositions Man8–9Xyl1–2Me3GlcNAc2. The present study is the first report of N-glycans with the terminal Xyl attached to the 6-mannose branch of the 6-antenna and to the 3-oxygen of the penultimate (core) GlcNAc. Another novel finding was that all four glycans contain three O-methylmannose residues in positions that have never been reported before. Although it is known that some lower organisms are able to methylate terminal monosaccharides in glycans, the present study on Porphyridium sp. is the first describing an organism that is able to methylate non-terminal mannose residues. This study will thus contribute to understanding of N-glycosylation in algae and might shed light on the evolutionary development from prokaryotes to multicellular organisms. It also may contribute to our understanding of the red algae polysaccharide formation. The additional importance of this research lies in its potential for biotechnological applications, especially in evaluating the use of microalgae as cell factories for the production of therapeutic proteins. PMID:21515680

  20. An update on post-translational modifications of hydroxyproline-rich glycoproteins: toward a model highlighting their contribution to plant cell wall architecture

    PubMed Central

    Hijazi, May; Velasquez, Silvia M.; Jamet, Elisabeth; Estevez, José M.; Albenne, Cécile

    2014-01-01

    Plant cell walls are composite structures mainly composed of polysaccharides, also containing a large set of proteins involved in diverse functions such as growth, environmental sensing, signaling, and defense. Research on cell wall proteins (CWPs) is a challenging field since present knowledge of their role into the structure and function of cell walls is very incomplete. Among CWPs, hydroxyproline (Hyp)-rich O-glycoproteins (HRGPs) were classified into three categories: (i) moderately glycosylated extensins (EXTs) able to form covalent scaffolds; (ii) hyperglycosylated arabinogalactan proteins (AGPs); and (iii) Hyp/proline (Pro)-Rich proteins (H/PRPs) that may be non-, weakly- or highly-glycosylated. In this review, we provide a description of the main features of their post-translational modifications (PTMs), biosynthesis, structure, and function. We propose a new model integrating HRGPs and their partners in cell walls. Altogether, they could form a continuous glyco-network with non-cellulosic polysaccharides via covalent bonds or non-covalent interactions, thus strongly contributing to cell wall architecture. PMID:25177325

  1. Cell wall integrity

    PubMed Central

    Pogorelko, Gennady; Lionetti, Vincenzo; Bellincampi, Daniela; Zabotina, Olga

    2013-01-01

    The plant cell wall, a dynamic network of polysaccharides and glycoproteins of significant compositional and structural complexity, functions in plant growth, development and stress responses. In recent years, the existence of plant cell wall integrity (CWI) maintenance mechanisms has been demonstrated, but little is known about the signaling pathways involved, or their components. Examination of key mutants has shed light on the relationships between cell wall remodeling and plant cell responses, indicating a central role for the regulatory network that monitors and controls cell wall performance and integrity. In this review, we present a short overview of cell wall composition and discuss post-synthetic cell wall modification as a valuable approach for studying CWI perception and signaling pathways. PMID:23857352

  2. Honey Glycoproteins Containing Antimicrobial Peptides, Jelleins of the Major Royal Jelly Protein 1, Are Responsible for the Cell Wall Lytic and Bactericidal Activities of Honey

    PubMed Central

    Brudzynski, Katrina; Sjaarda, Calvin

    2015-01-01

    We have recently identified the bacterial cell wall as the cellular target for honey antibacterial compounds; however, the chemical nature of these compounds remained to be elucidated. Using Concavalin A- affinity chromatography, we found that isolated glycoprotein fractions (glps), but not flow-through fractions, exhibited strong growth inhibitory and bactericidal properties. The glps possessed two distinct functionalities: (a) specific binding and agglutination of bacterial cells, but not rat erythrocytes and (b) non-specific membrane permeabilization of both bacterial cells and erythrocytes. The isolated glps induced concentration- and time-dependent changes in the cell shape of both E. coli and B. subtilis as visualized by light and SEM microscopy. The appearance of filaments and spheroplasts correlated with growth inhibition and bactericidal effects, respectively. The time-kill kinetics showed a rapid, >5-log10 reduction of viable cells within 15 min incubation at 1xMBC, indicating that the glps-induced damage of the cell wall was lethal. Unexpectedly, MALDI-TOF and electrospray quadrupole time of flight mass spectrometry, (ESI-Q-TOF-MS/MS) analysis of glps showed sequence identity with the Major Royal Jelly Protein 1 (MRJP1) precursor that harbors three antimicrobial peptides: Jelleins 1, 2, and 4. The presence of high-mannose structures explained the lectin-like activity of MRJP1, while the presence of Jelleins in MRJP1 may explain cell wall disruptions. Thus, the observed damages induced by the MRJP1 to the bacterial cell wall constitute the mechanism by which the antibacterial effects were produced. Antibacterial activity of MRJP1 glps directly correlated with the overall antibacterial activity of honey, suggesting that it is honey’s active principle responsible for this activity. PMID:25830314

  3. Honey glycoproteins containing antimicrobial peptides, Jelleins of the Major Royal Jelly Protein 1, are responsible for the cell wall lytic and bactericidal activities of honey.

    PubMed

    Brudzynski, Katrina; Sjaarda, Calvin

    2015-01-01

    We have recently identified the bacterial cell wall as the cellular target for honey antibacterial compounds; however, the chemical nature of these compounds remained to be elucidated. Using Concavalin A-affinity chromatography, we found that isolated glycoprotein fractions (glps), but not flow-through fractions, exhibited strong growth inhibitory and bactericidal properties. The glps possessed two distinct functionalities: (a) specific binding and agglutination of bacterial cells, but not rat erythrocytes and (b) non-specific membrane permeabilization of both bacterial cells and erythrocytes. The isolated glps induced concentration- and time-dependent changes in the cell shape of both E. coli and B. subtilis as visualized by light and SEM microscopy. The appearance of filaments and spheroplasts correlated with growth inhibition and bactericidal effects, respectively. The time-kill kinetics showed a rapid, >5-log10 reduction of viable cells within 15 min incubation at 1xMBC, indicating that the glps-induced damage of the cell wall was lethal. Unexpectedly, MALDI-TOF and electrospray quadrupole time of flight mass spectrometry, (ESI-Q-TOF-MS/MS) analysis of glps showed sequence identity with the Major Royal Jelly Protein 1 (MRJP1) precursor that harbors three antimicrobial peptides: Jelleins 1, 2, and 4. The presence of high-mannose structures explained the lectin-like activity of MRJP1, while the presence of Jelleins in MRJP1 may explain cell wall disruptions. Thus, the observed damages induced by the MRJP1 to the bacterial cell wall constitute the mechanism by which the antibacterial effects were produced. Antibacterial activity of MRJP1 glps directly correlated with the overall antibacterial activity of honey, suggesting that it is honey's active principle responsible for this activity. PMID:25830314

  4. The cell wall of Fusarium oxysporum.

    PubMed

    Schoffelmeer, E A; Klis, F M; Sietsma, J H; Cornelissen, B J

    1999-01-01

    Sugar analysis of isolated cell walls from three formae speciales of Fusarium oxysporum showed that they contained not only glucose and (N-acetyl)-glucosamine, but also mannose, galactose, and uronic acids, presumably originating from cell wall glycoproteins. Cell wall glycoproteins accounted for 50-60% of the total mass of the wall. X-ray diffraction studies showed the presence of alpha-1, 3-glucan in the alkali-soluble cell wall fraction and of beta-1, 3-glucan and chitin in the alkali-insoluble fraction. Electron microscopy and lectin binding studies indicated that glycoproteins form an external layer covering an inner layer composed of chitin and glucan. PMID:10441453

  5. Tunicamycins: translocase-I inhibitors that target bacterial cell wall and mammalian N-glycoproteins. The potential for selective inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tunicamycins are a heterologous family of nucleoside antibiotics that target the biosynthesis of bacterial peptidoglycan and eukaryotic N-glycoproteins. The mechanism of action is known, with the tunicamycin-Mg2+ complex established as a transition state analog for hexosamine-1-phosphate: prenol pho...

  6. DIVERSITY IN THE DISTRIBUTION OF POLYSACCHARIDE AND GLYCOPROTEIN EPITOPES IN THE CELL WALLS OF BRYOPHYTES: NEW EVIDENCE FOR THE MULTIPLE EVOLUTION OF WATER-CONDUCTING CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bryophyte water conducting cells produce some of the most unusual wall ultrastructures of any plant. In fact, the only similar sorts of wall structures are produced by herbicidal treatment of higher plants with herbicides that inhibit cellulose biosynthesis. To determine if the similar sorts of st...

  7. Immune Response Induced by an Immunodominant 60 kDa Glycoprotein of the Cell Wall of Sporothrix schenckii in Two Mice Strains with Experimental Sporotrichosis

    PubMed Central

    Alba-Fierro, Carlos A.; Pérez-Torres, Armando; Toriello, Conchita; Pulido-Camarillo, Evelyn; Romo-Lozano, Yolanda; Gutiérrez-Sánchez, Gerardo

    2016-01-01

    Cell wall (CW) components of fungus Sporothrix schenckii are the major inductors antigens of immune responses. The immunodominant 60 kDa glycoprotein (gp60) has been shown to be associated with the virulence of this fungus but its role in experimental sporotrichosis is unknown. In this work, the immunological effects of CW-purified gp60 were investigated in a model of experimental subcutaneous sporotrichosis in normal and gp60-preimmunized C57BL/6 and BALB/c mice strains which were then infected with S. schenckii conidia. Results showed that both mice strains use different cytokine profiles in order to fight S. schenckii infection; C57BL/6 mice seem to use a Th17 response while BALB/c mice tend to depend on a Th1 profile. Preimmunization with gp60 showed a downregulatory effect on the immune response since cytokines levels were diminished in both strains. There were no significant differences in the magnitude of dorsoplantar inflammation between gp60-preimmunized and nonimmunized mice of both strains. However, skin lesions due to the infection in gp60-preimmunized mice were more severe in BALB/c than in C57BL/6 mice, suggesting that the antigen exerts a higher downregulatory effect on the Th1 response. PMID:27051673

  8. Immune Response Induced by an Immunodominant 60 kDa Glycoprotein of the Cell Wall of Sporothrix schenckii in Two Mice Strains with Experimental Sporotrichosis.

    PubMed

    Alba-Fierro, Carlos A; Pérez-Torres, Armando; Toriello, Conchita; Pulido-Camarillo, Evelyn; López-Romero, Everardo; Romo-Lozano, Yolanda; Gutiérrez-Sánchez, Gerardo; Ruiz-Baca, Estela

    2016-01-01

    Cell wall (CW) components of fungus Sporothrix schenckii are the major inductors antigens of immune responses. The immunodominant 60 kDa glycoprotein (gp60) has been shown to be associated with the virulence of this fungus but its role in experimental sporotrichosis is unknown. In this work, the immunological effects of CW-purified gp60 were investigated in a model of experimental subcutaneous sporotrichosis in normal and gp60-preimmunized C57BL/6 and BALB/c mice strains which were then infected with S. schenckii conidia. Results showed that both mice strains use different cytokine profiles in order to fight S. schenckii infection; C57BL/6 mice seem to use a Th17 response while BALB/c mice tend to depend on a Th1 profile. Preimmunization with gp60 showed a downregulatory effect on the immune response since cytokines levels were diminished in both strains. There were no significant differences in the magnitude of dorsoplantar inflammation between gp60-preimmunized and nonimmunized mice of both strains. However, skin lesions due to the infection in gp60-preimmunized mice were more severe in BALB/c than in C57BL/6 mice, suggesting that the antigen exerts a higher downregulatory effect on the Th1 response. PMID:27051673

  9. A workflow for large-scale empirical identification of cell wall N-linked glycoproteins of tomato (Solanum lycopersicum) fruit by tandem mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glycosylation is a common post-translational modification of plant proteins that impacts a large number of important biological processes. Nevertheless, the impacts of differential site occupancy and the nature of specific glycoforms are obscure. Historically, characterization of glycoproteins has b...

  10. The Lamportian cell wall

    SciTech Connect

    Keiliszewski, M.; Lamport, D. )

    1991-05-01

    The Lamportian Warp-Weft hypothesis suggests a cellulose-extensin interpenetrating network where extensin mechanically couples the load-bearing cellulose microfibrils in a wall matrix that is best described as a microcomposite. This model is based on data gathered from the extensin-rich walls of tomato and sycamore cell suspension culture, wherein extensin precursors are insolubilized into the wall by undefined crosslinks. The authors recent work with cell walls isolated from intact tissue as well as walls from suspension cultured cells of the graminaceous monocots maize and rice, the non-graminaceous monocot asparagus, the primitive herbaceous dicot sugar beet, and the gymnosperm Douglas Fir indicate that although extensins are ubiquitous to all plant species examined, they are not the major structural protein component of most walls examined. Amino acid analyses of intact and HF-treated walls shows a major component neither an HRGP, nor directly comparable to the glycine-rich wall proteins such as those associated with seed coat walls or the 67 mole% glycine-rich proteins cloned from petunia and soybean. Clearly, structural wall protein alternatives to extensin exist and any cell wall model must take that into account. If we assume that extracellular matrices are a priori network structures, then new Hypless' structural proteins in the maize cell wall raise questions about the sort of network these proteins create: the kinds of crosslinks involved; how they are formed; and the roles played by the small amounts of HRGPs.

  11. Monoclonal antibodies against plant cell wall polysaccharides

    SciTech Connect

    Hahn, M.G.; Bucheli, E.; Darvill, A.; Albersheim, P. )

    1989-04-01

    Monoclonal antibodies (McAbs) are useful tools to probe the structure of plant cell wall polysaccharides and to localize these polysaccharides in plant cells and tissues. Murine McAbs were generated against the pectic polysaccharide, rhamnogalacturonan I (RG-I), isolated from suspension-cultured sycamore cells. The McAbs that were obtained were grouped into three classes based upon their reactivities with a variety of plant polysaccharides and membrane glycoproteins. Eleven McAbs (Class I) recognize epitope(s) that appear to be immunodominant and are found in RG-I from sycamore and maize, citrus pectin, polygalacturonic acid, and membrane glycoproteins from suspension-cultured cells of sycamore, maize, tobacco, parsley, and soybean. A second group of five McAbs (Class II) recognize epitope(s) present in sycamore RG-I, but do not bind to any of the other polysaccharides or glycoproteins recognized by Class I. Lastly, one McAb (Class III) reacts with sycamore RG-I, sycamore and tamarind xyloglucan, and sycamore and rice glucuronoarabinoxylan, but does not bind to maize RG-I, polygalacturonic acid or the plant membrane glycoproteins recognized by Class I. McAbs in Classes II and III are likely to be useful in studies of the structure, biosynthesis and localization of plant cell wall polysaccharides.

  12. Cell wall remodeling under abiotic stress

    PubMed Central

    Tenhaken, Raimund

    2015-01-01

    Plants exposed to abiotic stress respond to unfavorable conditions on multiple levels. One challenge under drought stress is to reduce shoot growth while maintaining root growth, a process requiring differential cell wall synthesis and remodeling. Key players in this process are the formation of reactive oxygen species (ROS) and peroxidases, which initially cross-link phenolic compounds and glycoproteins of the cell walls causing stiffening. The function of ROS shifts after having converted all the peroxidase substrates in the cell wall. If ROS-levels remain high during prolonged stress, OH°-radicals are formed which lead to polymer cleavage. In concert with xyloglucan modifying enzymes and expansins, the resulting cell wall loosening allows further growth of stressed organs. PMID:25709610

  13. Boron bridging of rhamnogalacturonan-II is promoted in vitro by cationic chaperones, including polyhistidine and wall glycoproteins.

    PubMed

    Chormova, Dimitra; Fry, Stephen C

    2016-01-01

    Dimerization of rhamnogalacturonan-II (RG-II) via boron cross-links contributes to the assembly and biophysical properties of the cell wall. Pure RG-II is efficiently dimerized by boric acid (B(OH)3 ) in vitro only if nonbiological agents for example Pb(2+) are added. By contrast, newly synthesized RG-II domains dimerize very rapidly in vivo. We investigated biological agents that might enable this. We tested for three such agents: novel enzymes, borate-transferring ligands and cationic 'chaperones' that facilitate the close approach of two polyanionic RG-II molecules. Dimerization was monitored electrophoretically. Parsley shoot cell-wall enzymes did not affect RG-II dimerization in vitro. Borate-binding ligands (apiose, dehydroascorbic acid, alditols) and small organic cations (including polyamines) also lacked consistent effects. Polylysine bound permanently to RG-II, precluding electrophoretic analysis. However, another polycation, polyhistidine, strongly promoted RG-II dimerization by B(OH)3 without irreversible polyhistidine-RG-II complexation. Likewise, partially purified spinach extensins (histidine/lysine-rich cationic glycoproteins), strongly promoted RG-II dimerization by B(OH)3 in vitro. Thus certain polycations, including polyhistidine and wall glycoproteins, can chaperone RG-II, manoeuvring this polyanionic polysaccharide domain such that boron-bridging is favoured. These chaperones dissociate from RG-II after facilitating its dimerization, indicating that they act catalytically rather than stoichiometrically. We propose a natural role for extensin-RG-II interaction in steering cell-wall assembly. PMID:26301520

  14. Identification of the Abundant Hydroxyproline-Rich Glycoproteins in the Root Walls of Wild-Type Arabidopsis, an ext3 Mutant Line, and Its Phenotypic Revertant

    PubMed Central

    Chen, Yuning; Ye, Dening; Held, Michael A.; Cannon, Maura C.; Ray, Tui; Saha, Prasenjit; Frye, Alexandra N.; Mort, Andrew J.; Kieliszewski, Marcia J.

    2015-01-01

    Extensins are members of the cell wall hydroxyproline-rich glycoprotein (HRGP) superfamily that form covalently cross-linked networks in primary cell walls. A knockout mutation in EXT3 (AT1G21310), the gene coding EXTENSIN 3 (EXT3) in Arabidopsis Landsberg erecta resulted in a lethal phenotype, although about 20% of the knockout plants have an apparently normal phenotype (ANP). In this study the root cell wall HRGP components of wild-type, ANP and the ext3 mutant seedlings were characterized by peptide fractionation of trypsin digested anhydrous hydrogen fluoride deglycosylated wall residues and by sequencing using LC-MS/MS. Several HRGPs, including EXT3, were identified in the wild-type root walls but not in walls of the ANP and lethal mutant. Indeed the ANP walls and walls of mutants displaying the lethal phenotype possessed HRGPs, but the profiles suggest that changes in the amount and perhaps type may account for the corresponding phenotypes. PMID:27135319

  15. Identification of the Abundant Hydroxyproline-Rich Glycoproteins in the Root Walls of Wild-Type Arabidopsis, an ext3 Mutant Line, and Its Phenotypic Revertant.

    PubMed

    Chen, Yuning; Ye, Dening; Held, Michael A; Cannon, Maura C; Ray, Tui; Saha, Prasenjit; Frye, Alexandra N; Mort, Andrew J; Kieliszewski, Marcia J

    2015-01-01

    Extensins are members of the cell wall hydroxyproline-rich glycoprotein (HRGP) superfamily that form covalently cross-linked networks in primary cell walls. A knockout mutation in EXT3 (AT1G21310), the gene coding EXTENSIN 3 (EXT3) in Arabidopsis Landsberg erecta resulted in a lethal phenotype, although about 20% of the knockout plants have an apparently normal phenotype (ANP). In this study the root cell wall HRGP components of wild-type, ANP and the ext3 mutant seedlings were characterized by peptide fractionation of trypsin digested anhydrous hydrogen fluoride deglycosylated wall residues and by sequencing using LC-MS/MS. Several HRGPs, including EXT3, were identified in the wild-type root walls but not in walls of the ANP and lethal mutant. Indeed the ANP walls and walls of mutants displaying the lethal phenotype possessed HRGPs, but the profiles suggest that changes in the amount and perhaps type may account for the corresponding phenotypes. PMID:27135319

  16. The effect of ginger extract on glycoproteins of Raji cells.

    PubMed

    Zamani, Zahra; Nassir-Ud-Din; Kohan, Haleemeh Kabini; Kadivar, Mehdi; Kalyee, Zahra; Rad, Behzad Laame; Iravani, Ayda; Rahimi, Nourooz Ali; Wahabi, Farideh; Sadeghi, Sedigheh; Pourfallah, Fatemeh; Arjmand, Mohammad

    2014-01-15

    Protein glycosylation is associated with the development and progression of specific diseases, including cancers. The ginger rhizome is known to have anti-cancer and anti-fungal properties. This investigation was carried out to study the effect of ginger on glycoproteins of Raji cells. A 10% yield of ginger extract was mixed with 0.01% DMSO and added to 6 x 10(4) Raji cells at different concentrations for 24, 48 and 72 h at 37 degrees C. Their half maximal inhibitory concentration (IC50) was determined and analyzed statistically using Graphpad prism software. Cell extracts were prepared and their glycoproteins purified using lectin-affinity chromatography (Q proteome total glycoprotein and O glycoprotein kits) and SDS PAGE was carried out. IC50 of ginger extract on Raji cells was 20 microg mL(-1) at 72 h with < 0.01 significance. Silver staining of purified glycoprotiens in Raji cells indicated the presence of O-glycans and N-glycans. N-linked mannose and N-linked sialic acids were detected with the total glycoprotein kit. O-linked galactose and O-linked sialic acids were identified with the O-glycoprotein. Ginger reduced the expression of O-linked sialic acid and also N-linked mannose on Raji cells but had no effect on other glycoproteins. Sialic acid is now well known as a cancer marker and investigations are on to use it as a drug-target in cancerous tissues. PMID:24783808

  17. Evidence for mucin-like glycoproteins that tether sporozoites of Cryptosporidium parvum to the inner surface of the oocyst wall.

    PubMed

    Chatterjee, Anirban; Banerjee, Sulagna; Steffen, Martin; O'Connor, Roberta M; Ward, Honorine D; Robbins, Phillips W; Samuelson, John

    2010-01-01

    Cryptosporidium parvum oocysts, which are spread by the fecal-oral route, have a single, multilayered wall that surrounds four sporozoites, the invasive form. The C. parvum oocyst wall is labeled by the Maclura pomifera agglutinin (MPA), which binds GalNAc, and the C. parvum wall contains at least two unique proteins (Cryptosporidium oocyst wall protein 1 [COWP1] and COWP8) identified by monoclonal antibodies. C. parvum sporozoites have on their surface multiple mucin-like glycoproteins with Ser- and Thr-rich repeats (e.g., gp40 and gp900). Here we used ruthenium red staining and electron microscopy to demonstrate fibrils, which appear to attach or tether sporozoites to the inner surface of the C. parvum oocyst wall. When disconnected from the sporozoites, some of these fibrillar tethers appear to collapse into globules on the inner surface of oocyst walls. The most abundant proteins of purified oocyst walls, which are missing the tethers and outer veil, were COWP1, COWP6, and COWP8, while COWP2, COWP3, and COWP4 were present in trace amounts. In contrast, MPA affinity-purified glycoproteins from C. parvum oocysts, which are composed of walls and sporozoites, included previously identified mucin-like glycoproteins, a GalNAc-binding lectin, a Ser protease inhibitor, and several novel glycoproteins (C. parvum MPA affinity-purified glycoprotein 1 [CpMPA1] to CpMPA4). By immunoelectron microscopy (immuno-EM), we localized mucin-like glycoproteins (gp40 and gp900) to the ruthenium red-stained fibrils on the inner surface wall of oocysts, while antibodies to the O-linked GalNAc on glycoproteins were localized to the globules. These results suggest that mucin-like glycoproteins, which are associated with the sporozoite surface, may contribute to fibrils and/or globules that tether sporozoites to the inner surface of oocyst walls. PMID:19949049

  18. Bacterial Cell Wall Components

    NASA Astrophysics Data System (ADS)

    Ginsberg, Cynthia; Brown, Stephanie; Walker, Suzanne

    Bacterial cell-surface polysaccharides cells are surrounded by a variety of cell-surface structures that allow them to thrive in extreme environments. Components of the cell envelope and extracellular matrix are responsible for providing the cells with structural support, mediating intercellular communication, allowing the cells to move or to adhere to surfaces, protecting the cells from attack by antibiotics or the immune system, and facilitating the uptake of nutrients. Some of the most important cell wall components are polysaccharide structures. This review discusses the occurrence, structure, function, and biosynthesis of the most prevalent bacterial cell surface polysaccharides: peptidoglycan, lipopolysaccharide, arabinogalactan, and lipoarabinomannan, and capsular and extracellular polysaccharides. The roles of these polysaccharides in medicine, both as drug targets and as therapeutic agents, are also described.

  19. High efficiency labeling of glycoproteins on living cells

    PubMed Central

    Zeng, Ying; Ramya, T. N. C.; Dirksen, Anouk; Dawson, Philip E.; Paulson, James C.

    2010-01-01

    We describe a simple method for efficiently labeling cell surface glycans on virtually any living animal cell. The method employs mild Periodate oxidation to generate an aldehyde on sialic acids, followed by Aniline-catalyzed oxime Ligation with a suitable tag (PAL). Aniline catalysis dramatically accelerates oxime ligation, allowing use of low concentrations of aminooxy-biotin at neutral pH to label the majority of cell surface glycoproteins while maintaining high cell viability. PMID:19234450

  20. Cell surface display of chimeric glycoproteins via the S-layer of Paenibacillus alvei

    PubMed Central

    Zarschler, Kristof; Janesch, Bettina; Kainz, Birgit; Ristl, Robin; Messner, Paul; Schäffer, Christina

    2015-01-01

    The Gram-positive, mesophilic bacterium Paenibacillus alvei CCM 2051T possesses a two-dimensional crystalline protein surface layer (S-layer) with oblique lattice symmetry composed of a single type of O-glycoprotein species. Herein, we describe a strategy for nanopatterned in vivo cell surface co-display of peptide and glycan epitopes based on this S-layer glycoprotein self-assembly system. The open reading frame of the corresponding structural gene spaA codes for a protein of 983 amino acids, including a signal peptide of 24 amino acids. The mature S-layer protein has a theoretical molecular mass of 105.95 kDa and a calculated pI of 5.83. It contains three S-layer homology domains at the N-terminus that are involved in anchoring of the glycoprotein via a non-classical, pyruvylated secondary cell wall polymer to the peptidoglycan layer of the cell wall. For this polymer, several putative biosynthesis enzymes were identified upstream of the spaA gene. For in vivo cell surface display, the hexahistidine tag and the enhanced green fluorescent protein, respectively, were translationally fused to the C-terminus of SpaA. Immunoblot analysis, immunofluorescence staining, and fluorescence microscopy revealed that the fused epitopes were efficiently expressed and successfully displayed via the S-layer glycoprotein matrix on the surface of P. alvei CCM 2051T cells. In contrast, exclusively non-glycosylated chimeric SpaA proteins were displayed, when the S-layer of the glycosylation-deficient wsfP mutant was used as a display matrix. PMID:20513375

  1. Cell surface display of chimeric glycoproteins via the S-layer of Paenibacillus alvei.

    PubMed

    Zarschler, Kristof; Janesch, Bettina; Kainz, Birgit; Ristl, Robin; Messner, Paul; Schäffer, Christina

    2010-07-01

    The Gram-positive, mesophilic bacterium Paenibacillus alvei CCM 2051(T) possesses a two-dimensional crystalline protein surface layer (S-layer) with oblique lattice symmetry composed of a single type of O-glycoprotein species. Herein, we describe a strategy for nanopatterned in vivo cell surface co-display of peptide and glycan epitopes based on this S-layer glycoprotein self-assembly system. The open reading frame of the corresponding structural gene spaA codes for a protein of 983 amino acids, including a signal peptide of 24 amino acids. The mature S-layer protein has a theoretical molecular mass of 105.95kDa and a calculated pI of 5.83. It contains three S-layer homology domains at the N-terminus that are involved in anchoring of the glycoprotein via a non-classical, pyruvylated secondary cell wall polymer to the peptidoglycan layer of the cell wall. For this polymer, several putative biosynthesis enzymes were identified upstream of the spaA gene. For in vivo cell surface display, the hexahistidine tag and the enhanced green fluorescent protein, respectively, were translationally fused to the C-terminus of SpaA. Immunoblot analysis, immunofluorescence staining, and fluorescence microscopy revealed that the fused epitopes were efficiently expressed and successfully displayed via the S-layer glycoprotein matrix on the surface of P. alvei CCM 2051(T) cells. In contrast, exclusively non-glycosylated chimeric SpaA proteins were displayed, when the S-layer of the glycosylation-deficient wsfP mutant was used as a display matrix. PMID:20513375

  2. Synthesis of cell envelope glycoproteins of Cryptococcus laurentii.

    PubMed

    Schutzbach, John; Ankel, Helmut; Brockhausen, Inka

    2007-05-21

    Fungi of the genus Cryptococcus are encapsulated basidiomycetes that are ubiquitously found in the environment. These organisms infect both lower and higher animals. Human infections that are common in immune-compromised individuals have proven difficult to cure or even control with currently available antimycotics that are quite often toxic to the host. The virulence of Cryptococcus has been linked primarily to its polysaccharide capsule, but also to cell-bound glycoproteins. In this review, we show that Cryptococcus laurentii is an excellent model for studies of polysaccharide and glycoprotein synthesis in the more pathogenic relative C. neoformans. In particular, we will discuss the structure and biosynthesis of O-linked carbohydrates on cell envelope glycoproteins of C. laurentii. These O-linked structures are synthesized by at least four mannosyltransferases, two galactosyltransferases, and at least one xylosyltransferase that have been characterized. These glycosyltransferases have no known homologues in human tissues. Therefore, enzymes involved in the synthesis of cryptococcal glycoproteins, as well as related enzymes involved in capsule synthesis, are potential targets for the development of specific inhibitors for treatment of cryptococcal disease. PMID:17316583

  3. Cell wall proteomics of crops

    PubMed Central

    Komatsu, Setsuko; Yanagawa, Yuki

    2012-01-01

    Cell wall proteins play key roles in cell structure and metabolism, cell enlargement, signal transduction, responses to environmental stress, and many other physiological events. Agricultural crops are often used for investigating stress tolerance because cultivars with differing degrees of tolerance are available. Abiotic and biotic stress factors markedly influence the geographical distribution and yields of many crop species. Crop cell wall proteomics is of particular importance for improving crop productivity, particularly under unfavorable environmental conditions. To better understand the mechanisms underlying stress response in crops, cell wall proteomic analyses are being increasingly utilized. In this review, the methods of purification and purity assays of cell wall protein fractions from crops are described, and the results of protein identification using gel-based and gel-free proteomic techniques are presented. Furthermore, protein composition of the cell walls of rice, wheat, maize, and soybean are compared, and the role of cell wall proteins in crops under flooding and drought stress is discussed. This review will be useful for clarifying the role of the cell wall of crops in response to environmental stresses. PMID:23403621

  4. Glycoproteins: Occurrence and Significance

    NASA Astrophysics Data System (ADS)

    Wittmann, Valentin

    Protein glycosylation is regarded as the most complex form of post-translational modification leading to a heterogeneous expression of glycoproteins as mixtures of glycoforms. This chapter describes the structure and occurrence of glycoproteins with respect to their glycan chains. Discussed are different carbohydrate-peptide linkages including GPI anchors, common structures of N- and O-glycans, and the structure of glycosaminoglycans contained in proteoglycans. Also covered are the bacterial cell wall polymer peptidoglycan and the glycopeptide antibiotics of the vancomycin group. Properties and functions of the glycans contained in glycoproteins are dealt with in the next chapter of this book.

  5. Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

    PubMed Central

    Orlean, Peter

    2012-01-01

    The wall gives a Saccharomyces cerevisiae cell its osmotic integrity; defines cell shape during budding growth, mating, sporulation, and pseudohypha formation; and presents adhesive glycoproteins to other yeast cells. The wall consists of β1,3- and β1,6-glucans, a small amount of chitin, and many different proteins that may bear N- and O-linked glycans and a glycolipid anchor. These components become cross-linked in various ways to form higher-order complexes. Wall composition and degree of cross-linking vary during growth and development and change in response to cell wall stress. This article reviews wall biogenesis in vegetative cells, covering the structure of wall components and how they are cross-linked; the biosynthesis of N- and O-linked glycans, glycosylphosphatidylinositol membrane anchors, β1,3- and β1,6-linked glucans, and chitin; the reactions that cross-link wall components; and the possible functions of enzymatic and nonenzymatic cell wall proteins. PMID:23135325

  6. Plant cell walls to ethanol.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Conversion of plant cell walls to ethanol constitutes generation 2 bioethanol production. The process consists of several steps: biomass selection/genetic modification, physiochemical pretreatment, enzymatic saccharification, fermentation, and separation. Ultimately, it is desired to combine as man...

  7. Nucleated assembly of Chlamydomonas and Volvox cell walls.

    PubMed

    Adair, W S; Steinmetz, S A; Mattson, D M; Goodenough, U W; Heuser, J E

    1987-11-01

    The Chlamydomonas reinhardtii cell wall is made up of hydroxyproline-rich glycoproteins, arranged in five distinct layers. The W6 (crystalline) layer contains three major glycoproteins (GP1, GP2, GP3), selectively extractable with chaotropic agents, that self-assemble into crystals in vitro. A system to study W6 assembly in a quantitative fashion was developed that employs perchlorate-extracted Chlamydomonas cells as nucleating agents. Wall reconstitution by biotinylated W6 monomers was monitored by FITC-streptavidin fluorescence and quick-freeze/deep-etch electron microscopy. Optimal reconstitution was obtained at monomer concentrations (0.2-0.3 mg/ml) well below those required for nonnucleated assembly. Assembly occurred from multiple nucleation sites, and faithfully reflected the structure of the intact W6 layer. Specificity of nucleated assembly was demonstrated using two cell-wall mutants (cw-2 and cw-15); neither served as a substrate for assembly of wild-type monomers. In addition, W6 sublayers were assembled from purified components: GP2 and GP3 coassembled to form the inner (W6A) sublayer; this then served as a substrate for self-assembly of GP1 into the outer (W6B) sublayer. Finally, evolutionary relationships between C. reinhardtii and two additional members of the Volvocales (Chlamydomonas eugametos and Volvox carteri) were explored by performing interspecific reconstitutions. Hybrid walls were obtained between C. reinhardtii and Volvox but not with C. eugametos, confirming taxonomic assignments based on structural criteria. PMID:3680387

  8. Cell surface and secreted protein profiles of human thyroid cancer cell lines reveal distinct glycoprotein patterns.

    PubMed

    Arcinas, Arthur; Yen, Ten-Yang; Kebebew, Electron; Macher, Bruce A

    2009-08-01

    Cell surface proteins have been shown to be effective therapeutic targets. In addition, shed forms of these proteins and secreted proteins can serve as biomarkers for diseases, including cancer. Thus, identification of cell surface and secreted proteins has been a prime area of interest in the proteomics field. Most cell surface and secreted proteins are known to be glycosylated, and therefore, a proteomics strategy targeting these proteins was applied to obtain proteomic profiles from various thyroid cancer cell lines that represent the range of thyroid cancers of follicular cell origin. In this study, we oxidized the carbohydrates of secreted proteins and those on the cell surface with periodate and isolated them via covalent coupling to hydrazide resin. The glycoproteins obtained were identified from tryptic peptides and N-linked glycopeptides released from the hydrazide resin using two-dimensional liquid chromatography-tandem mass spectrometry in combination with the gas phase fractionation. Thyroid cancer cell lines derived from papillary thyroid cancer (TPC-1), follicular thyroid cancer (FTC-133), Hurthle cell carcinoma (XTC-1), and anaplastic thyroid cancer (ARO and DRO-1) were evaluated. An average of 150 glycoproteins were identified per cell line, of which more than 57% are known cell surface or secreted glycoproteins. The usefulness of the approach for identifying thyroid cancer associated biomarkers was validated by the identification of glycoproteins (e.g., CD44, galectin 3 and metalloproteinase inhibitor 1) that have been found to be useful markers for thyroid cancer. In addition to glycoproteins that are commonly expressed by all of the cell lines, we identified others that are only expressed in the more well-differentiated thyroid cancer cell lines (follicular, Hurthle cell and papillary), or by cell lines derived from undifferentiated tumors that are uniformly fatal forms of thyroid cancer (i.e., anaplastic). On the basis of the results obtained, a

  9. Expression of membrane glycoproteins in normal keratinocytes and squamous carcinoma cell lines

    SciTech Connect

    Rayter, Z. ); McIlhinney, R. ); Gusterson, B. )

    1989-08-01

    Con A acceptor glycoproteins were analyzed by 2D-PAGE and {sup 125}I-Con A overlay in three squamous carcinoma cell lines and compared with those in the simian virus (SV40)-transformed keratinocyte cell line SVK-14 and in normal keratinocytes. The majority of the glycoproteins identified by this technique were expressed at similar levels in all of the cells examined, independent of the culture conditions used. A cell surface glycoprotein gp34 was increased in the tumor cells compared with normal keratinocytes and expression varied with the culture density. Another glycoprotein, gp21, was found to be increased in expression in normal keratinocytes and stratified hyperconfluent cultures of squamous carcinoma cell lines. This paper describes the potential of this technique to identify membrane glycoproteins which may be expressed as a function of proliferation or differentiation.

  10. Back wall solar cell

    NASA Technical Reports Server (NTRS)

    Brandhorst, H. W., Jr. (Inventor)

    1978-01-01

    A solar cell is disclosed which comprises a first semiconductor material of one conductivity type with one face having the same conductivity type but more heavily doped to form a field region arranged to receive the radiant energy to be converted to electrical energy, and a layer of a second semiconductor material, preferably highly doped, of opposite conductivity type on the first semiconductor material adjacent the first semiconductor material at an interface remote from the heavily doped field region. Instead of the opposite conductivity layer, a metallic Schottky diode layer may be used, in which case no additional back contact is needed. A contact such as a gridded contact, previous to the radiant energy may be applied to the heavily doped field region of the more heavily doped, same conductivity material for its contact.

  11. Some factors affecting the production, by cultured baby-hamster kidney cells, of BHK glycoprotein I which cross-reacts immunologically with Tamm-Horsfall glycoprotein.

    PubMed Central

    Bloomfield, F J; Dunstan, D R; Foster, C L; Serafini-Cessi, F; Marshall, R D

    1977-01-01

    Cultured baby-hamster kidney cells (BHK-21/C13), which are adapted to grow in suspension (strain 2P), roduce a glycoprotein, termed BHK glycoprotein I, which cross-reacts immunologically with hamster urinary (Tamm-Horsfall glycoprotein. BHK glycoprotein I was isolated in an electrophoretically (sodium dodecyl sulphate/polyacrylamide gel) homogeneous form by application of affinity chromatography to the medium in which cells had been cultured. Insolubilized anti-(Tamm-Horsfall glycoprotein immunoglobulin G) was used as the adsorbent. The amount of BHK glycoprotein I associated with the cultured cells was found by both radioimmunoassay and immunofluorescence to be related to the amount of Ca2+ in the medium and to the particular stage of the cell cycle. 5'-Nucleotidase was also shed by the cells into the culture medium in amounts related to the stage of the cell cycle. The turnover of hamster Tamm-Horsfall glycoprotein in vivo appeared to be considerably more rapid than can be accounted for by cell turnover. Hamster Tamm-Horsfall glycoprotein was shown to be ineffective in inhibiting agglutination of chicken erythrocytes caused by influenza virus. Images PLATE 1 PLATE 2 PLATE 3 PLATE 4 PMID:328011

  12. Selective binding of human cumulus cell-secreted glycoproteins to human spermatozoa during capacitation in vitro

    SciTech Connect

    Tesarik, J.; Kopecny, V.; Dvorak, M.

    1984-06-01

    The results of this study demonstrate that glycoproteins manufactured by human cumulus cells can be detected bound to human spermatozoa incubated in capacitational medium containing the labeled cumulus-cell secretions. Cumulus-cell-secreted glycoproteins were labeled with a mixture of /sup 3/H-methionine and /sup 3/H-tryptophan or with 3H-fucose, and the binding of the labeled compounds to spermatozoa was evaluated by autoradiography. The binding was highly selective, involving only approximately 1% of the samples of spermatozoa used. The results suggest that the binding of cumulus-cell-secreted glycoproteins to spermatozoa may represent a final and highly selective step in human sperm capacitation.

  13. Structural studies of complex carbohydrates of plant cell walls. Progress report, September 1985-July 1986

    SciTech Connect

    Albersheim, P.

    1986-07-01

    The polymers that have been identified as components of cell walls include two hemicelluloses (xyloglucan and xylan), three pectine polysaccharides (homogalacturonan, rhamnogalacturonan I, and rhamnogalacturonan II, and hydroxproline- and glycine-rich glycoproteins. This report discribes the results of studies into the molecular structure if these plant polysaccharides. 5 figs.

  14. Catalysts of plant cell wall loosening

    PubMed Central

    Cosgrove, Daniel J.

    2016-01-01

    The growing cell wall in plants has conflicting requirements to be strong enough to withstand the high tensile forces generated by cell turgor pressure while selectively yielding to those forces to induce wall stress relaxation, leading to water uptake and polymer movements underlying cell wall expansion. In this article, I review emerging concepts of plant primary cell wall structure, the nature of wall extensibility and the action of expansins, family-9 and -12 endoglucanases, family-16 xyloglucan endotransglycosylase/hydrolase (XTH), and pectin methylesterases, and offer a critical assessment of their wall-loosening activity PMID:26918182

  15. Unique aspects of the grass cell wall

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Grasses are amongst the most important crops worldwide, and the composition of their cell walls is critical for uses as food, feed, and energy crops. Grass cell walls differ dramatically from dicot cell walls in terms of the major structural polysaccharides present, how those polysaccharides are lin...

  16. Glucocorticoid-Dependent Complementation of a Hepatoma Cell Variant Defective in Viral Glycoprotein Sorting

    NASA Astrophysics Data System (ADS)

    John, Nancy J.; Bravo, Deborah A.; Haffar, Omar K.; Firestone, Gary L.

    1988-02-01

    We have utilized the rat hepatoma (HTC) cell sorting variant CR4 to examine the glucocorticoid-regulated pathways that localize mouse mammary tumor virus glycoproteins to the cell surface. The defective sorting of cell surface mouse mammary tumor virus glycoproteins in CR4 cells was complemented after fusion with either normal rat hepatocytes or uninfected HTC cells. Indirect immunofluorescence of transient heterokaryons revealed that the regulated localization of mouse mammary tumor virus glycoproteins was dependent upon glucocorticoid treatment and required de novo RNA and protein synthesis. Thus, a glucocorticoid-regulated trafficking activity, unrelated to mouse mammary tumor virus sequences, which is induced in both adult rat liver and cultured hepatoma cells, can act in trans to mediate an intracellular sorting pathway for membrane glycoproteins.

  17. Localization of P-glycoprotein at the nuclear envelope of rat brain cells

    SciTech Connect

    Babakhanian, Karlo; Bendayan, Moise; Bendayan, Reina . E-mail: r.bendayan@utoronto.ca

    2007-09-21

    P-Glycoprotein is a plasma membrane drug efflux protein implicated in extrusion of cytotoxic compounds out of a cell. There is now evidence that suggests expression of this transporter at several subcellular sites, including the nucleus, mitochondria, and Golgi apparatus. This study investigated the localization and expression of P-glycoprotein at the nuclear membrane of rat brain microvessel endothelial (RBE4) and microglial (MLS-9) cell lines. Immunocytochemistry at the light and electron microscope levels using P-glycoprotein monoclonals antibodies demonstrated the localization of the protein at the nuclear envelope of RBE4 and MLS-9 cells. Western blot analysis revealed a single band of 170-kDa in purified nuclear membranes prepared from isolated nuclei of RBE4 and MLS-9 cells. These findings indicate that P-glycoprotein is expressed at the nuclear envelope of rat brain cells and suggest a role in multidrug resistance at this subcellular site.

  18. Glycoprotein E of Varicella-Zoster Virus Enhances Cell-Cell Contact in Polarized Epithelial Cells

    PubMed Central

    Mo, Chengjun; Schneeberger, Eveline E.; Arvin, Ann M.

    2000-01-01

    Varicella-zoster virus (VZV) infection involves the cell-cell spread of virions, but how viral proteins interact with the host cell membranes that comprise intercellular junctions is not known. Madin-Darby canine kidney (MDCK) cells were constructed to express the glycoproteins gE, gI, or gE/gI constitutively and were used to examine the effects of these VZV glycoproteins in polarized epithelial cells. At low cell density, VZV gE induced partial tight junction (TJ) formation under low-calcium conditions, whether expressed alone or with gI. Although most VZV gE was intracellular, gE was also shown to colocalize with the TJ protein ZO-1 with or without concomitant expression of gI. Freeze fracture electron microscopy revealed normal TJ strand morphology in gE-expressing MDCK cells. Functionally, the expression of gE was associated with a marked acceleration in the establishment of maximum transepithelial electrical resistance (TER) in MDCK-gE cells; MDCK-gI and MDCK-gE/gI cells exhibited a similar pattern of early TER compared to MDCK cells, although peak resistances were lower than those of gE alone. VZV gE expression altered F-actin organization and lipid distribution, but coexpression of gI modulated these effects. Two regions of the gE ectodomain, amino acids (aa) 278 to 355 and aa 467 to 498, although lacking Ca2+ binding motifs, exhibit similarities with corresponding regions of the cell adhesion molecules, E-cadherin and desmocollin. These observations suggest that VZV gE and gE/gI may contribute to viral pathogenesis by facilitating epithelial cell-cell contacts. PMID:11070038

  19. KRE5 Suppression Induces Cell Wall Stress and Alternative ER Stress Response Required for Maintaining Cell Wall Integrity in Candida glabrata

    PubMed Central

    Sasaki, Masato; Ito, Fumie; Aoyama, Toshio; Sato-Okamoto, Michiyo; Takahashi-Nakaguchi, Azusa; Chibana, Hiroji; Shibata, Nobuyuki

    2016-01-01

    The maintenance of cell wall integrity in fungi is required for normal cell growth, division, hyphae formation, and antifungal tolerance. We observed that endoplasmic reticulum stress regulated cell wall integrity in Candida glabrata, which possesses uniquely evolved mechanisms for unfolded protein response mechanisms. Tetracycline-mediated suppression of KRE5, which encodes a predicted UDP-glucose:glycoprotein glucosyltransferase localized in the endoplasmic reticulum, significantly increased cell wall chitin content and decreased cell wall β-1,6-glucan content. KRE5 repression induced endoplasmic reticulum stress-related gene expression and MAP kinase pathway activation, including Slt2p and Hog1p phosphorylation, through the cell wall integrity signaling pathway. Moreover, the calcineurin pathway negatively regulated cell wall integrity, but not the reduction of β-1,6-glucan content. These results indicate that KRE5 is required for maintaining both endoplasmic reticulum homeostasis and cell wall integrity, and that the calcineurin pathway acts as a regulator of chitin-glucan balance in the cell wall and as an alternative mediator of endoplasmic reticulum stress in C. glabrata. PMID:27548283

  20. KRE5 Suppression Induces Cell Wall Stress and Alternative ER Stress Response Required for Maintaining Cell Wall Integrity in Candida glabrata.

    PubMed

    Tanaka, Yutaka; Sasaki, Masato; Ito, Fumie; Aoyama, Toshio; Sato-Okamoto, Michiyo; Takahashi-Nakaguchi, Azusa; Chibana, Hiroji; Shibata, Nobuyuki

    2016-01-01

    The maintenance of cell wall integrity in fungi is required for normal cell growth, division, hyphae formation, and antifungal tolerance. We observed that endoplasmic reticulum stress regulated cell wall integrity in Candida glabrata, which possesses uniquely evolved mechanisms for unfolded protein response mechanisms. Tetracycline-mediated suppression of KRE5, which encodes a predicted UDP-glucose:glycoprotein glucosyltransferase localized in the endoplasmic reticulum, significantly increased cell wall chitin content and decreased cell wall β-1,6-glucan content. KRE5 repression induced endoplasmic reticulum stress-related gene expression and MAP kinase pathway activation, including Slt2p and Hog1p phosphorylation, through the cell wall integrity signaling pathway. Moreover, the calcineurin pathway negatively regulated cell wall integrity, but not the reduction of β-1,6-glucan content. These results indicate that KRE5 is required for maintaining both endoplasmic reticulum homeostasis and cell wall integrity, and that the calcineurin pathway acts as a regulator of chitin-glucan balance in the cell wall and as an alternative mediator of endoplasmic reticulum stress in C. glabrata. PMID:27548283

  1. Modifying crops to increase cell wall digestibility.

    PubMed

    Jung, Hans-Joachim G; Samac, Deborah A; Sarath, Gautam

    2012-04-01

    Improving digestibility of roughage cell walls will improve ruminant animal performance and reduce loss of nutrients to the environment. The main digestibility impediment for dicotyledonous plants is highly lignified secondary cell walls, notably in stem secondary xylem, which become almost non-digestible. Digestibility of grasses is slowed severely by lignification of most tissues, but these cell walls remain largely digestible. Cell wall lignification creates an access barrier to potentially digestible wall material by rumen bacteria if cells have not been physically ruptured. Traditional breeding has focused on increasing total dry matter digestibility rather than cell wall digestibility, which has resulted in minimal reductions in cell wall lignification. Brown midrib mutants in some annual grasses exhibit small reductions in lignin concentration and improved cell wall digestibility. Similarly, transgenic approaches down-regulating genes in monolignol synthesis have produced plants with reduced lignin content and improved cell wall digestibility. While major reductions in lignin concentration have been associated with poor plant fitness, smaller reductions in lignin provided measurable improvements in digestibility without significantly impacting agronomic fitness. Additional targets for genetic modification to enhance digestibility and improve roughages for use as biofuel feedstocks are discussed; including manipulating cell wall polysaccharide composition, novel lignin structures, reduced lignin/polysaccharide cross-linking, smaller lignin polymers, enhanced development of non-lignified tissues, and targeting specific cell types. Greater tissue specificity of transgene expression will be needed to maximize benefits while avoiding negative impacts on plant fitness.cauliflower mosiac virus (CaMV) 35S promoter. PMID:22325867

  2. Changes in Cell Wall Properties Coincide with Overexpression of Extensin Fusion Proteins in Suspension Cultured Tobacco Cells

    DOE PAGESBeta

    Tan, Li; Pu, Yunqiao; Pattathil, Sivakumar; Avci, Utku; Qian, Jin; Arter, Allison; Chen, Liwei; Hahn, Michael G.; Ragauskas, Arthur J.; Kieliszewski, Marcia J.

    2014-12-23

    Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp4 glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increasedmore » wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. In conclusion, these data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production.« less

  3. Changes in Cell Wall Properties Coincide with Overexpression of Extensin Fusion Proteins in Suspension Cultured Tobacco Cells

    SciTech Connect

    Tan, Li; Pu, Yunqiao; Pattathil, Sivakumar; Avci, Utku; Qian, Jin; Arter, Allison; Chen, Liwei; Hahn, Michael G.; Ragauskas, Arthur J.; Kieliszewski, Marcia J.

    2014-12-23

    Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp4 glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increased wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. In conclusion, these data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production.

  4. Changes in Cell Wall Properties Coincide with Overexpression of Extensin Fusion Proteins in Suspension Cultured Tobacco Cells

    PubMed Central

    Tan, Li; Avci, Utku; Qian, Jin; Arter, Allison; Chen, Liwei; Hahn, Michael G.; Ragauskas, Arthur J.; Kieliszewski, Marcia J.

    2014-01-01

    Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp4 glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increased wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. These data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production. PMID:25536327

  5. Changes in cell wall properties coincide with overexpression of extensin fusion proteins in suspension cultured tobacco cells.

    PubMed

    Tan, Li; Pu, Yunqiao; Pattathil, Sivakumar; Avci, Utku; Qian, Jin; Arter, Allison; Chen, Liwei; Hahn, Michael G; Ragauskas, Arthur J; Kieliszewski, Marcia J

    2014-01-01

    Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp4 glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increased wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. These data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production. PMID:25536327

  6. Moss cell walls: structure and biosynthesis

    PubMed Central

    Roberts, Alison W.; Roberts, Eric M.; Haigler, Candace H.

    2012-01-01

    The genome sequence of the moss Physcomitrella patens has stimulated new research examining the cell wall polysaccharides of mosses and the glycosyl transferases that synthesize them as a means to understand fundamental processes of cell wall biosynthesis and plant cell wall evolution. The cell walls of mosses and vascular plants are composed of the same classes of polysaccharides, but with differences in side chain composition and structure. Similarly, the genomes of P. patens and angiosperms encode the same families of cell wall glycosyl transferases, yet, in many cases these families have diversified independently in each lineage. Our understanding of land plant evolution could be enhanced by more complete knowledge of the relationships among glycosyl transferase functional diversification, cell wall structural and biochemical specialization, and the roles of cell walls in plant adaptation. As a foundation for these studies, we review the features of P. patens as an experimental system, analyses of cell wall composition in various moss species, recent studies that elucidate the structure and biosynthesis of cell wall polysaccharides in P. patens, and phylogenetic analysis of P. patens genes potentially involved in cell wall biosynthesis. PMID:22833752

  7. Architecture of dermatophyte cell Walls: Electron microscopic and biochemical analysis

    NASA Technical Reports Server (NTRS)

    Nozawa, Y.; Kitajima, Y.

    1984-01-01

    A review with 83 references on the cell wall structure of dermatophytes is presented. Topics discussed include separation and preparation of cell walls; microstructure of cell walls by electron microscopy; chemical composition of cell walls; structural model of cell walls; and morphological structure of cell walls.

  8. Production of cell surface and secreted glycoproteins in mammalian cells.

    PubMed

    Seiradake, Elena; Zhao, Yuguang; Lu, Weixian; Aricescu, A Radu; Jones, E Yvonne

    2015-01-01

    Mammalian protein expression systems are becoming increasingly popular for the production of eukaryotic secreted and cell surface proteins. Here we describe methods to produce recombinant proteins in adherent or suspension human embryonic kidney cell cultures, using transient transfection or stable cell lines. The protocols are easy to scale up and cost-efficient, making them suitable for protein crystallization projects and other applications that require high protein yields. PMID:25502196

  9. Glycoprotein isolated from Solanum nigrum L. kills HT-29 cells through apoptosis.

    PubMed

    Lim, Kye-Taek

    2005-01-01

    Solanum nigrum L. (SNL) has been used in folk medicine for its anti-inflammatory activity. We previously isolated glycoprotein from SNL and observed that it decreased viable HT-29 cell numbers at a low concentration (60 microg/mL). This study investigated the apoptotic signal pathway triggered by glycoprotein isolated from SNL in HT-29 cells. Treatment of HT-29 cells with SNL glycoprotein (60 microg/mL) for 4 hours resulted in a cytotoxic effect of more than 60%, compared with the control. To explain the apoptotic effects of SNL glycoprotein, we investigated its effects on 12-O-tetradecanoylphorbol 13-acetate (TPA)-stimulated protein kinase C (PKC) alpha activity and DNA-binding activity of nuclear factor (NF) kappaB in HT-29 cells, using western blot analysis and electrophoretic mobility shift assays. Results from these experiments showed that SNL glycoprotein has remarkable inhibitory effects on the activities of TPA (100 nM)-stimulated PKCalpha and NF-kappaB in HT-29 cells. They also substantiated that PKCalpha is a part of the TPA-activated upstream signal pathway of NF-kappaB, since NF-kappaB activity was inhibited by staurosporine (a PKC inhibitor) and pyrrolidine dithiocarbamate (an NF-kappaB inhibitor) in a western blot analysis. Furthermore, to verify the triggering of apoptosis by the SNL glycoprotein, we performed DNA fragmentation, nuclear staining, and protein expression assays of apoptotic-related proteins. The amount of DNA fragmentation and apoptotic cell numbers increased in a dose-dependent manner after treatment with SNL glycoprotein. Apoptosis-related protein assays demonstrated that SNL glycoprotein-induced apoptosis is associated with the regulation of bcl-2 and Bax expression. Taken together, the results of this study showed that the activation of PKCalpha, NF-kappaB, and Bax expression by SNL glycoprotein is possibly involved in the apoptotic process. Consequently, these results indicate that SNL glycoprotein causes HT-29 cell death through

  10. Distinct P-glycoprotein precursors are overproduced in independently isolated drug-resistant cell lines.

    PubMed

    Greenberger, L M; Lothstein, L; Williams, S S; Horwitz, S B

    1988-06-01

    A family of P-glycoproteins are overproduced in multidrug-resistant cells derived from the murine macrophage-like line J774.2. To determine whether individual family members are overproduced in response to different drugs, the P-glycoprotein precursors in several independently isolated cell lines, which were selected for resistance to vinblastine or taxol, were compared. Individual cell lines selected with vinblastine overproduced P-glycoprotein precursors of either 120 or 125 kDa. Taxol-selected cell lines overproduced either the 125-kDa precursor or both precursors simultaneously. Two similar but distinct peptide maps for the mature P-glycoproteins were observed. These maps corresponded to each precursor regardless of the drug used for selection. One vinblastine-resistant cell line switched from the 125- to the 120-kDa precursor when grown in increasing concentrations of drug. This change coincided with the overexpression of a distinct subset of mRNA species that code for P-glycoprotein. It is concluded that precursor expression is not drug-specific. These data suggest that individual overproduced P-glycoprotein family members are translated as distinct polypeptides. The results may help to explain the diversity in the multidrug-resistant phenotype. PMID:2897689

  11. Impact of Cell Wall Composition on Maize Resistance to Pests and Diseases

    PubMed Central

    Santiago, Rogelio; Barros-Rios, Jaime; Malvar, Rosa A.

    2013-01-01

    In cereals, the primary cell wall is built of a skeleton of cellulosic microfibrils embedded in a matrix of hemicelluloses and smaller amounts of pectins, glycoproteins and hydroxycinnamates. Later, during secondary wall development, p-coumaryl, coniferyl and sinapyl alcohols are copolymerized to form mixed lignins. Several of these cell wall components show a determinative role in maize resistance to pest and diseases. However, defense mechanisms are very complex and vary among the same plant species, different tissues or even the same tissue at different developmental stages. Thus, it is important to highlight that the role of the cell wall components needs to be tested in diverse genotypes and specific tissues where the feeding or attacking by the pathogen takes place. Understanding the role of cell wall constituents as defense mechanisms may allow modifications of crops to withstand pests and diseases. PMID:23535334

  12. Natural Paradigms of Plant Cell Wall Degradation

    SciTech Connect

    Wei, H.; Xu, Q.; Taylor, L. E.; Baker, J. O.; Tucker, M. P.; Ding, S. Y.

    2009-01-01

    Natural processes of recycling carbon from plant cell walls are slow but very efficient, generally involving microbial communities and their secreted enzymes. Efficient combinations of microbial communities and enzymes act in a sequential and synergistic manner to degrade plant cell walls. Recent understanding of plant cell wall ultra-structure, as well as the carbon metabolism, ATP production, and ecology of participating microbial communities, and the biochemical properties of their cellulolytic enzymes have led to new perspectives on saccharification of biomass. Microbial communities are dynamic functions of the chemical and structural compositions of plant cell wall components. The primitive 'multicellularity' exhibited by certain cellulolytic microorganisms may play a role in facilitating cell-cell communication and cell-plant cell wall-substrate interaction.

  13. Method of using alpha-1 acid glycoprotein on T-cells as a marker for alzheimer's disease

    SciTech Connect

    Fudenberg, H.H.

    1989-01-31

    A method is described of diagnosing a dementia of the Alzheimer's type characterized by a change in the percentage of T-cells bearing surface membrane alpha-1 acid glycoprotein which comprises providing T-cells from a subject, determining the percentage of those T cells which bear surface membrane alpha-1 acid glycoprotein, and comparing that percentage of the percentage of T cells which bear the glycoprotein in a control, whereby the dementia is diagnosed.

  14. Growth and metabolism of fucosylated plasma-membrane glycoproteins in mouse neuroblastoma N2a cells

    PubMed Central

    Milenkovic, Ada G.; Rachmeler, Martin; Johnson, Terry C.

    1978-01-01

    The presence of 1.0mm-dibutyryl cyclic AMP (N6,O2′-dibutyryladenosine 3′:5′-cyclic monophosphate) and 1.5mm-theophylline completely inhibits the growth of mouse neuroblastoma N2a cells by 24–36h. When compared with N2a cultures without inhibitors (controls), the proportion of cells in S phase, measured by radioautography with [3H]-thymidine, was decreased from 55 to 12%. In addition, the presence of the inhibitors decreased apparent [3H]fucose incorporation into glycoproteins by 50%, and removing the inhibitors resulted in a rapid recovery of both DNA synthesis and glycoprotein metabolism. Measurement of intracellular acid-soluble radioactive fucose revealed that decreased fucose uptake could account for the apparent change in incorporation. Removing dibutyryl cyclic AMP and theophylline from the medium resulted in a rapid uptake of radioactive fucose to within control values, which illustrated that the inhibitors decreased transport of the carbohydrate, although the cells remained viable. Treatment with dibutyryl cyclic AMP and theophylline also reversibly inhibited glycoprotein degradation. Plasma membranes isolated from growing cells and from growth-inhibited cells labelled with [14C]fucose and [3H]fucose respectively were co-electrophoresed on sodium dodecyl sulphate/polyacrylamide gels. These displayed no apparent differences in synthesis of specific membrane glycoproteins. Electrophoresis of plasma membranes isolated from cultures pulse–chased with [14C]fucose and [3H]fucose was used to discern turnover patterns of specific plasma-membrane glycoproteins. High-molecular-weight glycoproteins exhibited rapid rates of turnover in membranes from growing cells, but moderate turnover rates in growth-inhibited cells and cells reversed from growth inhibition. These data indicate that growth arrest of N2a cells results in alterations in the metabolic turnover of plasma-membrane glycoproteins. PMID:218551

  15. How do plant cell walls extend?

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1993-01-01

    This article briefly summarizes recent work that identifies the biophysical and biochemical processes that give rise to the extension of plant cell walls. I begin with the biophysical notion of stress relaxation of the wall and follow with recent studies of wall enzymes thought to catalyze wall extension and relaxation. Readers should refer to detailed reviews for more comprehensive discussion of earlier literature (Taiz, 1984; Carpita and Gibeaut, 1993; Cosgrove, 1993).

  16. Fluorescent Labeling of Yeast Cell Wall Components.

    PubMed

    Okada, Hiroki; Ohya, Yoshikazu

    2016-01-01

    Yeast cells stained with a fluorescent dye that specifically binds to one of the cell wall components can be observed under a fluorescent microscope. Visualization of the components 1,3-β-glucan, mannoproteins, and/or chitin not only provides information concerning the cell wall, but also reveals clues about various cellular activities such as cell polarity, vesicular transport, establishment of budding pattern, apical and isotropic bud growth, and replicative cell age. This protocol describes a standard method for visualizing different components of the yeast cell wall. PMID:27480714

  17. Identification of a Streptococcus salivarius Cell Wall Component Mediating Coaggregation with Veillonella alcalescens VI

    PubMed Central

    Weerkamp, Anton H.; McBride, Barry C.

    1981-01-01

    Cell walls of Streptococcus salivarius HB aggregated Veillonella alcalescens V1, but cell walls of the mutant S. salivarius HB-V5 did not. We found no correlation between the presence of fimbriae on streptococcal walls and the ability to aggregate Veillonella strains. Treatment of the walls with lysozyme solubilized a fraction which possessed Veillonella-aggregating activity. Solubilized cell wall preparations of strain HB contained three major (glyco)proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at least four antigens as determined by immunoelectrophoresis with antiserum prepared against strain HB walls. A specific antiserum, which was obtained by adsorption of anti-HB serum on strain HB-V5 cells, contained monospecific antibody that reacted with the solubilized strain HB wall preparation. Similar fractions prepared from strain HB-V5 cell walls did not possess aggregating activity and lacked one protein band (protein I) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and one antigen (antigen b) after immunoelectrophoresis. The same antigen was absent when lysozyme-solubilized wall preparations of strain HB were reacted with anti-HB-V5 serum. Crossed-immunoisoelectric focusing indicated that this specific (glyco)protein and this antigen were identical and had an isoelectric point of 4.60. Protein I and antigen b were specifically adsorbed when solubilized strain HB cell walls were incubated with V. alcalescens V1 but were not adsorbed by nonaggregating Veillonella parvula ATCC 10790 cells. Culture supernatants of strain HB contained V. alcalescens V1-aggregating activity. Antigen b was present in the culture supernatant, but was not found in cultures of strain HB-V5. A total of 18 S. salivarius isolates possessing the streptococcal group K antigen released aggregating activity and antigen b into the culture medium, but 11 strains which lacked the K-antigen did not. Images PMID:7251145

  18. Cell Wall Remodeling Enzymes Modulate Fungal Cell Wall Elasticity and Osmotic Stress Resistance

    PubMed Central

    Ene, Iuliana V.; Walker, Louise A.; Schiavone, Marion; Lee, Keunsook K.; Martin-Yken, Hélène; Dague, Etienne; Gow, Neil A. R.; Munro, Carol A.

    2015-01-01

    ABSTRACT The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock. PMID:26220968

  19. Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry

    SciTech Connect

    Follis, Kathryn E.; York, Joanne; Nunberg, Jack H. . E-mail: jack.nunberg@umontana.edu

    2006-07-05

    The fusogenic potential of Class I viral envelope glycoproteins is activated by proteloytic cleavage of the precursor glycoprotein to generate the mature receptor-binding and transmembrane fusion subunits. Although the coronavirus (CoV) S glycoproteins share membership in this class of envelope glycoproteins, cleavage to generate the respective S1 and S2 subunits appears absent in a subset of CoV species, including that responsible for the severe acute respiratory syndrome (SARS). To determine whether proteolytic cleavage of the S glycoprotein might be important for the newly emerged SARS-CoV, we introduced a furin recognition site at single basic residues within the putative S1-S2 junctional region. We show that furin cleavage at the modified R667 position generates discrete S1 and S2 subunits and potentiates membrane fusion activity. This effect on the cell-cell fusion activity by the S glycoprotein is not, however, reflected in the infectivity of pseudotyped lentiviruses bearing the cleaved glycoprotein. The lack of effect of furin cleavage on virion infectivity mirrors that observed in the normally cleaved S glycoprotein of the murine coronavirus and highlights an additional level of complexity in coronavirus entry.

  20. EXPRESSION OF THE MAIZE MOSAIC VIRUS GLYCOPROTEIN IN INSECT CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize mosaic virus (genus Nucleorhabdovirus, family Rhabdoviridae) is transmitted in a persistent-propagative manner by Peregrinus maidis, the corn planthopper. Like other rhabdoviruses, the MMV genome encodes a surface glycoprotein that is likely involved in virus attachment and entry into host ce...

  1. An unusual dependence of human herpesvirus-8 glycoproteins-induced cell-to-cell fusion on heparan sulfate

    SciTech Connect

    Tiwari, Vaibhav; Darmani, Nissar A.; Thrush, Gerald R.; Shukla, Deepak

    2009-12-18

    Human herpesvirus-8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins-induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: (1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; (2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, (3) co-expression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis.

  2. Modifying crops to increase cell wall digestibility

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Improving digestibility of roughage cell walls will improve ruminant animal performance and reduce loss of nutrients to the environment. The main digestibility impediment for dicotyledonous plants are highly lignified secondary cell walls, notably in stem secondary xylem, which become almost non-dig...

  3. Altered intracellular pH regulation in cells with high levels of P-glycoprotein expression.

    PubMed

    Young, Gregory; Reuss, Luis; Altenberg, Guillermo A

    2011-01-01

    P-glycoprotein is an ATP-binding-cassette transporter that pumps many structurally unrelated drugs out of cells through an ATP-dependent mechanism. As a result, multidrug-resistant cells that overexpress P-glycoprotein have reduced intracellular steady-state levels of a variety of chemotherapeutic agents. In addition, increased cytosolic pH has been a frequent finding in multidrug-resistant cells that express P-glycoprotein, and it has been proposed that this consequence of P-glycoprotein expression may contribute to the lower intracellular levels of chemotherapeutic agents. In these studies, we measured intracellular pH and the rate of acid extrusion in response to an acid load in two cells with very different levels of P-glycoprotein expression: V79 parental cells and LZ-8 multidrug resistant cells. Compared to the wild-type V79 cells, LZ-8 cells have a lower intracellular pH and a slower recovery of intracellular pH after an acid load. The data also show that LZ-8 cells have reduced ability to extrude acid, probably due to a decrease in Na(+)/H(+) exchanger activity. The alterations in intracellular pH and acid extrusion in LZ-8 cells are reversed by 24-h exposure to the multidrug-resistance modulator verapamil. The lower intracellular pH in LZ-8 indicates that intracellular alkalinization is not necessary for multidrug resistance. The reversal by verapamil of the decreased acid-extrusion suggests that P-glycoprotein can affect other membrane transport mechanism. PMID:22003434

  4. Requirements for Cell Rounding and Surface Protein Down-Regulation by Ebola Virus Glycoprotein

    PubMed Central

    Francica, Joseph R.; Matukonis, Meghan K.; Bates, Paul

    2009-01-01

    Ebola virus causes an acute hemorrhagic fever that is associated with high morbidity and mortality. The viral glycoprotein is thought to contribute to pathogenesis, though precise mechanisms are unknown. Cellular pathogenesis can be modeled in vitro by expression of the Ebola viral glycoprotein (GP) in cells, which causes dramatic morphological changes, including cell rounding and surface protein down-regulation. These effects are known to be dependent on the presence of a highly glycosylated region of the glycoprotein, the mucin domain. Here we show that the mucin domain from the highly pathogenic Zaire subtype of Ebola virus is sufficient to cause characteristic cytopathology when expressed in the context of a foreign glycoprotein. Similarly to full length Ebola GP, expression of the mucin domain causes rounding, detachment from the extracellular matrix, and the down-regulation of cell surface levels of β1 integrin and major histocompatibility complex class 1. These effects were not seen when the mucin domain was expressed in the context of a glycophosphatidylinositol-anchored isoform of the foreign glycoprotein. In contrast to earlier analysis of full length Ebola glycoproteins, chimeras carrying the mucin domains from the Zaire and Reston strains appear to cause similar levels of down-modulation and cell detachment. Cytopathology associated with Ebola glycoprotein expression does not occur when GP expression is restricted to the endoplasmic reticulum. In contrast to a previously published report, our results demonstrate that GP-induced surface protein down-regulation is not mediated through a dynamin-dependent pathway. Overall, these results support a model in which the mucin domain of Ebola GP acts at the cell surface to induce protein down modulation and cytopathic effects. PMID:19013626

  5. Phosphatidylinositol-anchored glycoproteins of PC12 pheochromocytoma cells and brain

    SciTech Connect

    Margolis, R.K.; Goossen, B.; Margolis, R.U.

    1988-05-03

    PC12 pheochromocytoma cells and cultures of early postnatal rat cerebellium were labeled with (/sup 3/H)glucosamine, (/sup 3/H)fucose, (/sup 3/H)leucine, (/sup 3/H)ethanolamine, or sodium (/sup 35/S)sulfate and treated with a phosphatidylinositol-specific phospholipase C. Enzyme treatment of (/sup 3/H) glucosamine- or (/sup 3/H)fucose-labeled PC12 cells led to a 15-fold increase in released glycoproteins. On sodium dodecyl sulfate-polyacrylamide gel ectrophoresis, most of the released material migrated as a broad band with an apparent molecular size of 32,000 daltons (Da), which was specifically immunoprecipitated by a monoclonal antibody to the Thy-l glycoprotein. A second glycoprotein, with an apparent molecular size of 158,000 Da, was also released. After treatment with endo-..beta..-galactosidase, 40-45% of the (/sup 3/H)glucosamine of (/sup 3/H)fucose radioactivity in the phospholipase-released glycoproteins was converted to products of disaccharide size, and the molecular size of the 158-kDa glycoprotein decreased to 145 kDa, demonstrating that it contains fucosylated poly-(N-acetyllactosaminyl) oligosaccharides. The phospholipase also released labeled Thy-1 and the 158-kDa glycoprotein from PC12 cells cultured in the presence of (/sup 3/H)ethanolamine, which specifically labels this component of the phosphatidylinositol membrane-anchoring sequence,while in the lipid-free protein residue of cells not treated with phospholipase, Thy-1 and a doublet at 46/48 kDa were the only labeled proteins. Sulfated glycoproteins of 155, 132/134, 61, and 21 kDa are the predominant species released by phospholipase, which does not affect a major 44-kDa protein seen in (/sup 3/H)ethanolamine-labeled brain cultures. The 44-48- and 155/158-kDa proteins may be common to both PC12 cells and brain.

  6. Safranine fluorescent staining of wood cell walls.

    PubMed

    Bond, J; Donaldson, L; Hill, S; Hitchcock, K

    2008-06-01

    Safranine is an azo dye commonly used for plant microscopy, especially as a stain for lignified tissues such as xylem. Safranine fluorescently labels the wood cell wall, producing green/yellow fluorescence in the secondary cell wall and red/orange fluorescence in the middle lamella (ML) region. We examined the fluorescence behavior of safranine under blue light excitation using a variety of wood- and fiber-based samples of known composition to interpret the observed color differentiation of different cell wall types. We also examined the basis for the differences in fluorescence emission using spectral confocal microscopy to examine lignin-rich and cellulose-rich cell walls including reaction wood and decayed wood compared to normal wood. Our results indicate that lignin-rich cell walls, such as the ML of tracheids, the secondary wall of compression wood tracheids, and wood decayed by brown rot, tend to fluoresce red or orange, while cellulose-rich cell walls such as resin canals, wood decayed by white rot, cotton fibers and the G-layer of tension wood fibers, tend to fluoresce green/yellow. This variation in fluorescence emission seems to be due to factors including an emission shift toward red wavelengths combined with dye quenching at shorter wavelengths in regions with high lignin content. Safranine fluorescence provides a useful way to differentiate lignin-rich and cellulose-rich cell walls without counterstaining as required for bright field microscopy. PMID:18802812

  7. Shape dynamics of growing cell walls.

    PubMed

    Banerjee, Shiladitya; Scherer, Norbert F; Dinner, Aaron R

    2016-04-14

    We introduce a general theoretical framework to study the shape dynamics of actively growing and remodeling surfaces. Using this framework we develop a physical model for growing bacterial cell walls and study the interplay of cell shape with the dynamics of growth and constriction. The model allows us to derive constraints on cell wall mechanical energy based on the observed dynamics of cell shape. We predict that exponential growth in cell size requires a constant amount of cell wall energy to be dissipated per unit volume. We use the model to understand and contrast growth in bacteria with different shapes such as spherical, ellipsoidal, cylindrical and toroidal morphologies. Coupling growth to cell wall constriction, we predict a discontinuous shape transformation, from partial constriction to cell division, as a function of the chemical potential driving cell wall synthesis. Our model for cell wall energy and shape dynamics relates growth kinetics with cell geometry, and provides a unified framework to describe the interplay between shape, growth and division in bacterial cells. PMID:26953519

  8. Effect of the ionophore monensin on herpes simplex virus type 1-induced cell fusion, glycoprotein synthesis, and virion infectivity.

    PubMed

    Kousoulas, K G; Bzik, D J; Person, S

    1983-01-01

    The ionophore monensin inhibited the formation of mature, fully glycosylated glycoproteins gB, gC, and gD during herpes simplex virus type 1 infection of human embryonic lung cells. Underglycosylated forms, including the apparent high-mannose precursor forms of the major glycoproteins, appeared. Monensin inhibited virus-induced cell fusion. Infectious virions produced in the presence of monensin appeared to contain predominantly underglycosylated glycoproteins. PMID:6307921

  9. Binding partners for the myelin-associated glycoprotein of N2A neuroblastoma cells.

    PubMed

    Strenge, K; Schauer, R; Kelm, S

    1999-02-01

    The myelin-associated glycoprotein (MAG) has been proposed to be important for the integrity of myelinated axons. For a better understanding of the interactions involved in the binding of MAG to neuronal axons, we performed this study to identify the binding partners for MAG on neuronal cells. Experiments with glycosylation inhibitors revealed that sialylated N-glycans of glycoproteins represent the major binding sites for MAG on the neuroblastoma cell line N2A. From extracts of [3H]glucosamine-labelled N2A cells several glycoproteins with molecular weights between 20 and 230 kDa were affinity-precipitated using immobilised MAG. The interactions of these proteins with MAG were sialic acid-dependent and specific for MAG. PMID:10037148

  10. Molecular regulation of plant cell wall extensibility

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1998-01-01

    Gravity responses in plants often involve spatial and temporal changes in cell growth, which is regulated primarily by controlling the ability of the cell wall to extend. The wall is thought to be a cellulose-hemicellulose network embedded in a hydrated matrix of complex polysaccharides and a small amount of structural protein. The wall extends by a form of polymer creep, which is mediated by expansins, a novel group of wall-loosening proteins. Expansins were discovered during a molecular dissection of the "acid growth" behavior of cell walls. Expansin alters the rheology of plant walls in profound ways, yet its molecular mechanism of action is still uncertain. It lacks detectable hydrolytic activity against the major components of the wall, but it is able to disrupt noncovalent adhesion between wall polysaccharides. The discovery of a second family of expansins (beta-expansins) sheds light on the biological role of a major group of pollen allergens and implies that expansins have evolved for diverse developmental functions. Finally, the contribution of other processes to wall extensibility is briefly summarized.

  11. 2003 Plant Cell Walls Gordon Conference

    SciTech Connect

    Daniel J. Cosgrove

    2004-09-21

    This conference will address recent progress in many aspects of cell wall biology. Molecular, genetic, and genomic approaches are yielding major advances in our understanding of the composition, synthesis, and architecture of plant cell walls and their dynamics during growth, and are identifying the genes that encode the machinery needed to make their biogenesis possible. This meeting will bring together international scientists from academia, industry and government labs to share the latest breakthroughs and perspectives on polysaccharide biosynthesis, wood formation, wall modification, expansion and interaction with other organisms, and genomic & evolutionary analyses of wall-related genes, as well as to discuss recent ''nanotechnological'' advances that take wall analysis to the level of a single cell.

  12. Modulation of a Glycoprotein Recognition System on Rat Hepatic Endothelial Cells by Glucose and Diabetes Mellitus

    PubMed Central

    Summerfield, John A.; Vergalla, John; Jones, E. Anthony

    1982-01-01

    The cellular location and carbohydrate specificities of a glycoprotein recognition system on rat hepatic sinusoidal cells have been determined. Purified preparations of endothelial, Kupffer, and parenchymal cells were prepared by collagenase liver perfusion, centrifugation on Percoll gradients, and centrifugal elutriation. 125I-labeled agalactoorosomucoid, an N-acetylglucosamine-terminated glycoprotein, was selectively taken up in vitro by endothelial cells. Uptake was shown to be protein dependent, calcium ion dependent, and saturable, and could be described by Michaelis-Menten kinetics (apparent Km 0.29 μM; apparent maximum velocity 4.8 pmol/h per 5 × 106 cells). Uptake was inhibited not only by N-acetylglucosamine, mannose, and mannan but also by glucose, fructose, and a glucose-albumin conjugate. Inhibition by glucose was competitive over a wide range of concentrations and was almost 100% at a glucose concentration of 56 mM. Fasting and the induction of diabetes mellitus prior to isolation of cells was associated with 60% reductions in the recovery of endothelial cells. Uptake by cells isolated from fasted rats was enhanced (apparent maximum velocity 14.3 pmol/h per 5 × 106 cells without change in the apparent Km). These observations suggest that fasting is associated with a marked increase in the mean number of glycoprotein receptors per endothelial cell isolated from normal rats. This effect of fasting could be due to upregulation of glycoprotein receptors on endothelial cells or to the selective isolation of a subpopulation of endothelial cells from fasted animals that bears more glycoprotein receptors per cell than does another subpopulation of these cells. In addition, in vivo studies of the fate of intravenously administered 125I-agalactoorosomucoid indicated that its rate of disappearance from plasma, hepatic accumulation, and catabolism were slower in diabetic than in normal rats. The results suggest that modulation of a carbohydrate

  13. Refractive index of plant cell walls

    NASA Technical Reports Server (NTRS)

    Gausman, H. W.; Allen, W. A.; Escobar, D. E.

    1974-01-01

    Air was replaced with media of higher refractive indices by vacuum infiltration in leaves of cucumber, blackeye pea, tomato, and string bean plants, and reflectance of noninfiltrated and infiltrated leaves was spectrophotometrically measured. Infiltrated leaves reflected less light than noninfiltrated leaves over the 500-2500-nm wavelength interval because cell wall-air interfaces were partly eliminated. Minimal reflectance should occur when the average refractive index of plant cell walls was matched by the infiltrating fluid. Although refractive indices that resulted in minimal reflectance differed among the four plant genera, an average value of 1.425 approximates the refractive index of plant cell walls for the four plant genera.

  14. Tensile Strength of Cell Walls of Living Cells 1

    PubMed Central

    Carpita, Nicholas C.

    1985-01-01

    A gas decompression technique was used to determine the breaking strength of cell walls of single cells. Breaking strengths of the bacterium Salmonella typhimurium and the unicellular green alga Chlamydomonas eugametos were 100 and 95 atmospheres, respectively, while those of sporophytes of the water mold Blastocladiella emersonii were 65 atmospheres, and those of suspension cultured cells of carrot were only 30 atmospheres. Estimation of wall tensile stress based on breaking pressures, cell radii, and estimation of wall thickness, indicates that microfibrillar walls are not necessarily stronger than walls of primitive organisms. Hence, alternative hypotheses for their evolution must be considered. PMID:16664436

  15. An efficient platform for screening expression and crystallization of glycoproteins produced in human cells

    PubMed Central

    Lee, Jeffrey E.; Fusco, Marnie L.; Saphire, Erica Ollmann

    2010-01-01

    Glycoproteins mediate multiple, diverse and critical cellular functions, that are desirable to explore by structural analysis. However, structure determination of these molecules has been hindered by difficulties expressing milligram quantities of stable, homogeneous protein and in determining, which modifications will yield samples amenable to structural studies. We describe a platform proven effective for rapidly screening expression and crystallization of challenging glycoprotein targets produced in mammalian cells. Here, multiple glycoprotein constructs are produced in parallel by transient expression of adherent human embryonic kidney (HEK) 293T cells and subsequently screened in small quantities for crystallization by microfluidic free interface diffusion. As a result, recombinant proteins are produced and processed in a native, mammalian environment and crystallization screening can be accomplished with as little as 65 μg of protein. Moreover, large numbers of constructs can be screened for expression and crystallization and scaled up for structural studies in a matter of five weeks. PMID:19373230

  16. Proteomic dataset for altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells.

    PubMed

    Sheta, Razan; Roux-Dalvai, Florence; Woo, Christina M; Fournier, Frédéric; Bourassa, Sylvie; Bertozzi, Carolyn R; Droit, Arnaud; Bachvarov, Dimcho

    2016-09-01

    This article contains raw and processed data related to research published in "Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation" [1]. The data presented here was obtained with the application of a bioorthogonal chemical reporter strategy analyzing differential glycoprotein expression following the knock-down (KD) of the GALNT3 gene in the epithelial ovarian cancer (EOC) cell line A2780s. LC-MS/MS mass spectrometry analysis was then performed and the processed data related to the identified glycoproteins show that several hundred proteins are differentially expressed between control and GALNT3 KD A2780s cells. The obtained data also uncover numerous novel glycoproteins; some of which could represent new potential EOC biomarkers and/or therapeutic targets. PMID:27331112

  17. Membrane Glycoproteins Associated with Breast Tumor Cell Progression Identified by a Lectin Affinity Approach

    PubMed Central

    Wang, Yanfei; Ao, Xiaoping; Vuong, Huy; Konanur, Meghana; Miller, Fred R.; Goodison, Steve; Lubman, David M.

    2008-01-01

    The membrane glycoprotein component of the cellular proteome represents a promising source for potential disease biomarkers and therapeutic targets. Here we describe the development of a method that facilitates the analysis of membrane glycoproteins and apply it to the differential analysis of breast tumor cells with distinct malignant phenotypes. The approach combines two membrane extraction procedures, and enrichment using ConA and WGA lectin affinity columns, prior to digestion and analysis by LC–MS/MS. The glycoproteins are identified and quantified by spectral counting. Although the distribution of glycoprotein expression as a function of MW and pI was very similar between the two related cell lines tested, the approach enabled the identification of several distinct membrane glycoproteins with an expression index correlated with either a precancerous (MCF10AT1), or a malignant, metastatic cellular phenotype (MCF10CA1a). Among the proteins associated with the malignant phenotype, Gamma-glutamyl hydrolase, CD44, Galectin-3-binding protein, and Syndecan-1 protein have been reported as potential biomarkers of breast cancer. PMID:18729497

  18. Secondary cell walls: biosynthesis and manipulation.

    PubMed

    Kumar, Manoj; Campbell, Liam; Turner, Simon

    2016-01-01

    Secondary cell walls (SCWs) are produced by specialized plant cell types, and are particularly important in those cells providing mechanical support or involved in water transport. As the main constituent of plant biomass, secondary cell walls are central to attempts to generate second-generation biofuels. Partly as a consequence of this renewed economic importance, excellent progress has been made in understanding how cell wall components are synthesized. SCWs are largely composed of three main polymers: cellulose, hemicellulose, and lignin. In this review, we will attempt to highlight the most recent progress in understanding the biosynthetic pathways for secondary cell wall components, how these pathways are regulated, and how this knowledge may be exploited to improve cell wall properties that facilitate breakdown without compromising plant growth and productivity. While knowledge of individual components in the pathway has improved dramatically, how they function together to make the final polymers and how these individual polymers are incorporated into the wall remain less well understood. PMID:26663392

  19. Differential scanning calorimetry of plant cell walls

    SciTech Connect

    Lin, Liangshiou; Varner, J.E. ); Yuen, H.K. )

    1991-03-15

    High-sensitivity differential scanning calorimetry has been used to study the phase transition of cell wall preparations of the elongating and mature regions of soybean hypocotyls and of celery epidermis and collenchyma strands. A step-like transition believed to be glass transition was observed in walls isolated from the elongating region of soybean hypocotyls at 52.9C. Addition of 1 mM CaCl{sub 2} to the cell wall preparation increased the transition temperature to 60.8C and greatly reduced the transition magnitude. In walls from the mature region, the transition was small and occurred at a higher temperature (60.1C). Addition of calcium to the mature region cell wall had little effect on the transition. Based on the known interactions between calcium and pectin, the authors propose that calcium affects the glass transition by binding to the polygalacturonate backbone of wall pectin, resulting in a more rigid wall with a smaller transition at a higher temperature. The mature region either has more calcium in the wall or has more methyl-esterified pectin, making it less responsive to added calcium.

  20. Role of cell wall deconstructing enzymes in the proanthocyanidin-cell wall adsorption-desorption phenomena.

    PubMed

    Castro-López, Liliana del Rocío; Gómez-Plaza, Encarna; Ortega-Regules, Ana; Lozada, Daniel; Bautista-Ortín, Ana Belén

    2016-04-01

    The transference of proanthocyanidins from grapes to wine is quite low. This could be due, among other causes, to proanthocyanidins being bound to grape cell wall polysaccharides, which are present in high concentrations in the must. Therefore, the effective extraction of proanthocyanidins from grapes will depend on the ability to disrupt these associations, and, in this respect, enzymes that degrade these polysaccharides could play an important role. The main objective of this work was to test the behavior of proanthocyanidin-cell wall interactions when commercial maceration enzymes are present in the solution. The results showed that cell wall polysaccharides adsorbed a high amount of proanthocyanidins and only a limited quantity of proanthocyanidins could be desorbed from the cell walls after washing with a model solution. The presence of enzymes in the solution reduced the proanthocyanidin-cell wall interaction, probably through the elimination of pectins from the cell wall network. PMID:26593523

  1. Cell-wall dynamics in growing bacteria

    NASA Astrophysics Data System (ADS)

    Furchtgott, Leon; Wingreen, Ned; Huang, Kerwyn Casey

    2010-03-01

    Bacterial cells come in a large variety of shapes, and cell shape plays an important role in the regulation of many biological functions. Cell shape in bacterial cells is dictated by a cell wall composed of peptidoglycan, a polymer made up of long, stiff glycan strands and flexible peptide crosslinks. Although much is understood about the structural properties of peptidoglycan, little is known about the dynamics of cell wall organization in bacterial cells. In particular, during cell growth, how does the bacterial cell wall continuously expand and reorganize while maintaining cell shape? In order to investigate this question quantitatively, we model the cell wall of the Gram-negative bacterium Escherichia coli using a simple elastic model, in which glycan and peptide subunits are treated as springs with different spring constants and relaxed lengths. We consider the peptidoglycan network as a single-layered network of these springs under tension due to an internal osmotic pressure. Within this model, we simulate possible hypotheses for cell growth as different combinations of addition of new springs and breakage of old springs.

  2. Early Activation of Primary Brain Microvascular Endothelial Cells by Nipah Virus Glycoprotein-Containing Particles.

    PubMed

    Freitag, Tanja C; Maisner, Andrea

    2016-03-01

    Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes pronounced infection of brain endothelia and central nervous system (CNS) inflammation. Using primary porcine brain microvascular endothelial cells, we showed that upregulation of E-selectin precedes cytokine induction and is induced not only by infectious NiV but also by NiV-glycoprotein-containing virus-like particles. This demonstrates that very early events in NiV brain endothelial infection do not depend on NiV replication but can be triggered by the NiV glycoproteins alone. PMID:26676791

  3. Early Activation of Primary Brain Microvascular Endothelial Cells by Nipah Virus Glycoprotein-Containing Particles

    PubMed Central

    Freitag, Tanja C.

    2015-01-01

    Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes pronounced infection of brain endothelia and central nervous system (CNS) inflammation. Using primary porcine brain microvascular endothelial cells, we showed that upregulation of E-selectin precedes cytokine induction and is induced not only by infectious NiV but also by NiV-glycoprotein-containing virus-like particles. This demonstrates that very early events in NiV brain endothelial infection do not depend on NiV replication but can be triggered by the NiV glycoproteins alone. PMID:26676791

  4. Trophoblast glycoprotein promotes pancreatic ductal adenocarcinoma cell metastasis through Wnt/planar cell polarity signaling.

    PubMed

    He, Ping; Jiang, Shuheng; Ma, Mingze; Wang, Yang; Li, Rongkun; Fang, Fang; Tian, Guangang; Zhang, Zhigang

    2015-07-01

    Trophoblast glycoprotein (TPBG), a 72 kDa glycoprotein was identified using a monoclonal antibody, which specifically binds human trophoblast. The expression of TPBG in normal tissues is limited; however, it is upregulated in numerous types of cancer. When TPBG is expressed at a high level, this usually indicates a poor clinical outcome. In the present study, it was demonstrated that TPBG was more commonly observed in human pancreatic ductal adenocarcinoma (PDAC) compared with normal pancreatic tissue. Immunohistochemical analysis of PDAC tissue microarrays indicated that the expression of TPBG in PDAC tissues was closely correlated with the tumor-node-metastasis stage of the tumor. Silencing of TPBG in PDAC cell lines resulted in a decreased ability of cancer cell migration and invasion. Further investigation demonstrated that the Wnt/planar cell polarity signaling pathway was suppressed, as the expression of Wnt5a and the activation of c-Jun N-terminal kinase was inhibited following TPBG knockdown. In conclusion, the present study provided evidence that TPBG is involved in PDAC metastasis, and that TPBG and its associated signaling pathways may be a suitable target for PDAC therapy. PMID:25738465

  5. Vesicular transport across the fungal cell wall

    PubMed Central

    Casadevall, Arturo; Nosanchuk, Joshua D.; Williamson, Peter; Rodrigues, Marcio L.

    2014-01-01

    Recent findings indicate that fungi use vesicular transport to deliver substances across their cell walls. Fungal vesicles are similar to mammalian exosomes and could originate from cytoplasmic multivesicular bodies. Vesicular transport enables the export of large molecules across the cell wall, and vesicles contain lipids, proteins and polysaccharides, many of which are associated with virulence. Concentration of fungal products in vesicles could increase their efficiency in food acquisition and/or delivering potentially noxious substances to other cells, such as amoebae or phagocytes. The discovery of vesicular transport in fungi opens many new avenues for investigation in basic cell biology and pathogenesis. PMID:19299133

  6. Structure of Plant Cell Walls 1

    PubMed Central

    Ishii, Tadashi; Thomas, Jerry; Darvill, Alan; Albersheim, Peter

    1989-01-01

    Considerable information has been obtained about the primary structures of suspension-cultured sycamore (Acer pseudoplatanus) cell-wall pectic polysaccharides, i.e. rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan. However, these polysaccharides, which are solubilized from the walls by endo-α-1,4-polygalacturonase, account for only about half of the pectic polysaccharides known to be present in sycamore cell walls. We now report that, after exhaustive treatment with endo-α-1,4-polygalacturonase, additional pectic polysaccharides were extracted from sycamore cell walls by treatment with Na2CO3 at 1 and 22°C. These previously uncharacterized polysaccharides accounted for ∼4% of the cell wall. Based on the glycosyl and glycosyl-linkage compositions and the nature of the products obtained by treating the quantitatively predominant NaCO3-extracted polysaccharides with lithium metal dissolved in ethylenediamine, the polysaccharides were found to strongly resemble rhamnogalacturonan I. However, unlike rhamnogalacturonan I that characteristically had equal amounts of 2- and 2,4-linked rhamnosyl residues in its backbone, the polysaccharides extracted in Na2CO3 at 1°C had markedly disparate ratios of 2- to 2,4-linked rhamnosyl residues. We concluded that polysaccharides similar to rhamnogalacturonan I but with different degrees of branching are present in the walls of suspension-cultured sycamore cells. PMID:16666559

  7. Altering Entry Site Preference of Lentiviral Vectors into Neuronal Cells by Pseudotyping with Envelope Glycoproteins.

    PubMed

    Kobayashi, Kenta; Kato, Shigeki; Inoue, Ken-Ichi; Takada, Masahiko; Kobayashi, Kazuto

    2016-01-01

    A lentiviral vector system provides a powerful strategy for gene therapy trials against a variety of neurological and neurodegenerative disorders. Pseudotyping of lentiviral vectors with different envelope glycoproteins not only confers the neurotropism to the vectors, but also alters the preference of sites of vector entry into neuronal cells. One major group of lentiviral vectors is a pseudotype with vesicular stomatitis virus glycoprotein (VSV-G) that enters preferentially cell body areas (somata/dendrites) of neurons and transduces them. Another group contains lentiviral vectors pseudotyped with fusion envelope glycoproteins composed of different sets of rabies virus glycoprotein and VSV-G segments that enter predominantly axon terminals of neurons and are transported through axons retrogradely to their cell bodies, resulting in enhanced retrograde gene transfer. This retrograde gene transfer takes a considerable advantage of delivering the transgene into neuronal cell bodies situated in regions distant from the injection site of the vectors. The rational use of these two vector groups characterized by different entry mechanisms will further extend the strategy for gene therapy of neurological and neurodegenerative disorders. PMID:26611586

  8. Determining P-glycoprotein-drug interactions: evaluation of reconstituted P-glycoprotein in a liposomal system and LLC-MDR1 polarized cell monolayers

    PubMed Central

    Melchior, Donald L.; Sharom, Frances J.; Evers, Raymond; Wright, George E.; Chu, Joseph W.K.; Wright, Stephen E.; Chu, Xiaoyan; Yabut, Jocelyn

    2012-01-01

    Introduction P-Glycoprotein (ABCB1, MDR1) is a multidrug efflux pump that is a member of the ATP-binding cassette (ABC) superfamily. Many drugs in common clinical use are either substrates or inhibitors of this transporter. Quantitative details of P-glycoprotein inhibition by pharmaceutical agents are essential for assessment of their pharmacokinetic behavior and prevention of negative patient reactions. Cell-based systems have been widely used for determination of drug interactions with P-glycoprotein, but they suffer from several disadvantages, and results are often widely variable between laboratories. We aimed to demonstrate that a novel liposomal system employing contemporary biochemical methodologies could measure the ability of clinically used drugs to inhibit the P-glycoprotein pump. To accomplish this we compared results with those of cell-based approaches. Methods Purified transport-competent hamster Abcb1a P-glycoprotein was reconstituted into a unilamellar liposomal system, Fluorosome-trans-pgp, whose aqueous interior contains fluorescent drug sensors. This provides a well-defined system for measuring P-glycoprotein transport inhibition by test drugs in real time using rapid fluorescence-based technology. Results Inhibition of ATP-driven transport by Fluorosome-trans-pgp employed a panel of 46 representative drugs. Resulting IC50 values correlated well (r2 = 0.80) with Kd values for drug binding to purified P-glycoprotein. They also showed a similar trend to transport inhibition data obtained using LLC-MDR1 cell monolayers. Fluorosome-trans-pgp IC50 values were in agreement with published results of digoxin drug-drug interaction studies in humans. Discussion This novel approach using a liposomal system and fluorescence-based technology is shown to be suitable to study whether marketed drugs and drug candidates are P-glycoprotein inhibitors. The assay is rapid, allowing a 7-point IC50 determination in <6 minutes, and requires minimal quantities of test

  9. Sialic Acids on Varicella-Zoster Virus Glycoprotein B Are Required for Cell-Cell Fusion*

    PubMed Central

    Suenaga, Tadahiro; Matsumoto, Maki; Arisawa, Fuminori; Kohyama, Masako; Hirayasu, Kouyuki; Mori, Yasuko; Arase, Hisashi

    2015-01-01

    Varicella-zoster virus (VZV) is a member of the human Herpesvirus family that causes varicella (chicken pox) and zoster (shingles). VZV latently infects sensory ganglia and is also responsible for encephalomyelitis. Myelin-associated glycoprotein (MAG), a member of the sialic acid (SA)-binding immunoglobulin-like lectin family, is mainly expressed in neural tissues. VZV glycoprotein B (gB) associates with MAG and mediates membrane fusion during VZV entry into host cells. The SA requirements of MAG when associating with its ligands vary depending on the specific ligand, but it is unclear whether the SAs on gB are involved in the association with MAG. In this study, we found that SAs on gB are essential for the association with MAG as well as for membrane fusion during VZV infection. MAG with a point mutation in the SA-binding site did not bind to gB and did not mediate cell-cell fusion or VZV entry. Cell-cell fusion and VZV entry mediated by the gB-MAG interaction were blocked by sialidase treatment. N-glycosylation or O-glycosylation inhibitors also inhibited the fusion and entry mediated by gB-MAG interaction. Furthermore, gB with mutations in N-glycosylation sites, i.e. asparagine residues 557 and 686, did not associate with MAG, and the cell-cell fusion efficiency was low. Fusion between the viral envelope and cellular membrane is essential for host cell entry by herpesviruses. Therefore, these results suggest that SAs on gB play important roles in MAG-mediated VZV infection. PMID:26105052

  10. Sialic Acids on Varicella-Zoster Virus Glycoprotein B Are Required for Cell-Cell Fusion.

    PubMed

    Suenaga, Tadahiro; Matsumoto, Maki; Arisawa, Fuminori; Kohyama, Masako; Hirayasu, Kouyuki; Mori, Yasuko; Arase, Hisashi

    2015-08-01

    Varicella-zoster virus (VZV) is a member of the human Herpesvirus family that causes varicella (chicken pox) and zoster (shingles). VZV latently infects sensory ganglia and is also responsible for encephalomyelitis. Myelin-associated glycoprotein (MAG), a member of the sialic acid (SA)-binding immunoglobulin-like lectin family, is mainly expressed in neural tissues. VZV glycoprotein B (gB) associates with MAG and mediates membrane fusion during VZV entry into host cells. The SA requirements of MAG when associating with its ligands vary depending on the specific ligand, but it is unclear whether the SAs on gB are involved in the association with MAG. In this study, we found that SAs on gB are essential for the association with MAG as well as for membrane fusion during VZV infection. MAG with a point mutation in the SA-binding site did not bind to gB and did not mediate cell-cell fusion or VZV entry. Cell-cell fusion and VZV entry mediated by the gB-MAG interaction were blocked by sialidase treatment. N-glycosylation or O-glycosylation inhibitors also inhibited the fusion and entry mediated by gB-MAG interaction. Furthermore, gB with mutations in N-glycosylation sites, i.e. asparagine residues 557 and 686, did not associate with MAG, and the cell-cell fusion efficiency was low. Fusion between the viral envelope and cellular membrane is essential for host cell entry by herpesviruses. Therefore, these results suggest that SAs on gB play important roles in MAG-mediated VZV infection. PMID:26105052

  11. Regulation of Cell Wall Biogenesis in Saccharomyces cerevisiae: The Cell Wall Integrity Signaling Pathway

    PubMed Central

    Levin, David E.

    2011-01-01

    The yeast cell wall is a strong, but elastic, structure that is essential not only for the maintenance of cell shape and integrity, but also for progression through the cell cycle. During growth and morphogenesis, and in response to environmental challenges, the cell wall is remodeled in a highly regulated and polarized manner, a process that is principally under the control of the cell wall integrity (CWI) signaling pathway. This pathway transmits wall stress signals from the cell surface to the Rho1 GTPase, which mobilizes a physiologic response through a variety of effectors. Activation of CWI signaling regulates the production of various carbohydrate polymers of the cell wall, as well as their polarized delivery to the site of cell wall remodeling. This review article centers on CWI signaling in Saccharomyces cerevisiae through the cell cycle and in response to cell wall stress. The interface of this signaling pathway with other pathways that contribute to the maintenance of cell wall integrity is also discussed. PMID:22174182

  12. Modes of deformation of walled cells.

    PubMed

    Dumais, Jacques

    2013-11-01

    The bewildering morphological diversity found in cells is one of the starkest illustrations of life's ability to self-organize. Yet the morphogenetic mechanisms that produce the multifarious shapes of cells are still poorly understood. The shared similarities between the walled cells of prokaryotes, many protists, fungi, and plants make these groups particularly appealing to begin investigating how morphological diversity is generated at the cell level. In this review, I attempt a first classification of the different modes of surface deformation used by walled cells. Five modes of deformation were identified: inextensional bending, equi-area shear, elastic stretching, processive intussusception, and chemorheological growth. The two most restrictive modes-inextensional and equi-area deformations-are embodied in the exine of pollen grains and the wall-like pellicle of euglenoids, respectively. For these modes, it is possible to express the deformed geometry of the cell explicitly in terms of the undeformed geometry and other easily observable geometrical parameters. The greatest morphogenetic power is reached with the processive intussusception and chemorheological growth mechanisms that underlie the expansive growth of walled cells. A comparison of these two growth mechanisms suggests a possible way to tackle the complexity behind wall growth. PMID:24014868

  13. Identification of Novel Cell Wall Components

    SciTech Connect

    Michelle Momany

    2009-10-26

    Our DOE Biosciences-funded work focused on the fungal cell wall and morphogenesis. We are especially interested in how new cell wall material is targeted to appropriate areas for polar (asymmetric) growth. Polar growth is the only way that filamentous fungi explore the environment to find suitable substrates to degrade. Work funded by this grant has resulted in a total of twenty peer-reviewed publications. In work funded by this grant, we identified nine Aspergillus nidulans temperature-sensitive (ts) mutants that fail to send out a germ tube and show a swollen cell phenotype at restrictive temperature, the swo mutants. In other organisms, a swollen cell phenotype is often associated with misdirected growth or weakened cell walls. Our work shows that several of the A. nidulans swo mutants have defects in the establishment and maintenance of polarity. Cloning of several swo genes by complementation also showed that secondary modification of proteins seems is important in polarity. We also investigated cell wall biosynthesis and branching based on leads in literature from other organisms and found that branching and nuclear division are tied and that the cell wall reorganizes during development. In our most recent work we have focused on gene expression during the shift from isotropic to polar growth. Surprisingly we found that genes previously thought to be involved only in spore formation are important in early vegetative growth as well.

  14. Planctomycetes do possess a peptidoglycan cell wall

    PubMed Central

    Jeske, Olga; Schüler, Margarete; Schumann, Peter; Schneider, Alexander; Boedeker, Christian; Jogler, Mareike; Bollschweiler, Daniel; Rohde, Manfred; Mayer, Christoph; Engelhardt, Harald; Spring, Stefan; Jogler, Christian

    2015-01-01

    Most bacteria contain a peptidoglycan (PG) cell wall, which is critical for maintenance of shape and important for cell division. In contrast, Planctomycetes have been proposed to produce a proteinaceous cell wall devoid of PG. The apparent absence of PG has been used as an argument for the putative planctomycetal ancestry of all bacterial lineages. Here we show, employing multiple bioinformatic methods, that planctomycetal genomes encode proteins required for PG synthesis. Furthermore, we biochemically demonstrate the presence of the sugar and the peptide components of PG in Planctomycetes. In addition, light and electron microscopic experiments reveal planctomycetal PG sacculi that are susceptible to lysozyme treatment. Finally, cryo-electron tomography demonstrates that Planctomycetes possess a typical PG cell wall and that their cellular architecture is thus more similar to that of other Gram-negative bacteria. Our findings shed new light on the cellular architecture and cell division of the maverick Planctomycetes. PMID:25964217

  15. Genetic resources for maize cell wall biology.

    PubMed

    Penning, Bryan W; Hunter, Charles T; Tayengwa, Reuben; Eveland, Andrea L; Dugard, Christopher K; Olek, Anna T; Vermerris, Wilfred; Koch, Karen E; McCarty, Donald R; Davis, Mark F; Thomas, Steven R; McCann, Maureen C; Carpita, Nicholas C

    2009-12-01

    Grass species represent a major source of food, feed, and fiber crops and potential feedstocks for biofuel production. Most of the biomass is contributed by cell walls that are distinct in composition from all other flowering plants. Identifying cell wall-related genes and their functions underpins a fundamental understanding of growth and development in these species. Toward this goal, we are building a knowledge base of the maize (Zea mays) genes involved in cell wall biology, their expression profiles, and the phenotypic consequences of mutation. Over 750 maize genes were annotated and assembled into gene families predicted to function in cell wall biogenesis. Comparative genomics of maize, rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana) sequences reveal differences in gene family structure between grass species and a reference eudicot species. Analysis of transcript profile data for cell wall genes in developing maize ovaries revealed that expression within families differed by up to 100-fold. When transcriptional analyses of developing ovaries before pollination from Arabidopsis, rice, and maize were contrasted, distinct sets of cell wall genes were expressed in grasses. These differences in gene family structure and expression between Arabidopsis and the grasses underscore the requirement for a grass-specific genetic model for functional analyses. A UniformMu population proved to be an important resource in both forward- and reverse-genetics approaches to identify hundreds of mutants in cell wall genes. A forward screen of field-grown lines by near-infrared spectroscopic screen of mature leaves yielded several dozen lines with heritable spectroscopic phenotypes. Pyrolysis-molecular beam mass spectrometry confirmed that several nir mutants had altered carbohydrate-lignin compositions. PMID:19926802

  16. Cell Wall Development in Maize Coleoptiles 1

    PubMed Central

    Carpita, Nicholas C.

    1984-01-01

    The physical bases for enhancement of growth rates induced by auxin involve changes in cell wall structure. Changes in the chemical composition of the primary walls during maize (Zea mays L. cv WF9 × Bear 38) coleoptile development were examined to provide a framework to study the nature of auxin action. This report documents that the primary walls of maize cells vary markedly depending on developmental state; polymers synthesized and deposited in the primary wall during cell division are substantially different from those formed during cell elongation. The embryonal coleoptile wall is comprised of mostly glucuronoarabinoxylan (GAX), xyloglucan, and polymers enriched in 5-arabinosyl linkages. During development, both GAX and xyloglucan are synthesized, but the 5-arabinosyls are not. Rapid coleoptile elongation is accompanied by synthesis of a mixed-linked glucan that is nearly absent from the embryonal wall. A GAX highly substituted with mostly terminal arabinofuranosyl units is also synthesized during elongation and, based on pulse-chase studies, exhibits turnover possibly to xylans with less substitution via loss of the arabinosyl and glucuronosyl linkages. Images Fig. 2 PMID:16663799

  17. The Structure of Plant Cell Walls

    PubMed Central

    McNeil, Michael; Albersheim, Peter; Taiz, Lincoln; Jones, Russell L.

    1975-01-01

    The walls of barley (Hordeum vulgare var. Himalaya) aleurone cells are composed of two major polysaccharides, arabinoxylan (85%) and cellulose (8%). The cell wall preparations contain 6% protein, but this protein does not contain detectable amounts of hydroxyproline. The arabinoxylan has a linear 1,4-xylan backbone; 33% of the xylosyl residues are substituted at the 2 and/or 3 position with single arabinofuranosyl residues. The results of in vitro cellulose binding experiments support the hypothesis that noncovalent bonds between the arabinoxylan chains and cellulose fibers play a part in maintaining wall structure. It is suggested that bonding between the arabinoxylan chains themselves is also utilized in forming the walls. PMID:16659029

  18. Cell Wall Metabolism in Ripening Fruit

    PubMed Central

    Ahmed, Ahmed Elrayah; Labavitch, John M.

    1980-01-01

    Mature `Bartlett' pear (Pyrus communis) fruits were ripened at 20 C. Fruits at different stages of ripeness were homogenized, and extracts of the low speed pellet (crude cell wall) were prepared. These extracts contained polygalacturonase, pectin esterase, and activity against seven p-nitrophenyl glycoside substrates. Polygalacturonase, α-galactosidase, and α-mannosidase increased in activity as the fruit ripened. Cellulase and activities against pear wall xylan and arabinan were absent from the extracts. PMID:16661276

  19. Glycosylation Engineering of Glycoproteins

    NASA Astrophysics Data System (ADS)

    Sadamoto, Reiko; Nishimura, Shin-Ichiro

    Naturally occurring glycosylation of glycoproteins varies in glycosylation site and in the number and structure of glycans. The engineering of well-defined glycoproteins is an important technology for the preparation of pharmaceutically relevant glycoproteins and in the study of the relationship between glycans and proteins on a structure-function level. In pharmaceutical applications of glycoproteins, the presence of terminal sialic acids on glycans is particularly important for the in vivo circulatory half life, since sialic acid-terminated glycans are not recognized by asialoglycoprotein receptors. Therefore, there have been a number of attempts to control or modify cellular metabolism toward the expression of glycoproteins with glycosylation profiles similar to that of human glycoproteins. In this chapter, recent methods for glycoprotein engineering in various cell culture systems (mammalian cells, plant, yeast, and E. coli) and advances in the chemical approach to glycoprotein formation are described.

  20. Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production.

    PubMed

    Gerken, Henri G; Donohoe, Bryon; Knoshaug, Eric P

    2013-01-01

    Cell walls of microalgae consist of a polysaccharide and glycoprotein matrix providing the cells with a formidable defense against its environment. We characterized enzymes that can digest the cell wall and weaken this defense for the purpose of protoplasting or lipid extraction. A growth inhibition screen demonstrated that chitinase, lysozyme, pectinase, sulfatase, β-glucuronidase, and laminarinase had the broadest effect across the various Chlorella strains tested and also inhibited Nannochloropsis and Nannochloris strains. Chlorella is typically most sensitive to chitinases and lysozymes, both enzymes that degrade polymers containing N-acetylglucosamine. Using a fluorescent DNA stain, we developed rapid methodology to quantify changes in permeability in response to enzyme digestion and found that treatment with lysozyme in conjunction with other enzymes has a drastic effect on cell permeability. Transmission electron microscopy of enzymatically treated Chlorella vulgaris indicates that lysozyme degrades the outer surface of the cell wall and removes hair-like fibers protruding from the surface, which differs from the activity of chitinase. This action on the outer surface of the cell causes visible protuberances on the cell surface and presumably leads to the increased settling rate when cells are treated with lysozyme. We demonstrate radical ultrastructural changes to the cell wall in response to treatment with various enzyme combinations which, in some cases, causes a greater than twofold increase in the thickness of the cell wall. The enzymes characterized in this study should prove useful in the engineering and extraction of oils from microalgae. PMID:23011569

  1. A novel baculovirus vector for the production of nonfucosylated recombinant glycoproteins in insect cells

    PubMed Central

    Mabashi-Asazuma, Hideaki; Kuo, Chu-Wei; Khoo, Kay-Hooi; Jarvis, Donald L

    2014-01-01

    Glycosylation is an important attribute of baculovirus-insect cell expression systems, but some insect cell lines produce core α1,3-fucosylated N-glycans, which are highly immunogenic and render recombinant glycoproteins unsuitable for human use. To address this problem, we exploited a bacterial enzyme, guanosine-5′-diphospho (GDP)-4-dehydro-6-deoxy-d-mannose reductase (Rmd), which consumes the GDP-l-fucose precursor. We expected this enzyme to block glycoprotein fucosylation by blocking the production of GDP-l-fucose, the donor substrate required for this process. Initially, we engineered two different insect cell lines to constitutively express Rmd and isolated subclones with fucosylation-negative phenotypes. However, we found the fucosylation-negative phenotypes induced by Rmd expression were unstable, indicating that this host cell engineering approach is ineffective in insect systems. Thus, we constructed a baculovirus vector designed to express Rmd immediately after infection and facilitate the insertion of genes encoding any glycoprotein of interest for expression later after infection. We used this vector to produce a daughter encoding rituximab and found, in contrast to an Rmd-negative control, that insect cells infected with this virus produced a nonfucosylated form of this therapeutic antibody. These results indicate that our Rmd+ baculoviral vector can be used to solve the immunogenic core α1,3-fucosylation problem associated with the baculovirus-insect cell system. In conjunction with existing glycoengineered insect cell lines, this vector extends the utility of the baculovirus-insect cell system to include therapeutic glycoprotein production. This new vector also extends the utility of the baculovirus-insect cell system to include the production of recombinant antibodies with enhanced effector functions, due to its ability to block core α1,6-fucosylation. PMID:24362443

  2. The B-cell lymphoma 2 (BCL2)-inhibitors, ABT-737 and ABT-263, are substrates for P-glycoprotein

    SciTech Connect

    Vogler, Meike; Dickens, David; Dyer, Martin J.S.; Owen, Andrew; Pirmohamed, Munir; Cohen, Gerald M.

    2011-05-06

    Highlights: {yields} The BCL2-inhibitor ABT-263 is a substrate for P-glycoprotein. {yields} Apoptosis is inhibited by P-glycoprotein expression. {yields} Overexpression of P-glycoprotein may contribute to resistance to ABT-263 or ABT-737. -- Abstract: Inhibition of BCL2 proteins is one of the most promising new approaches to targeted cancer therapy resulting in the induction of apoptosis. Amongst the most specific BCL2-inhibitors identified are ABT-737 and ABT-263. However, targeted therapy is often only effective for a limited amount of time because of the occurrence of drug resistance. In this study, the interaction of BCL2-inhibitors with the drug efflux transporter P-glycoprotein was investigated. Using {sup 3}H labelled ABT-263, we found that cells with high P-glycoprotein activity accumulated less drug. In addition, cells with increased P-glycoprotein expression were more resistant to apoptosis induced by either ABT-737 or ABT-263. Addition of tariquidar or verapamil sensitized the cells to BCL2-inhibitor treatment, resulting in higher apoptosis. Our data suggest that the BCL2-inhibitors ABT-737 and ABT-263 are substrates for P-glycoprotein. Over-expression of P-glycoprotein may be, at least partly, responsible for resistance to these BCL2-inhibitors.

  3. Transient axonal glycoprotein-1 induces apoptosis-related gene expression without triggering apoptosis in U251 glioma cells

    PubMed Central

    Chang, Haigang; Song, Shanshan; Chen, Zhongcan; Wang, Yaxiao; Yang, Lujun; Du, Mouxuan; Ke, Yiquan; Xu, Ruxiang; Jin, Baozhe; Jiang, Xiaodan

    2014-01-01

    Previous studies show that transient axonal glycoprotein-1, a ligand of amyloid precursor protein, increases the secretion of amyloid precursor protein intracellular domain and is involved in apoptosis in Alzheimer's disease. In this study, we examined the effects of transient axonal glycoprotein-1 on U251 glioma cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that transient axonal glycoprotein-1 did not inhibit the proliferation of U251 cells, but promoted cell viability. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that transient axonal glycoprotein-1 did not induce U251 cell apoptosis. Real-time PCR revealed that transient axonal glycoprotein-1 substantially upregulated levels of amyloid precursor protein intracellular C-terminal domain, and p53 and epidermal growth factor receptor mRNA expression. Thus, transient axonal glycoprotein-1 increased apoptosis-related gene expression in U251 cells without inducing apoptosis. Instead, transient axonal glycoprotein-1 promoted the proliferation of these glioma cells. PMID:25206849

  4. The charophycean green algae provide insights into the early origins of plant cell walls.

    PubMed

    Sørensen, Iben; Pettolino, Filomena A; Bacic, Antony; Ralph, John; Lu, Fachuang; O'Neill, Malcolm A; Fei, Zhangzhun; Rose, Jocelyn K C; Domozych, David S; Willats, William G T

    2011-10-01

    Numerous evolutionary innovations were required to enable freshwater green algae to colonize terrestrial habitats and thereby initiate the evolution of land plants (embryophytes). These adaptations probably included changes in cell-wall composition and architecture that were to become essential for embryophyte development and radiation. However, it is not known to what extent the polymers that are characteristic of embryophyte cell walls, including pectins, hemicelluloses, glycoproteins and lignin, evolved in response to the demands of the terrestrial environment or whether they pre-existed in their algal ancestors. Here we show that members of the advanced charophycean green algae (CGA), including the Charales, Coleochaetales and Zygnematales, but not basal CGA (Klebsormidiales and Chlorokybales), have cell walls that are comparable in several respects to the primary walls of embryophytes. Moreover, we provide both chemical and immunocytochemical evidence that selected Coleochaete species have cell walls that contain small amounts of lignin or lignin-like polymers derived from radical coupling of hydroxycinnamyl alcohols. Thus, the ability to synthesize many of the components that characterize extant embryophyte walls evolved during divergence within CGA. Our study provides new insight into the evolutionary window during which the structurally complex walls of embryophytes originated, and the significance of the advanced CGA during these events. PMID:21707800

  5. Preliminary characterization of an epitope involved in neutralization and cell attachment that is located on the major bovine rotavirus glycoprotein.

    PubMed Central

    Sabara, M; Gilchrist, J E; Hudson, G R; Babiuk, L A

    1985-01-01

    The 38,200-molecular weight (unreduced)/41,900-molecular-weight (reduced) glycoprotein of bovine rotavirus, isolate C486, was identified as the major neutralizing antigen. This glycoprotein as well as the corresponding glycoprotein of another bovine rotavirus serotype also specifically attached to cell monolayers under normal conditions for virus adsorption in vitro. Further support for this glycoprotein being directly responsible for virus attachment to cells was that (i) infectious virus of both serotypes could compete with the C486 glycoprotein for cell surface receptors, and (ii) neutralizing monospecific antiserum and neutralizing monoclonal antibodies directed toward the glycoprotein could block this virus-cell interaction. Preliminary epitope mapping of the glycoprotein with monoclonal antibodies further localized the neutralization-adsorption domain to a peptide with an approximate molecular weight of 14,000. The effect of two protein modifications, glycosylation and disulfide bridging, on the reactivity of this peptide with antibodies and cell surface receptors was investigated. It was demonstrated that, whereas glycosylation did not appear to affect these reactivities, disulfide bridging seemed to be essential. Images PMID:2578197

  6. Aberrant trafficking of hepatitis B virus glycoproteins in cells in which N-glycan processing is inhibited

    PubMed Central

    Lu, Xuanyong; Mehta, Anand; Dadmarz, Mitra; Dwek, Raymond; Blumberg, Baruch S.; Block, Timothy M.

    1997-01-01

    The role of N-glycan trimming in glycoprotein fate and function is unclear. We have recently shown that hepatitis B virus (HBV) DNA is not efficiently secreted from cells in which α-glucosidase mediated N-glycan trimming is inhibited. Here it is shown that, in cells in glucosidase-inhibited cells, viral DNA, accompanied by envelope and core proteins, most likely accumulate within lysosomal compartments. Pulse–chase experiments show that although the viral glycoproteins (L, M, and S) are dysfunctional, in the sense that they do not mediate virion egress and are not efficiently secreted from the cell, they all still leave the endoplasmic reticulum (ER). Surprisingly, however, the glycoproteins retained within the cell were not rapidly degraded, appearing as aggregates, enriched for L and M, with intracellular half-lives exceeding 20 h. Moreover, by 24 h after synthesis, a substantial fraction of the detained glycoproteins appeared to return to the ER, although a considerable amount was also found in the lysosomes. To our knowledge, this is the first report that shows, as a consequence of inhibiting glycosylation processing, certain glycoproteins (i) become dysfunctional and aggregate, yet still depart from the ER, and (ii) have extended rather than shortened half-lives. Taken together, these data suggest that proper intracellular routing of HBV glycoproteins requires ER glucosidase function. It is hypothesized that failure to process N-glycan causes HBV glycoproteins to aggregate and that impaired protein–protein interactions and trafficking are the result of misfolding. PMID:9122203

  7. Separation of bivalent anti-T cell immunotoxin from Pichia pastoris glycoproteins by borate anion exchange.

    PubMed

    Woo, Jung Hee; Neville, David M

    2003-08-01

    A major problem encountered in the large-scale purification of the bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(G4S), from Pichia pastoris supernatants was the presence of host glycoproteins exhibiting similar charge, size, and hydrophobicity characteristics. We overcame this problem by employing borate anion exchange chromatography. The borate anion has an affinity for carbohydrates and imparts negative charges to these structures. We found that at a concentration of sodium borate between 50 and 100 mM, the nonglycosylated immunotoxin did not bind to Poros 50 HQ anion exchanger resin, but glycoproteins, including aggregates related to the immunotoxin, did. By using this property of the immunotoxin in the presence of sodium borate, we successfully developed a 3-step purification procedure: (i) Butyl-650M hydrophobic interaction chromatography, (ii) Poros 50 HQ anion exchange chromatography in the presence of borate, and (iii) HiTrap Q anion exchange chromatography. The final preparation exhibited a purity of greater than 98% and a yield of greater than 50% from the supernatant. Previously, boronic acid resins have been used to separate glycoproteins from proteins. However, combining borate anion with conventional anion exchange resins accomplishes the separation of the immunotoxin from glycoproteins and eliminates the need to evaluate nonstandard resins with respect to good manufacturing practice guidelines. PMID:12951782

  8. Roles of membrane trafficking in plant cell wall dynamics

    PubMed Central

    Ebine, Kazuo; Ueda, Takashi

    2015-01-01

    The cell wall is one of the characteristic components of plant cells. The cell wall composition differs among cell types and is modified in response to various environmental conditions. To properly generate and modify the cell wall, many proteins are transported to the plasma membrane or extracellular space through membrane trafficking, which is one of the key protein transport mechanisms in eukaryotic cells. Given the diverse composition and functions of the cell wall in plants, the transport of the cell wall components and proteins that are involved in cell wall-related events could be specialized for each cell type, i.e., the machinery for cell wall biogenesis, modification, and maintenance could be transported via different trafficking pathways. In this review, we summarize the recent progress in the current understanding of the roles and mechanisms of membrane trafficking in plant cells and focus on the biogenesis and regulation of the cell wall. PMID:26539200

  9. Cell Wall and Extensin mRNA Changes during Cold Acclimation of Pea Seedlings

    PubMed Central

    Weiser, Russell L.; Wallner, Stephen J.; Waddell, John W.

    1990-01-01

    During exposure to 2 °C, pea (Pisum sativum) seedlings cold acclimated to a killing temperature of −6 °C. Associated with this increase in freezing resistance was an increase in the weight of cell walls and changes in wall composition. Arabinosyl content increased by 100%, while other cell wall glycosyl residues and cellulose increased by about 20%. The cell wall hydroxyproline content increased by 80%. Arabinose and hydroxyproline are both major components of the structural cell wall glycoprotein, extensin. The increase in these components indicates that the level of extensin in the cell wall increases during cold acclimation. Northern blot analysis, using the pDC5A1 genomic clone as a probe, revealed a more than three-fold increase in total extensin mRNA during exposure to cold temperature. Specific extensin transcripts of 6.0, 4.5, 3.5, 2.6, 2.3, 1.8, and 1.5 kilobases were identified. Those at 6.0, 2.6, and 1.5 kilobases were especially promoted by low temperature treatment. The rise in extensin during cold acclimation may be regulated, at least in part, at the gene level. The possible structural role of this protein in freezing protection is discussed. Images Figure 7 PMID:16667551

  10. Evidence for P-Glycoprotein Involvement in Cell Volume Regulation Using Coulter Sizing in Flow Cytometry.

    PubMed

    Pasquier, Jennifer; Rioult, Damien; Abu-Kaoud, Nadine; Hoarau-Véchot, Jessica; Marin, Matthieu; Le Foll, Frank

    2015-01-01

    The regulation of cell volume is an essential function that is coupled to a variety of physiological processes such as receptor recycling, excitability and contraction, cell proliferation, migration, and programmed cell death. Under stress, cells undergo emergency swelling and respond to such a phenomenon with a regulatory volume decrease (RVD) where they release cellular ions, and other osmolytes as well as a concomitant loss of water. The link between P-glycoprotein, a transmembrane transporter, and cell volume regulation is controversial, and changes in cells volume are measured using microscopy or electrophysiology. For instance, by using the patch-clamp method, our team demonstrated that chloride currents activated in the RVD were more intense and rapid in a breast cancer cell line overexpressing the P-glycoprotein (P-gp). The Cell Lab Quanta SC is a flow cytometry system that simultaneously measures electronic volume, side scatter and three fluorescent colors; altogether this provides unsurpassed population resolution and accurate cell counting. Therefore, here we propose a novel method to follow cellular volume. By using the Coulter-type channel of the cytometer Cell Lab Quanta SC MPL (multi-platform loading), we demonstrated a role for the P-gp during different osmotic treatments, but also a differential activity of the P-gp through the cell cycle. Altogether, our data strongly suggests a role of P-gp in cell volume regulation. PMID:26114386

  11. Extracellular Vesicle-Associated Transitory Cell Wall Components and Their Impact on the Interaction of Fungi with Host Cells.

    PubMed

    Nimrichter, Leonardo; de Souza, Marcio M; Del Poeta, Maurizio; Nosanchuk, Joshua D; Joffe, Luna; Tavares, Patricia de M; Rodrigues, Marcio L

    2016-01-01

    Classic cell wall components of fungi comprise the polysaccharides glucans and chitin, in association with glycoproteins and pigments. During the last decade, however, system biology approaches clearly demonstrated that the composition of fungal cell walls include atypical molecules historically associated with intracellular or membrane locations. Elucidation of mechanisms by which many fungal molecules are exported to the extracellular space suggested that these atypical components are transitorily located to the cell wall. The presence of extracellular vesicles (EVs) at the fungal cell wall and in culture supernatants of distinct pathogenic species suggested a highly functional mechanism of molecular export in these organisms. Thus, the passage of EVs through fungal cell walls suggests remarkable molecular diversity and, consequently, a potentially variable influence on the host antifungal response. On the basis of information derived from the proteomic characterization of fungal EVs from the yeasts Cryptoccocus neoformans and Candida albicans and the dimorphic fungi Histoplasma capsulatum and Paracoccidioides brasiliensis, our manuscript is focused on the clear view that the fungal cell wall is much more complex than previously thought. PMID:27458437

  12. Extracellular Vesicle-Associated Transitory Cell Wall Components and Their Impact on the Interaction of Fungi with Host Cells

    PubMed Central

    Nimrichter, Leonardo; de Souza, Marcio M.; Del Poeta, Maurizio; Nosanchuk, Joshua D.; Joffe, Luna; Tavares, Patricia de M.; Rodrigues, Marcio L.

    2016-01-01

    Classic cell wall components of fungi comprise the polysaccharides glucans and chitin, in association with glycoproteins and pigments. During the last decade, however, system biology approaches clearly demonstrated that the composition of fungal cell walls include atypical molecules historically associated with intracellular or membrane locations. Elucidation of mechanisms by which many fungal molecules are exported to the extracellular space suggested that these atypical components are transitorily located to the cell wall. The presence of extracellular vesicles (EVs) at the fungal cell wall and in culture supernatants of distinct pathogenic species suggested a highly functional mechanism of molecular export in these organisms. Thus, the passage of EVs through fungal cell walls suggests remarkable molecular diversity and, consequently, a potentially variable influence on the host antifungal response. On the basis of information derived from the proteomic characterization of fungal EVs from the yeasts Cryptoccocus neoformans and Candida albicans and the dimorphic fungi Histoplasma capsulatum and Paracoccidioides brasiliensis, our manuscript is focused on the clear view that the fungal cell wall is much more complex than previously thought. PMID:27458437

  13. Direct Involvement of HERV-W Env Glycoprotein in Human Trophoblast Cell Fusion and Differentiation

    PubMed Central

    Frendo, Jean-Louis; Olivier, Delphine; Cheynet, Valérie; Blond, Jean-Luc; Bouton, Olivier; Vidaud, Michel; Rabreau, Michèle; Evain-Brion, Danièle; Mallet, François

    2003-01-01

    We recently demonstrated that the product of the HERV-W env gene, a retroviral envelope protein also dubbed syncytin, is a highly fusogenic membrane glycoprotein inducing the formation of syncytia on interaction with the type D mammalian retrovirus receptor. In addition, the detection of HERV-W Env protein (Env-W) expression in placental tissue sections led us to propose a role for this fusogenic glycoprotein in placenta formation. To evaluate this hypothesis, we analyzed the involvement of Env-W in the differentiation of primary cultures of human villous cytotrophoblasts that spontaneously differentiate by cell fusion into syncytiotrophoblasts in vitro. First, we observed that HERV-W env mRNA and glycoprotein expression are colinear with primary cytotrophoblast differentiation and with expression of human chorionic gonadotropin (hCG), a marker of syncytiotrophoblast formation. Second, we observed that in vitro stimulation of trophoblast cell fusion and differentiation by cyclic AMP is also associated with a concomitant increase in HERV-W env and hCG mRNA and protein expression. Finally, by using specific antisense oligonucleotides, we demonstrated that inhibition of Env-W protein expression leads to a decrease of trophoblast fusion and differentiation, with the secretion of hCG in culture medium of antisense oligonucleotide-treated cells being decreased by fivefold. Taken together, these results strongly support a direct role for Env-W in human trophoblast cell fusion and differentiation. PMID:12724415

  14. A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin.

    PubMed Central

    Lu, C F; Kurjan, J; Lipke, P N

    1994-01-01

    Saccharomyces cerevisiae alpha-agglutinin is a cell wall-anchored adhesion glycoprotein. The previously identified 140-kDa form, which contains a glycosyl-phosphatidylinositol (GPI) anchor (D. Wojciechowicz, C.-F. Lu, J. Kurjan, and P. N. Lipke, Mol. Cell. Biol. 13:2554-2563, 1993), and additional forms of 80, 150, 250 to 300, and > 300 kDa had the properties of intermediates in a transport and cell wall anchorage pathway. N glycosylation and additional modifications resulted in successive increases in size during transport. The 150- and 250- to 300-kDa forms were membrane associated and are likely to be intermediates between the 140-kDa form and a cell surface GPI-anchored form of > 300 kDa. A soluble form of > 300 kDa that lacked the GPI anchor had properties of a periplasmic intermediate between the plasma membrane form and the > 300-kDa cell wall-anchored form. These results constitute experimental support for the hypothesis that GPI anchors act to localize alpha-agglutinin to the plasma membrane and that cell wall anchorage involves release from the GPI anchor to produce a periplasmic intermediate followed by linkage to the cell wall. Images PMID:8007981

  15. Cell wall peptidoglycan architecture in Bacillus subtilis

    PubMed Central

    Hayhurst, Emma J.; Kailas, Lekshmi; Hobbs, Jamie K.; Foster, Simon J.

    2008-01-01

    The bacterial cell wall is essential for viability and shape determination. Cell wall structural dynamics allowing growth and division, while maintaining integrity is a basic problem governing the life of bacteria. The polymer peptidoglycan is the main structural component for most bacteria and is made up of glycan strands that are cross-linked by peptide side chains. Despite study and speculation over many years, peptidoglycan architecture has remained largely elusive. Here, we show that the model rod-shaped bacterium Bacillus subtilis has glycan strands up to 5 μm, longer than the cell itself and 50 times longer than previously proposed. Atomic force microscopy revealed the glycan strands to be part of a peptidoglycan architecture allowing cell growth and division. The inner surface of the cell wall has a regular macrostructure with ≈50 nm-wide peptidoglycan cables [average 53 ± 12 nm (n = 91)] running basically across the short axis of the cell. Cross striations with an average periodicity of 25 ± 9 nm (n = 96) along each cable are also present. The fundamental cabling architecture is also maintained during septal development as part of cell division. We propose a coiled-coil model for peptidoglycan architecture encompassing our data and recent evidence concerning the biosynthetic machinery for this essential polymer. PMID:18784364

  16. Cell wall peptidoglycan architecture in Bacillus subtilis.

    PubMed

    Hayhurst, Emma J; Kailas, Lekshmi; Hobbs, Jamie K; Foster, Simon J

    2008-09-23

    The bacterial cell wall is essential for viability and shape determination. Cell wall structural dynamics allowing growth and division, while maintaining integrity is a basic problem governing the life of bacteria. The polymer peptidoglycan is the main structural component for most bacteria and is made up of glycan strands that are cross-linked by peptide side chains. Despite study and speculation over many years, peptidoglycan architecture has remained largely elusive. Here, we show that the model rod-shaped bacterium Bacillus subtilis has glycan strands up to 5 microm, longer than the cell itself and 50 times longer than previously proposed. Atomic force microscopy revealed the glycan strands to be part of a peptidoglycan architecture allowing cell growth and division. The inner surface of the cell wall has a regular macrostructure with approximately 50 nm-wide peptidoglycan cables [average 53 +/- 12 nm (n = 91)] running basically across the short axis of the cell. Cross striations with an average periodicity of 25 +/- 9 nm (n = 96) along each cable are also present. The fundamental cabling architecture is also maintained during septal development as part of cell division. We propose a coiled-coil model for peptidoglycan architecture encompassing our data and recent evidence concerning the biosynthetic machinery for this essential polymer. PMID:18784364

  17. Glycoconjugates and polysaccharides of fungal cell wall and activation of immune system

    PubMed Central

    Pinto, M.R.; Barreto-Bergter, E.; Taborda, C.P.

    2008-01-01

    Glycoproteins, glycosphingolipids and polysaccharides exposed at the most external layers of the wall are involved in several types of interactions of fungal cells with the exocellular environment. These molecules are fundamental building blocks of organisms, contributing to the structure, integrity, cell growth, differentiation and signaling. Several of them are immunologically active compounds with potential as regulators of pathogenesis and the immune response of the host. Some of these structures can be specifically recognized by antibodies from patients’ sera, suggesting that they can be also useful in the diagnosis of fungal infections. PMID:24031202

  18. Expression and Purification of E2 Glycoprotein from Insect Cells (Sf9) for Use in Serology.

    PubMed

    Chua, Chong Long; Sam, I-Ching; Chan, Yoke Fun

    2016-01-01

    Chikungunya virus (CHIKV) is a mosquito-borne arbovirus which poses a major threat to global public health. Definitive CHIKV diagnosis is crucial, especially in distinguishing the disease from dengue virus, which co-circulates in endemic areas and shares the same mosquito vectors. Laboratory diagnosis is mainly based on serological or molecular approaches. The E2 glycoprotein is a good candidate for serological diagnosis since it is the immunodominant antigen during the course of infection, and reacts with seropositive CHIKV sera. In this chapter, we describe the generation of stable clone Sf9 (Spodoptera frugiperda) cells expressing secreted, soluble, and native recombinant CHIKV E2 glycoprotein. We use direct plasmid expression in insect cells, rather than the traditional technique of generating recombinant baculovirus. This recombinant protein is useful for serological diagnosis of CHIKV infection. PMID:27233260

  19. Baculovirus Coinfection Strategy for Improved Galactosylation of Recombinant Glycoprotein Produced by Insect Cell Culture

    NASA Astrophysics Data System (ADS)

    Ney, Yap Wei; Rahman, Badarulhisam Abdul; Aziz, Azila Abdul

    Baculovirus Expression Vector System (BEVS) is widely used for the production of recombinant glycoproteins, but it is not ideal for pharmaceutical glycoprotein production due to incomplete glycosylation. The factors that ensure successful glycosylation are the presence of sufficient amount of glycosyltransferases, sugar nucleotides as the substrate donor and the recombinant protein as the substrate acceptor. In this study, we analyzed the galactosylation process by the introduction of ß-1,4galactosyltransferase (ß-1,4GalT) as the glycosyltransferase of interest and uridine-5`-diphosphogalactose (UDP-Gal) as the substrate donor. Recombinant human transferrin (rhTf) as a model protein was used as the substrate acceptor. Insect cell lines have been reported to produce a small amount of ß-1,4GalT and thus insufficient for effective galactosylation. In this study, we developed a method to produce galactosylated rhTf and optimized the expression of rhTf with better N-glycan quality. Recombinant ß-1,4GalT was introduced during protein expression by the coinfection of the BEVS with baculovirus carrying bovine ß-1,4GalT. To evaluate the extent of galactosylation by the coinfection strategy, a binding assay was established. In this binding assay, glycoprotein acceptor was absorbed onto ELISA plate surface. A lectin known as Ricinus communis agglutinin-I (RCA-I) labeled with peroxidase, was added and allowed to recognize Gal ß1>4GlcNAc group on the N-glycan of the glycoprotein, followed by appropriate color reaction measurements. Coexpression between rhTf and ß-1,4GalT did not show encouraging results due to the reduction of UDP-Gal upon baculovirus infection. This interesting finding suggested that the introduction of ß-1,4GalT alone was not sufficient for successful galactosylation. Alternatively, post harvest glycosylation method strategy seems to be a promising technique in the improvement of glycoprotein quality.

  20. Glucocorticoid-regulated and constitutive trafficking of proteolytically processed cell surface-associated glycoproteins in wild type and variant rat hepatoma cells

    SciTech Connect

    Amacher, S.L.; Goodman, L.J.; Bravo, D.A.; Wong, K.Y.; Goldfine, I.D.; Hawley, D.M.; Firestone, G.L. )

    1989-10-01

    Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by (125I) insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways.

  1. The effect of the state of differentiation on labeling of epidermal cell surface glycoproteins

    SciTech Connect

    Brysk, M.M.; Snider, J.M.

    1982-05-01

    Epidermal cells were grown in a medium in which the Ca++ concentration controlled the stage of differentiation. Cell surface molecules of differentiated and undifferentiated cells were compared by lactoperoxidase-catalyzed iodination, by the interaction with /sup 125/I-lectins, and by the metabolic incorporation of L-(/sup 3/H)-fucose. Molecular weights of the labeled components were determined by SDS-PAGE and autoradiography. After lactoperoxidase iodination, most of the radioactivity was found in polypeptide bands of 79,000, 65,000 and 56,000 daltons. The 79,000 band is the most intense for undifferentiated cells but disappears as differentiation proceeds. The 56,000 band is present in normal cells at all stages of differentiation but is absent from neoplastic cells. Glycoproteins reacted with /sup 125/I-lectins were found at 180,000, 130,000 and 85,000 daltons. The 130,000 band was the most prominent for differentiated cells labeled with wheat germ agglutinin but was essentially absent from the undifferentiated cells. With Ricinus communis agglutinin, this band was weaker for undifferentiated than for differentiated cells but was the most intense for both. After metabolic incorporation of tritiated fucose, radioactive glycoproteins were found at 130,000 and 85,000 daltons, with comparable intensities for differentiated and undifferentiated cells.

  2. Characterization of the Sclerotinia sclerotiorum cell wall proteome.

    PubMed

    Liu, Longzhou; Free, Stephen J

    2016-08-01

    We used a proteomic analysis to identify cell wall proteins released from Sclerotinia sclerotiorum hyphal and sclerotial cell walls via a trifluoromethanesulfonic acid (TFMS) digestion. Cell walls from hyphae grown in Vogel's glucose medium (a synthetic medium lacking plant materials), from hyphae grown in potato dextrose broth and from sclerotia produced on potato dextrose agar were used in the analysis. Under the conditions used, TFMS digests the glycosidic linkages in the cell walls to release intact cell wall proteins. The analysis identified 24 glycosylphosphatidylinositol (GPI)-anchored cell wall proteins and 30 non-GPI-anchored cell wall proteins. We found that the cell walls contained an array of cell wall biosynthetic enzymes similar to those found in the cell walls of other fungi. When comparing the proteins in hyphal cell walls grown in potato dextrose broth with those in hyphal cell walls grown in the absence of plant material, it was found that a core group of cell wall biosynthetic proteins and some proteins associated with pathogenicity (secreted cellulases, pectin lyases, glucosidases and proteases) were expressed in both types of hyphae. The hyphae grown in potato dextrose broth contained a number of additional proteins (laccases, oxalate decarboxylase, peroxidase, polysaccharide deacetylase and several proteins unique to Sclerotinia and Botrytis) that might facilitate growth on a plant host. A comparison of the proteins in the sclerotial cell wall with the proteins in the hyphal cell wall demonstrated that sclerotia formation is not marked by a major shift in the composition of cell wall protein. We found that the S. sclerotiorum cell walls contained 11 cell wall proteins that were encoded only in Sclerotinia and Botrytis genomes. PMID:26661933

  3. The cell surface expressed nucleolin is a glycoprotein that triggers calcium entry into mammalian cells

    SciTech Connect

    Losfeld, Marie-Estelle; Khoury, Diala El; Mariot, Pascal; Carpentier, Mathieu; Krust, Bernard; Briand, Jean-Paul; Mazurier, Joel; Hovanessian, Ara G.; Legrand, Dominique

    2009-01-15

    Nucleolin is an ubiquitous nucleolar phosphoprotein involved in fundamental aspects of transcription regulation, cell proliferation and growth. It has also been described as a shuttling molecule between nucleus, cytosol and the cell surface. Several studies have demonstrated that surface nucleolin serves as a receptor for various extracellular ligands implicated in cell proliferation, differentiation, adhesion, mitogenesis and angiogenesis. Previously, we reported that nucleolin in the extranuclear cell compartment is a glycoprotein containing N- and O-glycans. In the present study, we show that glycosylation is an essential requirement for surface nucleolin expression, since it is prevented when cells are cultured in the presence of tunicamycin, an inhibitor of N-glycosylation. Accordingly, surface but not nuclear nucleolin is radioactively labeled upon metabolic labeling of cells with [{sup 3}H]glucosamine. Besides its well-demonstrated role in the internalization of specific ligands, here we show that ligand binding to surface nucleolin could also induce Ca{sup 2+} entry into cells. Indeed, by flow cytometry, microscopy and patch-clamp experiments, we show that the HB-19 pseudopeptide, which binds specifically surface nucleolin, triggers rapid and intense membrane Ca{sup 2+} fluxes in various types of cells. The use of several drugs then indicated that Store-Operated Ca{sup 2+} Entry (SOCE)-like channels are involved in the generation of these fluxes. Taken together, our findings suggest that binding of an extracellular ligand to surface nucleolin could be involved in the activation of signaling pathways by promoting Ca{sup 2+} entry into cells.

  4. Cell Wall Heterogeneity in Root Development of Arabidopsis

    PubMed Central

    Somssich, Marc; Khan, Ghazanfar Abbas; Persson, Staffan

    2016-01-01

    Plant cell walls provide stability and protection to plant cells. During growth and development the composition of cell walls changes, but provides enough strength to withstand the turgor of the cells. Hence, cell walls are highly flexible and diverse in nature. These characteristics are important during root growth, as plant roots consist of radial patterns of cells that have diverse functions and that are at different developmental stages along the growth axis. Young stem cell daughters undergo a series of rapid cell divisions, during which new cell walls are formed that are highly dynamic, and that support rapid anisotropic cell expansion. Once the cells have differentiated, the walls of specific cell types need to comply with and support different cell functions. For example, a newly formed root hair needs to be able to break through the surrounding soil, while endodermal cells modify their walls at distinct positions to form Casparian strips between them. Hence, the cell walls are modified and rebuilt while cells transit through different developmental stages. In addition, the cell walls of roots readjust to their environment to support growth and to maximize nutrient uptake. Many of these modifications are likely driven by different developmental and stress signaling pathways. However, our understanding of how such pathways affect cell wall modifications and what enzymes are involved remain largely unknown. In this review we aim to compile data linking cell wall content and re-modeling to developmental stages of root cells, and dissect how root cell walls respond to certain environmental changes. PMID:27582757

  5. Cell Wall Heterogeneity in Root Development of Arabidopsis.

    PubMed

    Somssich, Marc; Khan, Ghazanfar Abbas; Persson, Staffan

    2016-01-01

    Plant cell walls provide stability and protection to plant cells. During growth and development the composition of cell walls changes, but provides enough strength to withstand the turgor of the cells. Hence, cell walls are highly flexible and diverse in nature. These characteristics are important during root growth, as plant roots consist of radial patterns of cells that have diverse functions and that are at different developmental stages along the growth axis. Young stem cell daughters undergo a series of rapid cell divisions, during which new cell walls are formed that are highly dynamic, and that support rapid anisotropic cell expansion. Once the cells have differentiated, the walls of specific cell types need to comply with and support different cell functions. For example, a newly formed root hair needs to be able to break through the surrounding soil, while endodermal cells modify their walls at distinct positions to form Casparian strips between them. Hence, the cell walls are modified and rebuilt while cells transit through different developmental stages. In addition, the cell walls of roots readjust to their environment to support growth and to maximize nutrient uptake. Many of these modifications are likely driven by different developmental and stress signaling pathways. However, our understanding of how such pathways affect cell wall modifications and what enzymes are involved remain largely unknown. In this review we aim to compile data linking cell wall content and re-modeling to developmental stages of root cells, and dissect how root cell walls respond to certain environmental changes. PMID:27582757

  6. Lipopolysaccharide increases cell surface P-glycoprotein that exhibits diminished activity in intestinal epithelial cells.

    PubMed

    Mishra, Jayshree; Zhang, Qiuye; Rosson, Jessica L; Moran, John; Dopp, John M; Neudeck, Brien L

    2008-10-01

    Increasingly, it is recognized that commensal microflora regulate epithelial cell processes through the dynamic interaction of pathogen-associated molecular patterns and host pattern recognition receptors such as Toll-like receptor 4 (TLR4). We therefore investigated the effects of bacterial lipopolysaccharide (LPS) on intestinal P-glycoprotein (P-gp) expression and function. Human SW480 (P-gp+/TLR4+) and Caco-2 (P-gp+/TLR4-) cells were treated with medium control or LPS (100 ng/ml) for 24 h prior to study. P-gp function was assessed by measuring the intracellular concentration of rhodamine 123 (Rh123). To confirm P-gp-specific effects, breast cancer resistance protein (BCRP/ABCG2) and multidrug resistance-associated protein 2 (MRP-2/ABCC2) were also analyzed. Treatment of SW480 cells with LPS led to diminished P-gp activity, which could be prevented with polymyxin B (control: 207+/-16 versus LPS: 402+/-22 versus LPS+polymyxin B: 238+/-26 pmoles Rh123/mg protein, p<0.05 control versus LPS). These effects could be blocked by using polymyxin B and were not seen in the P-gp+/TLR4--Caco-2 cell line (control: 771+/-28 versus LPS: 775+/-59 pmoles Rh123/mg protein). Total cellular levels of P-gp did not change in LPS-treated SW480 cells; however, a significant increase in cell surface P-gp was detected. No change in activity, total protein, or apically located MRP-2 was detected following LPS treatment. Sequence analysis confirmed wild-type status of SW480 cells. These data suggest that activation of TLR4 in intestinal epithelial cells leads to an increase in plasma membrane P-gp that demonstrates a diminished capacity to transport substrate. PMID:18687802

  7. Pulse-chase analysis of N-linked sugar chains from glycoproteins in mammalian cells.

    PubMed

    Avezov, Edward; Ron, Efrat; Izenshtein, Yana; Adan, Yosef; Lederkremer, Gerardo Z

    2010-01-01

    Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-(3)H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2

  8. Fluorescent tags to explore cell wall structure and dynamics

    PubMed Central

    Gonneau, Martine; Höfte, Herman; Vernhettes, Samantha

    2012-01-01

    Plant cell walls are highly dynamic and heterogeneous structures, which vary between cell types, growth stages but also between microdomains within a single cell wall. In this review, we summarize the imaging techniques using fluorescent tags that are currently being used and which should in the coming years revolutionize our understanding of the dynamics of cell wall architecture and the cellular processes involved in the synthesis of cell wall components. PMID:22783266

  9. The degradation and turnover of fucosylated glycoproteins in the plasma membrane of a neuroblastoma-cell line

    PubMed Central

    Hudson, James E.; Johnson, Terry C.

    1977-01-01

    When monolayer cultures of neuroblastoma N2a cells were prelabelled with [3H]fucose to steady state, and then reincubated in complete medium in the presence of unlabelled 40mm-l-fucose, there was a rapid metabolism of fucosylated cellular macromolecules and the specific radioactivity of the acid-insoluble material decreased by 22% within 2h. After this period of time the remaining radioactive glycoproteins appeared to be more stable and the rate of loss of specific radioactivity markedly decreased. Since fucose is known to be associated predominantly with plasma-membrane components, the analysis of fucosylated glycoproteins was characterized in plasma-membrane fractions by polyacrylamide-gel electrophoresis. Two experimental approaches were used to measure glycoprotein degradation and turnover in the cell-surface membranes. In one set of experiments, with a similar incubation procedure to that used with intact cells, three membrane components were rapidly degraded (150000, 130000 and 48000 daltons), but another surface glycoprotein (68000 daltons) appeared to be more slowly metabolized than the mean rate of glycoprotein degradation. The relationship of the degradation of membrane glycoproteins to their turnover was analysed by dual-label experiments that used both [14C]fucose and [3H]fucose. Glycoproteins of the surface membrane of neuroblastoma cells were found to turn over at heterogeneous rates. The components mentioned above that exhibited significantly rapid rates of degradation, were also shown to turn over more rapidly than the average surface component. In addition to the membrane components detected by the use of only [3H]fucose, dual-label experiments illustrated that numerous surface glycoproteins were metabolized more rapidly or slowly than most of the cell-surface constituents. PMID:911319

  10. A viral regulator of glycoprotein complexes contributes to human cytomegalovirus cell tropism

    PubMed Central

    Li, Gang; Nguyen, Christopher C.; Ryckman, Brent J.; Britt, William J.; Kamil, Jeremy P.

    2015-01-01

    Viral glycoproteins mediate entry of enveloped viruses into cells and thus play crucial roles in infection. In herpesviruses, a complex of two viral glycoproteins, gH and gL (gH/gL), regulates membrane fusion events and influences virion cell tropism. Human cytomegalovirus (HCMV) gH/gL can be incorporated into two different protein complexes: a glycoprotein O (gO)-containing complex known as gH/gL/gO, and a complex containing UL128, UL130, and UL131 known as gH/gL/UL128-131. Variability in the relative abundance of the complexes in the virion envelope correlates with differences in cell tropism exhibited between strains of HCMV. Nonetheless, the mechanisms underlying such variability have remained unclear. We have identified a viral protein encoded by the UL148 ORF (UL148) that influences the ratio of gH/gL/gO to gH/gL/UL128-131 and the cell tropism of HCMV virions. A mutant disrupted for UL148 showed defects in gH/gL/gO maturation and enhanced infectivity for epithelial cells. Accordingly, reintroduction of UL148 into an HCMV strain that lacked the gene resulted in decreased levels of gH/gL/UL128-131 on virions and, correspondingly, decreased infectivity for epithelial cells. UL148 localized to the endoplasmic reticulum, but not to the cytoplasmic sites of virion envelopment. Coimmunoprecipitation results indicated that gH, gL, UL130, and UL131 associate with UL148, but that gO and UL128 do not. Taken together, the findings suggest that UL148 modulates HCMV tropism by regulating the composition of alternative gH/gL complexes. PMID:25831500

  11. Chemical and immunological analysis of the Aspergillus fumigatus cell wall.

    PubMed

    Hearn, V M; Sietsma, J H

    1994-04-01

    Hyphal-wall preparations of Aspergillus fumigatus have been analysed by sequential treatment with KOH, nitrous acid and again with KOH. By acidification of the alkali-soluble extract, a polyglucose was precipitated which showed an X-ray diffraction pattern similar to that of (1-->3)-alpha-glucan. The remainder of the alkali-soluble fraction was precipitated with ethanol; it contained all the mannose, galactose and protein of the wall and, in addition, 6.2% of the amino sugars. This wall-associated glycoprotein, following SDS-PAGE and immunoblotting, reacted with antisera raised against several mycelial extracts of A. fumigatus. Sera from patients with aspergilloma have antibodies which recognize components of this glycoprotein. The glycoprotein nature of these antigens was shown by their ability to bind Lens culinaris lectin. In addition, the antigen/antibody binding could be disrupted by exposure of antigen to periodate oxidation, hydrolysis with dilute acid or pretreatment with a large excess of an exo-beta-D-galactofuranosidase. The alkali-insoluble fraction consisted of a covalently linked glucan-chitin complex. Nitrous acid treatment, which specifically disrupts glycosidic linkages involving glucosamine, did not solubilize much material but changed the X-ray diffraction pattern from diffuse to a pattern showing the characteristic lines of crystalline (1-->3)-beta-glucan and chitin. Most of the glucan became alkali-soluble after this treatment, and the insoluble residue appeared to contain crystalline chitin. PMID:8012598

  12. A novel human T-leukemia virus type 1 cell-to-cell transmission assay permits definition of SU glycoprotein amino acids important for infectivity.

    PubMed Central

    Delamarre, L; Rosenberg, A R; Pique, C; Pham, D; Dokhélar, M C

    1997-01-01

    Human T-leukemia virus type 1 (HTLV-1) envelope glycoproteins play a major role in viral transmission, which in the case of this virus occurs almost exclusively via cell-to-cell contact. Until very recently, the lack of an HTLV-1 infectivity assay precluded the determination of the HTLV-1 protein domains required for infectivity. Here, we describe an assay which allows the quantitative evaluation of HTLV-1 cell-to-cell transmission in a single round of infection. Using this assay, we demonstrate that in this system, cell-to-cell transmission is at least 100 times more efficient than transmission with free viral particles. We have examined 46 surface (SU) glycoprotein mutants in order to define the amino acids of the HTLV-1 SU glycoprotein required for full infectivity. We demonstrate that these amino acids are distributed along the entire length of the SU glycoprotein, including the N-terminus and C-terminus regions, which have not been previously defined as being important for HTLV-1 glycoprotein function. For most of the mutated glycoproteins, the capacity to mediate cell-to-cell transmission is correlated with the ability to induce formation of syncytia. This result indicates that the fusion capacity is the main factor responsible for infectivity mediated by the HTLV-1 SU envelope glycoprotein, as is the case for other retroviral glycoproteins. However, other factors must also intervene, since two of the mutated glycoproteins were correctly fusogenic but could not mediate cell-to-cell transmission. Existence of this phenotype shows that capacity for fusion is not sufficient to confer infectivity, even in cell-to-cell transmission, and could suggest that postfusion events involve the SU. PMID:8985345

  13. A recombinant measles vaccine virus expressing wild-type glycoproteins: consequences for viral spread and cell tropism.

    PubMed

    Johnston, I C; ter Meulen, V; Schneider-Schaulies, J; Schneider-Schaulies, S

    1999-08-01

    Wild-type, lymphotropic strains of measles virus (MV) and tissue culture-adapted MV vaccine strains possess different cell tropisms. This observation has led to attempts to identify the viral receptors and to characterize the functions of the MV glycoproteins. We have functionally analyzed the interactions of MV hemagglutinin (H) and fusion (F) proteins of vaccine (Edmonston) and wild-type (WTF) strains in different combinations in transfected cells. Cell-cell fusion occurs when both Edmonston F and H proteins are expressed in HeLa or Vero cells. The expression of WTF glycoproteins in HeLa cells did not result in syncytia, yet they fused efficiently with cells of lymphocytic origin. To further investigate the role of the MV glycoproteins in virus cell entry and also the role of other viral proteins in cell tropism, we generated recombinant vaccine MVs containing one or both glycoproteins from WTF. These viruses were viable and grew similarly in lymphocytic cells. Recombinant viruses expressing the WTFH protein showed a restricted spread in HeLa cells but spread efficiently in Vero cells. Parental WTF remained restricted in both cell types. Therefore, not only differential receptor usage but also other cell-specific factors are important in determining MV cell tropism. PMID:10400788

  14. Lactobacillus plantarum L67 glycoprotein protects against cadmium chloride toxicity in RAW 264.7 cells.

    PubMed

    Song, Sooyeon; Oh, Sejong; Lim, Kye-Taek

    2016-03-01

    The food and water we consume may be contaminated with a range of chemicals and heavy metals, such as lead, cadmium, arsenic, chromium, and mercury by accumulation through the food chain. Cadmium is known to be one of the major components in cigarette smoke and can cause lesions in many organs. Some lactobacilli can bind and remove heavy metals such as cadmium, lead, and copper. However, the mechanisms of cadmium toxicity and inhibition by probiotics are not clear. In this study, we demonstrated that glycoprotein (18 kDa) isolated from Lactobacillus plantarum L67 protected RAW 264.7 cells from expression of inflammation-related factors stimulated by cadmium chloride (100 µM). Furthermore, we evaluated the cytotoxicity of cadmium using the MTT assay and intracellular Ca(2+) using fluorescence, and assessed activities of activator protein kinase C (PKC-α), inducible nitric oxide synthase, activator protein (AP)-1, and mitogen-activated protein kinases using immunoblot. Our results indicated that glycoprotein isolated from L. plantarum L67 inhibited intracellular Ca(2+) mobilization. It also significantly suppressed inflammatory factors such as AP-1 (c-Jun and c-Fos), mitogen-activated protein kinases (ERK, JNK, and p38), and inducible nitric oxide synthase. Our findings suggest that the 24-kDa glycoprotein isolated from L. plantarum L67 might be used as a food component for protection of inflammation caused by cadmium ion. PMID:26774722

  15. STREAMLINED METHOD FOR BIOMASS WHOLE-CELL-WALL STRUCTURAL PROFILING

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In wide-ranging research aimed at altering plant cell wall characteristics by conventional breeding or modern genetic methods, one of the biggest problems is in delineating the effects on the cell wall. Plant cell walls are a complex conglomerate of a variety of polysaccharides and lignin. Each comp...

  16. Wall relaxation and the driving forces for cell expansive growth

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1987-01-01

    When water uptake by growing cells is prevented, the turgor pressure and the tensile stress in the cell wall are reduced by continued wall loosening. This process, termed in vivo stress relaxation, provides a new way to study the dynamics of wall loosening and to measure the wall yield threshold and the physiological wall extensibility. Stress relaxation experiments indicate that wall stress supplies the mechanical driving force for wall yielding. Cell expansion also requires water absorption. The driving force for water uptake during growth is created by wall relaxation, which lowers the water potential of the expanding cells. New techniques for measuring this driving force show that it is smaller than believed previously; in elongating stems it is only 0.3 to 0.5 bar. This means that the hydraulic resistance of the water transport pathway is small and that rate of cell expansion is controlled primarily by wall loosening and yielding.

  17. Isolation of human alpha 2HS-glycoprotein synthesized by Sf9 cells.

    PubMed

    Lörincz, Z; Kalabay, L; Cseh, S; Závodszky, P; Arnaud, P; Jakab, L

    1998-01-01

    The human alpha 2HS-glycoprotein is a negative acute phase protein synthesized by hepatocytes. Because of its fragility and the difficulty of its purification, we used recombinant DNA techniques to produce the protein in order to investigate its biological effects. The cDNA coding for the whole alpha 2HS-glycoprotein from human liver library had been cloned into the baculovirus expression vector system using the pVL 1392 transfer vector and Sf9 cells. The recombinant protein was synthesized in the Sf9 cells and was isolated on a hydroxiapatite column from the culture medium. Western blot analysis indicated that the cells synthesized large quantities of the recombinant human protein. The molecular mass of the recombinant AHSG was the same as that of the protein in human plasma but was slightly lower than that of AHSG in the cell culture supernatants of HepG2 and higher than that of AHSG from Hep3B cell cultures, respectively. PMID:9873947

  18. Plant cell wall proteomics: the leadership of Arabidopsis thaliana

    PubMed Central

    Albenne, Cécile; Canut, Hervé; Jamet, Elisabeth

    2013-01-01

    Plant cell wall proteins (CWPs) progressively emerged as crucial components of cell walls although present in minor amounts. Cell wall polysaccharides such as pectins, hemicelluloses, and cellulose represent more than 90% of primary cell wall mass, whereas hemicelluloses, cellulose, and lignins are the main components of lignified secondary walls. All these polymers provide mechanical properties to cell walls, participate in cell shape and prevent water loss in aerial organs. However, cell walls need to be modified and customized during plant development and in response to environmental cues, thus contributing to plant adaptation. CWPs play essential roles in all these physiological processes and particularly in the dynamics of cell walls, which requires organization and rearrangements of polysaccharides as well as cell-to-cell communication. In the last 10 years, plant cell wall proteomics has greatly contributed to a wider knowledge of CWPs. This update will deal with (i) a survey of plant cell wall proteomics studies with a focus on Arabidopsis thaliana; (ii) the main protein families identified and the still missing peptides; (iii) the persistent issue of the non-canonical CWPs; (iv) the present challenges to overcome technological bottlenecks; and (v) the perspectives beyond cell wall proteomics to understand CWP functions. PMID:23641247

  19. Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

    PubMed Central

    Veillette, Maxime; Désormeaux, Anik; Roger, Michel; Finzi, Andrés

    2014-01-01

    HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies. PMID:25286159

  20. Plant cell wall lignification and monolignol metabolism

    PubMed Central

    Wang, Yin; Chantreau, Maxime; Sibout, Richard; Hawkins, Simon

    2013-01-01

    Plants are built of various specialized cell types that differ in their cell wall composition and structure. The cell walls of certain tissues (xylem, sclerenchyma) are characterized by the presence of the heterogeneous lignin polymer that plays an essential role in their physiology. This phenolic polymer is composed of different monomeric units – the monolignols – that are linked together by several covalent bonds. Numerous studies have shown that monolignol biosynthesis and polymerization to form lignin are tightly controlled in different cell types and tissues. However, our understanding of the genetic control of monolignol transport and polymerization remains incomplete, despite some recent promising results. This situation is made more complex since we know that monolignols or related compounds are sometimes produced in non-lignified tissues. In this review, we focus on some key steps of monolignol metabolism including polymerization, transport, and compartmentation. As well as being of fundamental interest, the quantity of lignin and its nature are also known to have a negative effect on the industrial processing of plant lignocellulose biomass. A more complete view of monolignol metabolism and the relationship that exists between lignin and other monolignol-derived compounds thereby appears essential if we wish to improve biomass quality. PMID:23847630

  1. Analysis of COPII Vesicles Indicates a Role for the Emp47-Ssp120 Complex in Transport of Cell Surface Glycoproteins.

    PubMed

    Margulis, Neil G; Wilson, Joshua D; Bentivoglio, Christine M; Dhungel, Nripesh; Gitler, Aaron D; Barlowe, Charles

    2016-03-01

    Coat protein complex II (COPII) vesicle formation at the endoplasmic reticulum (ER) transports nascent secretory proteins forward to the Golgi complex. To further define the machinery that packages secretory cargo and targets vesicles to Golgi membranes, we performed a comprehensive proteomic analysis of purified COPII vesicles. In addition to previously known proteins, we identified new vesicle proteins including Coy1, Sly41 and Ssp120, which were efficiently packaged into COPII vesicles for trafficking between the ER and Golgi compartments. Further characterization of the putative calcium-binding Ssp120 protein revealed a tight association with Emp47 and in emp47Δ cells Ssp120 was mislocalized and secreted. Genetic analyses demonstrated that EMP47 and SSP120 display identical synthetic positive interactions with IRE1 and synthetic negative interactions with genes involved in cell wall assembly. Our findings support a model in which the Emp47-Ssp120 complex functions in transport of plasma membrane glycoproteins through the early secretory pathway. PMID:26650540

  2. Differential Expression of O-glycoprotein Glycans in Cholangiocarcinoma Cell Lines

    PubMed Central

    Talabnin, Krajang; Talabnin, Chutima; Ishihara, Mayumi; Azadi, Parastoo; Wongkham, Sopit; Sripa, Banchob

    2016-01-01

    Protein glycosylation is the most common posttranslational modification in mammalian cells. Aberrant protein glycosylation has been reported in various diseases, including cancer. We identified and quantified the glycan structures of O-linked glycoprotein from cholangiocarcinoma (CCA) cell lines from different histological types and compared their profiles by nanospray ionization-linear ion trap mass spectrometry (NSI-MSn). Five human CCA cell lines, K100, M055, M139, M213 and M214 were characterized. The results showed that the O-linked glycans of the CCA cell lines comprised tri- to hexa-saccharides with terminal galactose and sialic acids: NeuAc1Gal1GalNAc1, Gal2GlcNAc1GalNAc1, NeuAc2Gal1GalNAc1 NeuAc1Gal2GlcNAc1GalNAc1 and NeuAc2Gal2GlcNAc1GalNAc1 All five CCA cell lines showed a similar glycan pattern, but with differences in their quantities. NeuAc1Gal1GalNAc1 proved to be the most abundant structure in poorly differentiated adenocarcinoma (K100; 57.1%), moderately differentiated adenocarcinoma (M055; 42.6%) and squamous cell carcinoma (M139; 43.0%), while moderately to poorly differentiated adenocarcinoma (M214; 40.1%) and adenosquamous cell carcinoma (M213; 34.7%) appeared dominated by NeuAc2Gal1GalNAc1. These results demonstrate differential expression of the O-linked glycans in the different histological types of CCA. All five CCA cell lines have abundant terminal sialic acid (NeuAc) O-linked glycans, suggesting an important role for sialic acid in cancer cells. Our structural analyses of glycans may provide important information regarding physiology of disease-related glycoproteins in CCA. PMID:26925665

  3. IRES-mediated translation of membrane proteins and glycoproteins in eukaryotic cell-free systems.

    PubMed

    Brödel, Andreas K; Sonnabend, Andrei; Roberts, Lisa O; Stech, Marlitt; Wüstenhagen, Doreen A; Kubick, Stefan

    2013-01-01

    Internal ribosome entry site (IRES) elements found in the 5' untranslated region of mRNAs enable translation initiation in a cap-independent manner, thereby representing an alternative to cap-dependent translation in cell-free protein expression systems. However, IRES function is largely species-dependent so their utility in cell-free systems from different species is rather limited. A promising approach to overcome these limitations would be the use of IRESs that are able to recruit components of the translation initiation apparatus from diverse origins. Here, we present a solution to this technical problem and describe the ability of a number of viral IRESs to direct efficient protein expression in different eukaryotic cell-free expression systems. The IRES from the intergenic region (IGR) of the Cricket paralysis virus (CrPV) genome was shown to function efficiently in four different cell-free systems based on lysates derived from cultured Sf21, CHO and K562 cells as well as wheat germ. Our results suggest that the CrPV IGR IRES-based expression vector is universally applicable for a broad range of eukaryotic cell lysates. Sf21, CHO and K562 cell-free expression systems are particularly promising platforms for the production of glycoproteins and membrane proteins since they contain endogenous microsomes that facilitate the incorporation of membrane-spanning proteins and the formation of post-translational modifications. We demonstrate the use of the CrPV IGR IRES-based expression vector for the enhanced synthesis of various target proteins including the glycoprotein erythropoietin and the membrane proteins heparin-binding EGF-like growth factor receptor as well as epidermal growth factor receptor in the above mentioned eukaryotic cell-free systems. CrPV IGR IRES-mediated translation will facilitate the development of novel eukaryotic cell-free expression platforms as well as the high-yield synthesis of desired proteins in already established systems. PMID

  4. Cell wall sorting of lipoproteins in Staphylococcus aureus.

    PubMed Central

    Navarre, W W; Daefler, S; Schneewind, O

    1996-01-01

    Many surface proteins are thought to be anchored to the cell wall of gram-positive organisms via their C termini, while the N-terminal domains of these molecules are displayed on the bacterial surface. Cell wall anchoring of surface proteins in Staphylococcus aureus requires both an N-terminal leader peptide and a C-terminal cell wall sorting signal. By fusing the cell wall sorting of protein A to the C terminus of staphylococcal beta-lactamase, we demonstrate here that lipoproteins can also be anchored to the cell wall of S. aureus. The topology of cell wall-anchored beta-lactamase is reminiscent of that described for Braun's murein lipoprotein in that the N terminus of the polypeptide chain is membrane anchored whereas the C-terminal end is tethered to the bacterial cell wall. PMID:8550464

  5. Atypical nuclear localization of CD133 plasma membrane glycoprotein in rhabdomyosarcoma cell lines.

    PubMed

    Nunukova, Alena; Neradil, Jakub; Skoda, Jan; Jaros, Josef; Hampl, Ales; Sterba, Jaroslav; Veselska, Renata

    2015-07-01

    CD133 (also known as prominin-1) is a cell surface glycoprotein that is widely used for the identification of stem cells. Furthermore, its glycosylated epitope, AC133, has recently been discussed as a marker of cancer stem cells in various human malignancies. During our recent experiments on rhabdomyosarcomas (RMS), we unexpectedly identified an atypical nuclear localization of CD133 in a relatively stable subset of cells in five RMS cell lines established in our laboratory. To the best of our knowledge, this atypical localization of CD133 has not yet been proven or analyzed in detail in cancer cells. In the present study, we verified the nuclear localization of CD133 in RMS cells using three independent anti-CD133 antibodies, including both rabbit polyclonal and mouse monoclonal antibodies. Indirect immunofluorescence and confocal microscopy followed by software cross-section analysis, transmission electron microscopy and cell fractionation with immunoblotting were also employed, and all the results undeniably confirmed the presence of CD133 in the nuclei of stable minor subpopulations of all five RMS cell lines. The proportion of cells showing an exclusive nuclear localization of CD133 ranged from 3.4 to 7.5%, with only minor differences observed among the individual anti-CD133 antibodies. Although the role of CD133 in the cell nucleus remains unclear, these results clearly indicate that this atypical nuclear localization of CD133 in a minor subpopulation of cancer cells is a common phenomenon in RMS cell lines. PMID:25977066

  6. Revealing Glycoproteins in the Secretome of MCF-7 Human Breast Cancer Cells

    PubMed Central

    Tan, Aik-Aun; Phang, Wai-Mei; Gopinath, Subash C. B.; Hashim, Onn H.; Kiew, Lik Voon; Chen, Yeng

    2015-01-01

    Breast cancer is one of the major issues in the field of oncology, reported with a higher prevalence rate in women worldwide. In attempt to reveal the potential biomarkers for breast cancer, the findings of differentially glycosylated haptoglobin and osteonectin in previous study have drawn our attention towards glycoproteins of secretome from the MCF-7 cancer cell line. In the present study, further analyses were performed on the medium of MCF-7 cells by subjecting it to two-dimensional analyses followed by image analysis in contrast to the medium of human mammary epithelial cells (HMEpC) as a negative control. Carboxypeptidase A4 (CPA4), alpha-1-antitrypsin (AAT), haptoglobin (HP), and HSC70 were detected in the medium of MCF-7, while only CPA4 and osteonectin (ON) were detected in HMEpC medium. In addition, CPA4 was detected as upregulated in the MCF-7 medium. Further analysis by lectin showed that CPA4, AAT, HP, and HSC70 were secreted as N-glycan in the medium of MCF-7, with HP also showing differentially N-glycosylated isoforms. For the HMEpC, only CPA4 was detected as N-glycan. No O-glycan was detected in the medium of HMEpC but MCF-7 expressed O-glycosylated CPA4 and HSC70. All these revealed that glycoproteins could be used as glycan-based biomarkers for the prognosis of breast cancer. PMID:26167486

  7. Exploiting fungal cell wall components in vaccines

    PubMed Central

    Levitz, Stuart M.; Huang, Haibin; Ostroff, Gary R.; Specht, Charles A.

    2014-01-01

    Innate recognition of fungi leads to strong adaptive immunity. Investigators are trying to exploit this observation in vaccine development by combining antigens with evolutionarily conserved fungal cell wall carbohydrates to induce protective responses. Best studied is β-1,3-glucan, a glycan that activates complement and is recognized by Dectin-1. Administration of antigens in association with β-1,3-glucan, either by direct conjugation or complexed in glucan particles, results in robust humoral and cellular immune responses. While the host has a host of mannose receptors, responses to fungal mannoproteins generally are amplified if cells are cooperatively stimulated with an additional danger signal such as a toll-like receptor agonist. Chitosan, a polycationic homopolymer of glucosamine manufactured by the deacetylation of chitin, is being studied as an adjuvant in DNA and protein-based vaccines. It appears particularly promising in mucosal vaccines. Finally, universal and organism-specific fungal vaccines have been formulated by conjugating fungal cell wall glycans to carrier proteins. A major challenge will be to advance these experimental findings so that at risk patients can be protected. PMID:25404118

  8. Hormonal regulation of secondary cell wall formation.

    PubMed

    Didi, Vojtěch; Jackson, Phil; Hejátko, Jan

    2015-08-01

    Secondary cell walls (SCWs) have critical functional importance but also constitute a high proportion of the plant biomass and have high application potential. This is true mainly for the lignocellulosic constituents of the SCWs in xylem vessels and fibres, which form a structured layer between the plasma membrane and the primary cell wall (PCW). Specific patterning of the SCW thickenings contributes to the mechanical properties of the different xylem cell types, providing the plant with mechanical support and facilitating the transport of solutes via vessels. In the last decade, our knowledge of the basic molecular mechanisms controlling SCW formation has increased substantially. Several members of the multi-layered regulatory cascade participating in the initiation and transcriptional regulation of SCW formation have been described, and the first cellular components determining the pattern of SCW at the subcellular resolution are being uncovered. The essential regulatory role of phytohormones in xylem development is well known and the molecular mechanisms that link hormonal signals to SCW formation are emerging. Here, we review recent knowledge about the role of individual plant hormones and hormonal crosstalk in the control over the regulatory cascades guiding SCW formation and patterning. Based on the analogy between many of the mechanisms operating during PCW and SCW formation, recently identified mechanisms underlying the hormonal control of PCW remodelling are discussed as potentially novel mechanisms mediating hormonal regulatory inputs in SCW formation. PMID:26002972

  9. Exploiting fungal cell wall components in vaccines.

    PubMed

    Levitz, Stuart M; Huang, Haibin; Ostroff, Gary R; Specht, Charles A

    2015-03-01

    Innate recognition of fungi leads to strong adaptive immunity. Investigators are trying to exploit this observation in vaccine development by combining antigens with evolutionarily conserved fungal cell wall carbohydrates to induce protective responses. Best studied is β-1,3-glucan, a glycan that activates complement and is recognized by dectin-1. Administration of antigens in association with β-1,3-glucan, either by direct conjugation or complexed in glucan particles, results in robust humoral and cellular immune responses. While the host has a host of mannose receptors, responses to fungal mannoproteins generally are amplified if cells are cooperatively stimulated with an additional danger signal such as a toll-like receptor agonist. Chitosan, a polycationic homopolymer of glucosamine manufactured by the deacetylation of chitin, is being studied as an adjuvant in DNA and protein-based vaccines. It appears particularly promising in mucosal vaccines. Finally, universal and organism-specific fungal vaccines have been formulated by conjugating fungal cell wall glycans to carrier proteins. A major challenge will be to advance these experimental findings so that at risk patients can be protected. PMID:25404118

  10. Accessory human cytomegalovirus glycoprotein US9 in the unique short component of the viral genome promotes cell-to-cell transmission of virus in polarized epithelial cells.

    PubMed Central

    Maidji, E; Tugizov, S; Jones, T; Zheng, Z; Pereira, L

    1996-01-01

    Human cytomegalovirus (CMV) encodes accessory glycoproteins that are dispensable for virus growth in nonpolarized cells in culture. We report that CMV deletion mutants lacking the gene for accessory glycoprotein US9 in the unique short component of the viral genome are impaired in plaque formation in polarized human retinal pigment epithelial (ARPE-19) cells. Comparison of CMV deletion mutants in US9 with herpes simplex virus type 1 deletion mutants lacking glycoproteins gE and gI showed that both of these mutants are impaired in altering junctional complexes and increasing paracellular permeability in polarized ARPE-19 cells cultured on permeable filter supports. Results of functional studies indicate that CMV US9 and homologs of gE have analogous roles in promoting virus spread across lateral membranes of polarized epithelial cells. PMID:8970961

  11. Plant and algal cell walls: diversity and functionality

    PubMed Central

    Popper, Zoë A.; Ralet, Marie-Christine; Domozych, David S.

    2014-01-01

    Background Although plants and many algae (e.g. the Phaeophyceae, brown, and Rhodophyceae, red) are only very distantly related they are united in their possession of carbohydrate-rich cell walls, which are of integral importance being involved in many physiological processes. Furthermore, wall components have applications within food, fuel, pharmaceuticals, fibres (e.g. for textiles and paper) and building materials and have long been an active topic of research. As shown in the 27 papers in this Special Issue, as the major deposit of photosynthetically fixed carbon, and therefore energy investment, cell walls are of undisputed importance to the organisms that possess them, the photosynthetic eukaryotes (plants and algae). The complexities of cell wall components along with their interactions with the biotic and abiotic environment are becoming increasingly revealed. Scope The importance of plant and algal cell walls and their individual components to the function and survival of the organism, and for a number of industrial applications, are illustrated by the breadth of topics covered in this issue, which includes papers concentrating on various plants and algae, developmental stages, organs, cell wall components, and techniques. Although we acknowledge that there are many alternative ways in which the papers could be categorized (and many would fit within several topics), we have organized them as follows: (1) cell wall biosynthesis and remodelling, (2) cell wall diversity, and (3) application of new technologies to cell walls. Finally, we will consider future directions within plant cell wall research. Expansion of the industrial uses of cell walls and potentially novel uses of cell wall components are both avenues likely to direct future research activities. Fundamentally, it is the continued progression from characterization (structure, metabolism, properties and localization) of individual cell wall components through to defining their roles in almost every

  12. Metabolic Labeling of Caenorhabditis elegans Primary Embryonic Cells with Azido-Sugars as a Tool for Glycoprotein Discovery

    PubMed Central

    Burnham-Marusich, Amanda R.; Snodgrass, Casey J.; Johnson, Anna M.; Kiyoshi, Conrad M.; Buzby, Sarah E.; Gruner, Matt R.; Berninsone, Patricia M.

    2012-01-01

    Glycobiology research with Caenorhabditis elegans (C. elegans) has benefitted from the numerous genetic and cell biology tools available in this system. However, the lack of a cell line and the relative inaccessibility of C. elegans somatic cells in vivo have limited the biochemical approaches available in this model. Here we report that C. elegans primary embryonic cells in culture incorporate azido-sugar analogs of N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc), and that the labeled glycoproteins can be analyzed by mass spectrometry. By using this metabolic labeling approach, we have identified a set of novel C. elegans glycoprotein candidates, which include several mitochondrially-annotated proteins. This observation was unexpected given that mitochondrial glycoproteins have only rarely been reported, and it suggests that glycosylation of mitochondrially-annotated proteins might occur more frequently than previously thought. Using independent experimental strategies, we validated a subset of our glycoprotein candidates. These include a mitochondrial, atypical glycoprotein (ATP synthase α-subunit), a predicted glycoprotein (aspartyl protease, ASP-4), and a protein family with established glycosylation in other species (actin). Additionally, we observed a glycosylated isoform of ATP synthase α-subunit in bovine heart tissue and a primate cell line (COS-7). Overall, our finding that C. elegans primary embryonic cells are amenable to metabolic labeling demonstrates that biochemical studies in C. elegans are feasible, which opens the door to labeling C. elegans cells with other radioactive or azido-substrates and should enable the identification of additional post-translationally modified targets and analysis of the genes required for their modification using C. elegans mutant libraries. PMID:23152843

  13. Enzymes and other agents that enhance cell wall extensibility

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1999-01-01

    Polysaccharides and proteins are secreted to the inner surface of the growing cell wall, where they assemble into a network that is mechanically strong, yet remains extensible until the cells cease growth. This review focuses on the agents that directly or indirectly enhance the extensibility properties of growing walls. The properties of expansins, endoglucanases, and xyloglucan transglycosylases are reviewed and their postulated roles in modulating wall extensibility are evaluated. A summary model for wall extension is presented, in which expansin is a primary agent of wall extension, whereas endoglucanases, xyloglucan endotransglycosylase, and other enzymes that alter wall structure act secondarily to modulate expansin action.

  14. Transient expression of Fc-fused human glycoprotein 130 in Expi293F suspension cells.

    PubMed

    Zhao, Xiaozhi; Chen, Wei; Ge, Liyuan; Jiang, Wei; Tang, Bo; Zhang, Qing; Xu, Xiaoyu; Wang, Chong; Cao, Lin; Guo, Hongqian

    2016-08-01

    Human glycoprotein 130 (gp130) is a signal-transducing receptor for interleukin 6 (IL-6), whose signaling plays a critical role in chronic inflammation and cancer. The soluble form of gp130 specifically inhibits IL-6 trans-signaling. However, achieving high-level expression of a large glycoprotein such as gp130 is difficult. Here, we designed and constructed one Fc-gp130-pcDNA mammalian expression vector, with the mouse IgG2a Fc fragment added to the N-terminus of human gp130, which greatly increased the secretion of recombinant gp130 protein from Expi293F suspension cells. Recombinant fusion Fc-gp130 was easily and efficiently purified from the supernatant of transfected cells by one-step affinity chromatography. Moreover, Fc-gp130 could automatically form dimers by the disulfide bond. Fc-gp130 was confirmed as a more efficient IL-6 trans-signaling blocker by its higher biological activity against signal transducer and activator of transcription 3 (STAT3). This purified active Fc-gp130 could be used to develop valuable therapeutic agents against inflammatory diseases and cancers. PMID:27113713

  15. Anthocyanins influence tannin-cell wall interactions.

    PubMed

    Bautista-Ortín, Ana Belén; Martínez-Hernández, Alejandro; Ruiz-García, Yolanda; Gil-Muñoz, Rocío; Gómez-Plaza, Encarna

    2016-09-01

    The rate of tannin extraction was studied in a vinification of red grapes and the results compared with another vinification made with white grapes fermented as for typical red wine, in the presence of skins and seeds. Even though the grapes presented a quite similar skin and seed tannin content, the differences in tannin concentration between both vinifications was very large, despite the fact that the only apparent difference between the phenolic composition of both wines was the anthocyanin content. This suggests that anthocyanins play an important role in tannin extractability, perhaps because they affect the extent of the tannin-cell wall interaction, a factor that largely controls the resulting quantity of tannins in wines. To confirm this observation, the effect of anthocyanins on the tannin extractability from grape seeds and skin and on the interaction between tannins and grape cell walls suspended in model solutions were studied. The results indicated that anthocyanins favored skin and seed tannin extraction and that there is a competition for the adsorption sites between anthocyanins and tannins that increases the tannin content when anthocyanins are present. PMID:27041322

  16. Disruption of cell walls for enhanced lipid recovery

    SciTech Connect

    Knoshaug, Eric P; Donohoe, Bryon S; Gerken, Henri; Laurens, Lieve; Van Wychen, Stefanie Rose

    2015-03-24

    Presented herein are methods of using cell wall degrading enzymes for recovery of internal lipid bodies from biomass sources such as algae. Also provided are algal cells that express at least one exogenous gene encoding a cell wall degrading enzyme and methods for recovering lipids from the cells.

  17. Plant cell wall dynamics and wall-related susceptibility in plant–pathogen interactions

    PubMed Central

    Bellincampi, Daniela; Cervone, Felice; Lionetti, Vincenzo

    2014-01-01

    The cell wall is a dynamic structure that often determines the outcome of the interactions between plants and pathogens. It is a barrier that pathogens need to breach to colonize the plant tissue. While fungal necrotrophs extensively destroy the integrity of the cell wall through the combined action of degrading enzymes, biotrophic fungi require a more localized and controlled degradation of the cell wall in order to keep the host cells alive and utilize their feeding structures. Also bacteria and nematodes need to degrade the plant cell wall at a certain stage of their infection process, to obtain nutrients for their growth. Plants have developed a system for sensing pathogens and monitoring the cell wall integrity, upon which they activate defense responses that lead to a dynamic cell wall remodeling required to prevent the disease. Pathogens, on the other hand, may exploit the host cell wall metabolism to support the infection. We review here the strategies utilized by both plants and pathogens to prevail in the cell wall battleground. PMID:24904623

  18. Shifting foundations: the mechanical cell wall and development.

    PubMed

    Braybrook, Siobhan A; Jönsson, Henrik

    2016-02-01

    The cell wall has long been acknowledged as an important physical mediator of growth in plants. Recent experimental and modelling work has brought the importance of cell wall mechanics into the forefront again. These data have challenged existing dogmas that relate cell wall structure to cell/organ growth, that uncouple elasticity from extensibility, and those which treat the cell wall as a passive and non-stressed material. Within this review we describe experiments and models which have changed the ways in which we view the mechanical cell wall, leading to new hypotheses and research avenues. It has become increasingly apparent that while we often wish to simplify our systems, we now require more complex multi-scale experiments and models in order to gain further insight into growth mechanics. We are currently experiencing an exciting and challenging shift in the foundations of our understanding of cell wall mechanics in growth and development. PMID:26799133

  19. Plant cell wall extensibility: connecting plant cell growth with cell wall structure, mechanics, and the action of wall-modifying enzymes.

    PubMed

    Cosgrove, Daniel J

    2016-01-01

    The advent of user-friendly instruments for measuring force/deflection curves of plant surfaces at high spatial resolution has resulted in a recent outpouring of reports of the 'Young's modulus' of plant cell walls. The stimulus for these mechanical measurements comes from biomechanical models of morphogenesis of meristems and other tissues, as well as single cells, in which cell wall stress feeds back to regulate microtubule organization, auxin transport, cellulose deposition, and future growth directionality. In this article I review the differences between elastic modulus and wall extensibility in the context of cell growth. Some of the inherent complexities, assumptions, and potential pitfalls in the interpretation of indentation force/deflection curves are discussed. Reported values of elastic moduli from surface indentation measurements appear to be 10- to >1000-fold smaller than realistic tensile elastic moduli in the plane of plant cell walls. Potential reasons for this disparity are discussed, but further work is needed to make sense of the huge range in reported values. The significance of wall stress relaxation for growth is reviewed and connected to recent advances and remaining enigmas in our concepts of how cellulose, hemicellulose, and pectins are assembled to make an extensible cell wall. A comparison of the loosening action of α-expansin and Cel12A endoglucanase is used to illustrate two different ways in which cell walls may be made more extensible and the divergent effects on wall mechanics. PMID:26608646

  20. Alteration of N-glycoproteins/N-glycosites in human hepatic stellate cells activated with transforming growth factor-β1.

    PubMed

    Qin, Y; Wang, Q; Zhong, Y; Zhao, F; Wu, F; Wang, Y; Ma, T; Liu, C; Bian, H; Li, Z

    2016-01-01

    Proteins N-glycosylation is significantly increased in the activated human hepatic stellate cells (HSCs) stimulated by transforming growth factor-β1 (TGF-β1) compared to the quiescent HSCs according to our previous study. However, little is known about the alteration of N-glycoprotein profiles in the activated HSCs. Profiles of N-glycopeptides / N-glycoproteins / N-glycosites in LX-2 cells, with and without activation by TGF-β1, were identified and compared using hydrazide chemistry enrichment coupled with liquid chromatography - mass spectrometry analysis. Western blot and immunohistochemistry were further used for validation. A total of 103 non-redundant N-glycopeptides, with 107 glycosylation sites from 86 N-glycoproteins, were identified in activated and quiescent LX-2 cells respectively. Among these, 23 proteins were known N-glycoproteins, and 58 were newly identified N-glycoproteins. In addition, 43 proteins (e.g., pigment epithelium-derived factor and clathrin heavy chain 1) were solely identified or up-regulated in the activated LX-2 cells, which participated in focal adhesion and glycosaminoglycan degradation pathways and were involved in interaction clusters of cytoskeletal proteins (e.g., myosin light chains and keratins). The increased expression of glucosamine (N-acetyl)-6-sulfatase and phospholipase C beta 2 and the decreased expression of zinc finger and BTB domain-containing protein 1 were validated in the activated compared to the quiescent LX-2 cells. In conclusion, increased expression of N-glycoproteins and N-glycosites play important roles in cellular contractility, signal transduction, and responses to stimuli in the activated HSCs, which might provide useful information for discovering novel molecular mechanism of HSC activation and therapeutic targets in liver fibrosis. PMID:27064874

  1. Disintegrin-like domain of glycoprotein B regulates Kaposi's sarcoma-associated herpesvirus infection of cells.

    PubMed

    Walker, Lia R; Hussein, Hosni A M; Akula, Shaw M

    2014-08-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) glycoprotein B (gB) is a lytic structural protein expressed on the envelope of mature virions and on the membrane of cells supporting lytic infection. In addition to this viral glycoprotein's interaction with integrins via its RGD (Arg-Gly-Asp) motif, KSHV gB possesses a disintegrin-like domain (DLD), which binds integrins as well. Prior to this study, there has been minimal research involving the less common integrin-binding motif, DLD, of gB as it pertains to herpesvirus infection. By using phage display peptide library screening and molecular biology techniques, the DLD of KSHV gB was shown to interact specifically with non-RGD binding α9β1 integrins. Similarly, monitoring wild-type infection confirmed α9β1:DLD interactions to be critical to successful KSHV infection of human foreskin fibroblast (HFF) cells and human dermal microvascular endothelial cells (HMVEC-d) compared with 293 cells. To further demonstrate the importance of the DLD of gB in KSHV infection, two recombinant virus constructs were generated using a bacterial artificial chromosome (BAC) system harbouring the KSHV genome (BAC36): BAC36ΔD-KSHV (lacking a functionally intact DLD of gB and containing an introduced tetracycline cassette) and BAC36.T-KSHV (containing an intact DLD sequence and an introduced tetracycline cassette). Accordingly, BAC36ΔD-KSHV presented significantly lower infection rates in HFF and HMVEC-d cells compared with the comparable infection rates achieved by wild-type BAC36-KSHV and BAC36.T-KSHV. Thus, the present report has delineated a critical role for the DLD of gB in KSHV infection, which may lead to a broader knowledge regarding the sophisticated mechanisms utilized by virus-encoded structural proteins in KSHV entry and infection. PMID:24814923

  2. Studies on N-linked glycoprotein synthesis in differentiating muscle cells

    SciTech Connect

    Miller, K.R.

    1986-01-01

    All N-linked glycoproteins share a common pathway with respect to the acquisition of their oligosaccharide chains. Isolated membranes from undifferentiated (UND) and differentiated (DIF) C/sub 2/ cells, which have the capability of differentiating from mononucleated myoblasts to contracting myotubes, were utilized to examine dolichol-linked oligosaccharide synthesis. A characterization of the glycosyltransferases involved in the early stages of lipid-linked oligosaccaride synthesis revealed that while UND cells demonstrated a greater ability to synthesize Dol-PP-GN/sub (1-2), Dol-P-Man, and Dol-P-Glc than did DIF cells, the presence of exogenous Dol-P plus detergent either reversed or equalized product formation. The ability to synthesize the larger dolichol-oligosaccharides was demonstrated both in whole cells and in isolated membranes from UND and DIF cells. Pulse-chase experiments, using (/sup 3/H)-glucosamine to metabolically label the N-acetylglucosamine residues demonstrated the precursor-product relationship between the dolichol-oligosaccharide intermediates in whole cell studies. DIF cells appear to be more efficient than UND cells for extending the smaller oligosaccharide intermediates to the tetradecasaccharide which would be transferred to protein. Membranes isolated from cells metabolically labeled with (/sup 3/H)-mannose were subject to pronase digestion, and the resulting glycopeptide analyzed by serial lectin affinity chromatography.

  3. Capsosiphon fulvescens glycoprotein inhibits AGS gastric cancer cell proliferation by downregulating Wnt-1 signaling.

    PubMed

    Kim, Young-Min; Kim, In-Hye; Nam, Taek-Jeong

    2013-11-01

    Previously, we examined various apoptosis pathways in the AGS gastric cancer cell line using Capsosiphon fulvescens glycoprotein (Cf-GP). In this study, we focused on the downregulation of the Wnt-1 signaling pathway and cell cycle arrest. Upregulation of the Wnt signaling pathway has been observed in various cancer cells. The Wnt signal ligand acts in both canonical and non-canonical pathways. Among them, Wnt-1 was dependent on the canonical pathway. Here, we show inhibition of Wnt-1 signaling, β-catenin and transcription factors in AGS cells via Cf-GP. First, we examined the Frizzled receptor and Wnt-1 signal-related proteins including Axin, LRP, β-catenin, APC and GSK-3β. In addition, the expression levels of transcription factors Tcf/LEF were determined by western blot analysis and RT-PCR. Based on the data, we confirmed downregulation of the Wnt-1 signaling pathway by Cf-GP. Also, we determined the expression levels of cell cycle-related proteins cyclin D and c-myc, and looked for cell cycle arrest by cell cycle test analysis. We found that AGS cells arrested in the G0/G1 phase by Cf-GP. These results provide a mechanism of AGS cell inhibition through the downregulation of Wnt-1 signaling by Cf-GP. PMID:23982808

  4. DUSP1 induces paclitaxel resistance through the regulation of p-glycoprotein expression in human ovarian cancer cells.

    PubMed

    Kang, Yu-Seon; Seok, Hyun-Jeong; Jeong, Eun-Jeong; Kim, Yuna; Yun, Seok-Joong; Min, Jeong-Ki; Kim, Sun Jin; Kim, Jang-Seong

    2016-09-01

    The heterogeneity and genetic instability of ovarian cancer cells often lead to the development of drug resistance, closely related with the increased cancer-related mortality. In this study, we investigated the role of dual-specificity phosphatase 1 (DUSP1) in the development of the resistance in human ovarian cancer cells against paclitaxel. Overexpression of DUSP1 in HeyA8 human ovarian cancer cells (HeyA8-DUSP1) up-regulated the expression of the drug efflux pump, p-glycoprotein. Consequently, HeyA8-DUSP1 cells are highly resistant to paclitaxel, with the resistance comparable to that of a multi-drug resistance cell line (HeyA8-MDR). Moreover, over expression of DUSP1 significantly increased the activation of p38 MAPK, leaving the activation of ERK1/2 and JNK1/2 unaffected. Pharmacological suppression of p38 MAPK activity prevents the up-regulation of p-glycoprotein expression and the consequent resistance against paclitaxel in HeyA8-DUSP1 cells. By contrast, HeyA8-MDR cells expressed a significantly higher level of DUSP1, but treatment with small interference RNA against DUSP1 significantly suppressed the expression of p-glycoprotein and the resistance against paclitaxel in HeyA8-MDR cells. Ectopic expression of MKK3, an upstream activator of p38 MAPK, significantly up-regulated the expression of p-glycoprotein and increased the consequent resistance against paclitaxel in HeyA8 cells. Collectively, these data indicated that DUSP1 may induce the resistance against paclitaxel through the p38 MAPK-mediated overexpression of p-glycoprotein in human ovarian cancer cells. PMID:27422607

  5. The Insulin Degrading Enzyme Binding Domain of Varicella-Zoster Virus (VZV) Glycoprotein E is Important for Cell-to-Cell Spread and VZV Infectivity, while a Glycoprotein I Binding Domain is Essential for Infection

    PubMed Central

    Ali, Mir A.; Li, Qingxue; Fischer, Elizabeth R.; Cohen, Jeffrey I.

    2009-01-01

    Varicella-zoster virus (VZV) glycoprotein E (gE) interacts with glycoprotein I and with insulin degrading enzyme (IDE), which is a receptor for the virus. We found that a VZV gE deletion mutant could only be grown in cells expressing gE. Expression of VZV gE on the surface of cells did not interfere with VZV infection. HSV deleted for gE is impaired for cell-to-cell spread; VZV gE could not complement this activity in an HSV gE null mutant. VZV lacking the IDE binding domain of gE grew to peak titers nearly equivalent to parental virus; however, it was impaired for cell-to-cell spread and for infectivity with cell-free virus. VZV deleted for a region of gE that binds glycoprotein I could not replicate in cell culture unless grown in cells expressing gE. We conclude that the IDE binding domain is important for efficient cell-to-cell spread and infectivity of cell-free virus. PMID:19233447

  6. Two endogenous proteins that induce cell wall extension in plants

    NASA Technical Reports Server (NTRS)

    McQueen-Mason, S.; Durachko, D. M.; Cosgrove, D. J.

    1992-01-01

    Plant cell enlargement is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive "wall-loosening" enzymes. By employing a reconstitution approach, we found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicot stems and the leaves of amaryllidaceous monocots, but was less effective on grass coleoptile walls. Endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions, thiol reducing agents, proteases, and boiling in methanol or water. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall. These proteins appear to mediate "acid growth" responses of isolated walls and may catalyze plant cell wall extension by a novel biochemical mechanism.

  7. Broad target cell selectivity of Kaposi's sarcoma-associated herpesvirus glycoprotein-mediated cell fusion and virion entry

    SciTech Connect

    Kaleeba, Johnan A.R.; Berger, Edward A. . E-mail: edward_berger@nih.gov

    2006-10-10

    The molecular mechanism of Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus 8) entry is poorly understood. We tested a broad variety of cell types of diverse species and tissue origin for their ability to function as targets in a quantitative reporter gene assay for KSHV-glycoprotein-mediated cell fusion. Several human, non-human primate, and rabbit cell lines were efficient targets, whereas rodent and all human lymphoblastoid cell lines were weak targets. Parallel findings were obtained with a virion entry assay using a recombinant KSHV encoding a reporter gene. No correlation was observed between target cell activity and surface expression of {alpha}3{beta}1 integrin, a proposed KSHV receptor. We hypothesize that target cell permissiveness in both the cell fusion and virion entry assays reflects the presence of a putative KSHV fusion-entry receptor.

  8. Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites

    SciTech Connect

    Cordon-Cardo, C.; O'Brien, J.P.; Casals, D.; Biedler, J.L.; Melamed, M.R.; Bertino, J.R. ); Rittman-Grauer, L. )

    1989-01-01

    Endothelial cells of human capillary blood vessels at the blood-brain and other blood-tissue barrier sites express P-glycoprotein as detected by mouse monoclonal antibodies against the human multidrug-resistance gene product. This pattern of endothelial cell expression may indicate a physiological role for P-glycoprotein in regulating the entry of certain molecules into the central nervous system and other anatomic compartments, such as the testes. These tissues, which limit the access of systemic drugs, are known pharmacologic sanctuaries for metastatic cancer. P-glycoprotein expression in capillary endothelium of brain and testes and not other tissues (i.e., kidney and placenta) may in part explain this phenomenon and could have important implications in cancer chemotherapy.

  9. A low-toxic artificial fluorescent glycoprotein can serve as an efficient cytoplasmic labeling in living cell.

    PubMed

    Si, Jiangju; Liang, Dawei; Kong, Dan; Wu, Sufang; Yuan, Lan; Xiang, Yan; Jiang, Lei

    2015-03-01

    To maintain the virtue of good optical property and discard the dross of conventional fluorescent staining dyes, we provide a strategy for designing new fluorescent scaffolds. In this study, a novel fluorescent labeling glycoprotein (chitosan-poly-L-cysteine, CPC) was synthesized through graft copolymerization. CPC gives emission peak at 465-470 nm when excited at 386 nm. The submicro-scale CPC microspheres could be localized and persisted specifically in the cytoplasm of living cells, with strong blue fluorescence. Moreover, CPC was highly resistant to photo bleaching, the fluorescence was remained stable for up to 72 h as the cells grew and developed. The glycoprotein CPC was bio-compatible and in zero grade cytotoxicity as quantified by MTT assay. The fluorescent labeling process with our newly designed glycoprotein CPC is exceptionally efficient. PMID:25498627

  10. Signal transduction in endothelial cells by the angiogenesis inhibitor histidine-rich glycoprotein targets focal adhesions

    SciTech Connect

    Lee, Chunsik; Dixelius, Johan; Thulin, Asa; Kawamura, Harukiyo; Claesson-Welsh, Lena; Olsson, Anna-Karin . E-mail: Anna-Karin.Olsson@genpat.uu.se

    2006-08-01

    Histidine-rich glycoprotein (HRGP) is an abundant heparin-binding plasma protein. We have shown that a fragment released from the central histidine/proline-rich (His/Pro-rich) domain of HRGP blocks endothelial cell migration in vitro and vascularization and growth of murine fibrosarcoma in vivo. The minimal active HRGP domain exerting the anti-angiogenic effect was recently narrowed down to a 35 amino acid peptide, HRGP330, derived from the His/Pro-rich domain of HRGP. By use of a signal transduction antibody array representing 400 different signal transduction molecules, we now show that HRGP and the synthetic peptide HRGP330 specifically induce tyrosine phosphorylation of focal adhesion kinase and its downstream substrate paxillin in endothelial cells. HRGP/HRGP330 treatment of endothelial cells induced disruption of actin stress fibers, a process reversed by treatment of cells with the FAK inhibitor geldanamycin. In addition, VEGF-mediated endothelial cell tubular morphogenesis in a three-dimensional collagen matrix was inhibited by HRGP and HRGP330. In contrast, VEGF-induced proliferation was not affected by HRGP or HRGP330, demonstrating the central role of cell migration during tube formation. In conclusion, our data show that HRGP targets focal adhesions in endothelial cells, thereby disrupting the cytoskeletal organization and the ability of endothelial cells to assemble into vessel structures.

  11. Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells

    PubMed Central

    2011-01-01

    Background Chikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively. Results Expression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits. Conclusions Chikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections. PMID:21762510

  12. (The structure of pectins from cotton suspension culture cell walls)

    SciTech Connect

    Mort, A.

    1990-01-01

    We have made progress on several projects to do with determining the structure of pectins. These include: (1) Devising a new sensitive method to determine the degree of methyl esterification (DOM) of pectins; (2) solubilization of all of RGI from cotton cell walls; (3) solubilization of RGII from cotton cell walls; (4) characterization of xyloglucan from cotton cell walls; and (5) investigation giving an indication of a cross-link between extension and pectin.

  13. Lignin Formation in Wheat Coleoptile Cell Walls

    PubMed Central

    Whitmore, F. W.

    1971-01-01

    Four growth-influencing compounds—hydroxyproline, 2,2′-dipyridyl, 2-chloroethylphosphonic acid, and indoleacetic acid—were used to examine the relationship between lignin formation and growth of wheat coleoptile sections. Hydroxyproline and 2-chloroethylphosphonic acid, at low concentrations, inhibited growth and increased lignin content. Dipyridyl, which promoted coleoptile elongation, decreased lignin content. Indoleacetic acid caused a 300% increase in growth at 0.1 mm but resulted in lignin content no different from controls with no auxin. Chemical and anatomical evidence is given which indicates that lignin is present in the epidermal cell walls of the wheat coleoptile. It is thus possible that bonding between lignin and hemicellulose may have some influence on coleoptile growth. Images PMID:16657843

  14. P-glycoprotein expression in Ehrlich ascites tumour cells after in vitro and in vivo selection with daunorubicin.

    PubMed Central

    Nielsen, D.; Eriksen, J.; Maare, C.; Jakobsen, A. H.; Skovsgaard, T.

    1998-01-01

    Fluctuation analysis experiments were performed to assess whether selection or induction determines expression of P-glycoprotein and resistance in the murine Ehrlich ascites tumour cell line (EHR2) after exposure to daunorubicin. Thirteen expanded populations of EHR2 cells were exposed to daunorubicin 7.5 x 10(-9) M or 10(-8) M for 2 weeks. Surviving clones were scored and propagated. Only clones exposed to daunorubicin 7.5 x 10(-9) M could be expanded for investigation. Drug resistance was assessed by the tetrazolium dye (MTT) cytotoxicity assay. Western blot was used for determination of P-glycoprotein. Compared with EHR2, the variant cells were 2.5- to 5.2-fold resistant to daunorubicin (mean 3.6-fold). P-glycoprotein was significantly increased in 11 of 25 clones (44%). Analysis of variance supported the hypothesis that spontaneous mutations conferred drug resistance in EHR2 cells exposed to daunorubicin 7.5 x 10(-9) M. At this level (5 log cell killing) of drug exposure, the mutation rate was estimated at 4.1 x 10(-6) per cell generation. In contrast, induction seemed to determine resistance in EHR2 cells in vitro exposed to daunorubicin 10(-8) M. The revertant EHR2/0.8/R was treated in vivo with daunorubicin for 24 h. After treatment, P-glycoprotein increased in EHR2/0.8/R (sevenfold) and the cell line developed resistance to daunorubicin (12-fold), suggesting that in EHR2/0.8/R the mdr1 gene was activated by induction. In conclusion, our study demonstrates that P-glycoprotein expression and daunorubicin resistance are primarily acquired by selection of spontaneously arising mutants. However, under certain conditions the mdr1 gene may be activated by induction. PMID:9820176

  15. An arabidopsis gene regulatory network for secondary cell wall synthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The plant cell wall is an important factor for determining cell shape, function and response to the environment. Secondary cell walls, such as those found in xylem, are composed of cellulose, hemicelluloses and lignin and account for the bulk of plant biomass. The coordination between transcriptiona...

  16. Localization of cell surface glycoproteins in membrane domains associated with the underlying filament network.

    PubMed

    Roos, E; Spiele, H; Feltkamp, C A; Huisman, H; Wiegant, F A; Traas, J; Mesland, D A

    1985-11-01

    To visualize the localization of cell surface constituents in relation to the plasma membrane-associated filament network, we developed a method based on a combination of immunogold labeling and dry-cleaving. For labeling we used trinitrophenyl-derivatized ligand, anti-TNP antibodies, and protein A-coated colloidal gold. Dry-cleaving (Mesland, D. A. M., H. Spiele, and E. Roos, 1981, Exp. Cell Res., 132: 169-184) involves cleavage of lightly fixed critical point-dried cells by means of adhesive tape. Since cells cleave close to the cell surface, the remaining layer is thin enough to be examined in transmission electron microscopy. Using this method, we studied concanavalin A-binding constituents on the medium-facing surface of H35 hepatoma cells. The distribution of the gold particles, which was partly dispersed and partly patchy, coincided strikingly with membrane-associated filaments, and label was virtually absent from areas overlying openings in the filament network. In stereo pairs we observed the label to be localized to areas of somewhat enhanced electron density at the plane of the membrane. These areas were interconnected in a pattern congruent with the filament network. Preliminary observations on wheat germ agglutinin receptors on the hepatoma cells as well as concanavalin A receptors on isolated hepatocytes yielded comparable results. It thus appears that surface glycoproteins, although seemingly randomly distributed as observed in thin sections, may actually be localized to particular membrane domains associated with underlying filaments. PMID:3902855

  17. Selenorhodamine Photosensitizers for Photodynamic Therapy of P-Glycoprotein-Expressing Cancer Cells

    PubMed Central

    2015-01-01

    We examined a series of selenorhodamines with amide and thioamide functionality at the 5-position of a 9-(2-thienyl) substituent on the selenorhodamine core for their potential as photosensitizers for photodynamic therapy (PDT) in P-glycoprotein (P-gp) expressing cells. These compounds were examined for their photophysical properties (absorption, fluorescence, and ability to generate singlet oxygen), for their uptake into Colo-26 cells in the absence or presence of verapamil, for their dark and phototoxicity toward Colo-26 cells, for their rates of transport in monolayers of multidrug-resistant, P-gp-overexpressing MDCKII-MDR1 cells, and for their colocalization with mitochondrial specific agents in Colo-26 cells. Thioamide derivatives 16b and 18b were more effective photosensitizers than amide derivatives 15b and 17b. Selenorhodamine thioamides 16b and 18b were useful in a combination therapy to treat Colo-26 cells in vitro: a synergistic therapeutic effect was observed when Colo-26 cells were exposed to PDT and treatment with the cancer drug doxorubicin. PMID:25250825

  18. CXCR4 mediated chemotaxis is regulated by 5T4 oncofetal glycoprotein in mouse embryonic cells.

    PubMed

    Southgate, Thomas D; McGinn, Owen J; Castro, Fernanda V; Rutkowski, Andrzej J; Al-Muftah, Mariam; Marinov, Georgi; Smethurst, Graeme J; Shaw, David; Ward, Christopher M; Miller, Crispin J; Stern, Peter L

    2010-01-01

    5T4 oncofetal molecules are highly expressed during development and upregulated in cancer while showing only low levels in some adult tissues. Upregulation of 5T4 expression is a marker of loss of pluripotency in the early differentiation of embryonic stem (ES) cells and forms an integrated component of an epithelial-mesenchymal transition, a process important during embryonic development and metastatic spread of epithelial tumors. Investigation of the transcriptional changes in early ES differentiation showed upregulation of CXCL12 and down-regulation of a cell surface protease, CD26, which cleaves this chemokine. CXCL12 binds to the widely expressed CXCR4 and regulates key aspects of development, stem cell motility and tumour metastasis to tissues with high levels of CXCL12. We show that the 5T4 glycoprotein is required for optimal functional cell surface expression of the chemokine receptor CXCR4 and CXCL12 mediated chemotaxis in differentiating murine embryonic stem cells and embryo fibroblasts (MEF). Cell surface expression of 5T4 and CXCR4 molecules is co-localized in differentiating ES cells and MEF. By contrast, differentiating ES and MEF derived from 5T4 knockout (KO) mice show only intracellular CXCR4 expression but infection with adenovirus encoding mouse 5T4 restores CXCL12 chemotaxis and surface co-localization with 5T4 molecules. A series of chimeric constructs with interchanged domains of 5T4 and the glycoprotein CD44 were used to map the 5T4 sequences relevant for CXCR4 membrane expression and function in 5T4KO MEF. These data identified the 5T4 transmembrane domain as sufficient and necessary to enable CXCR4 cell surface expression and chemotaxis. Furthermore, some monoclonal antibodies against m5T4 can inhibit CXCL12 chemotaxis of differentiating ES cells and MEF which is not mediated by simple antigenic modulation. Collectively, these data support a molecular interaction of 5T4 and CXCR4 occurring at the cell surface which directly facilitates

  19. Processing And Presentation of Variant Surface Glycoprotein Molecules to T Cells In African Trypanosomiasis1

    PubMed Central

    Dagenais, Taylor R.; Freeman, Bailey E.; Demick, Karen M.; Paulnock, Donna M.; Mansfield, John M.

    2009-01-01

    Th1 cell responses to the variant surface glycoprotein (VSG) of African trypanosomes play a critical role in controlling infection through the production of IFN-γ, but the role of antigen presenting cells (APCs) in the induction and regulation of T cell mediated protection is poorly understood. In this study, we have investigated the antigen presentation capabilities of dendritic cells (DCs) and macrophages (MPs) during early trypanosome infection in relatively resistant responder and susceptible non-responder mouse strains. Splenic DCs appeared to be the primary cell responsible for activating naive VSG-specific Th cell responses in resistant responder animals through the coordinated up-regulation of costimulatory molecules, secretion of IL-12, and presentation of VSG peptides to T cells in vivo. Splenic DC depletion and the down-regulation of costimulatory markers on splenic MPs were observed in susceptible animals and may be associated with the inability of these animals to elicit a significant VSG-specific T cell response. In contrast to splenic APCs, peritoneal MPs secreted NO, failed to activate naïve Th cells in vitro, and presented relatively low levels of VSG peptides to T cells in vivo. Thus, VSG-specific Th1 cell responses may be determined by tissue- and cell-specific differences in antigen presentation. Additionally, all APCs from resistant and susceptible strains displayed a reduced ability to process and present newly encountered exogenous antigen, including new VSG molecules, during high parasitemia. Thus, initial uptake of VSG (or other trypanosome factors) may interfere with antigen presentation and have dramatic consequences for subsequent T cell responses to other proteins. PMID:19675169

  20. Apoptosis induced by glycoprotein (150-kDa) isolated from Solanum nigrum L. is not related to intracellular reactive oxygen species (ROS) in HCT-116 cells.

    PubMed

    Lee, Sei-Jung; Lim, Kye-Taek

    2006-04-01

    This study was carried out to investigate the apoptotic effects of glycoprotein [Solanum nigrum L. (SNL) glycoprotein, 150-kDa] isolated from Solanum nigrum L., which has been used as an antipyretic and anticancer agent in folk medicine. With the purified SNL glycoprotein, we evaluated the cytotoxic and apoptotic effects of SNL glycoprotein on HCT-116 cells, DNA fragmentation and nuclear staining assays, respectively. SNL glycoprotein has an apparent cytotoxic and apoptotic effect at a concentration of 40 microg/ml after 4 h. To further verify the apoptotic effect, we investigated the changes in activity of the apoptotic-related proteins [Bid, cytochrome c, caspases and poly(ADP-ribose)polymerase (PARP)] triggered by SNL glycoprotein, using a western blot analysis. The results in this study indicated that SNL glycoprotein has a stimulatory effect on Bid activation, resulting in the release of cytochrome c, the stimulation of caspase-8, -9 and -3 activities, and the cleavage of PARP in HCT-116 cells. However, SNL glycoprotein did not significantly stimulate an increase in levels of intracellular reactive oxygen species (ROS). From the results in this experiment, it is suggested that SNL glycoprotein induces apoptosis through the mitochondrial apoptotic signal pathway in HCT-116 cells, rather than through intracellular ROS. PMID:16208518

  1. Detailed characterization of a high-molecular-weight glycoprotein secreted by lung cancer cells.

    PubMed

    Nonaka, N; Manaka, K; Kobayashi, K; Hirai, H

    1993-09-01

    A cancer-associated, high-molecular-weight glycoprotein antigen (6B3.Ag) recognized by monoclonal antibody 6B3 was purified from culture medium of human large cell lung carcinoma cell line (HLC-2) and characterized biochemically and immunochemically. The 6B3.Ag was purified more than 1,200-fold with a yield of 30% by salting out, precipitation by acidification at pH 4.5, and chromatographies on Sepharose 4B and concanavalin A-Sepharose. The molecular weight of 6B3.Ag is approximately 1,000,000 and the molecule is a homodecamer of 94,000 subunits. The 6B3.Ag is a glycoprotein containing 22.9% sugars, consisting of both N- and O-glycoside chains. The N-terminal 19 amino acids were determined and only 4 out of 19 amino acid residues were different from those of an antigen, L3, secreted by lung carcinoma cell line Calu-1. The serum level of 6B3.Ag was determined in normal adults as well as patients with various diseases by enzyme-linked immunosorbent assay. The mean serum level of 6B3.Ag was 3.1 micrograms/ml, ranging from 1.6 to 6.2 micrograms/ml in 131 healthy adults. When the cut-off value was set at 6.2 micrograms/ml, the incidence of positive values in the sera was elevated not only in malignant diseases such as hepatoma (73%) and leukemia (62%), but also in benign diseases such as chronic hepatitis (42%) and liver cirrhosis (63%). While the incidence of positive values was elevated in advanced liver diseases, namely, chronic hepatitis, liver cirrhosis and hepatoma, the cancer specificity of 6B3.Ag did not appear to be high. PMID:8407567

  2. The plant cell wall integrity maintenance mechanism--a case study of a cell wall plasma membrane signaling network.

    PubMed

    Hamann, Thorsten

    2015-04-01

    Some of the most important functions of plant cell walls are protection against biotic/abiotic stress and structural support during growth and development. A prerequisite for plant cell walls to perform these functions is the ability to perceive different types of stimuli in both qualitative and quantitative manners and initiate appropriate responses. The responses in turn involve adaptive changes in cellular and cell wall metabolism leading to modifications in the structures originally required for perception. While our knowledge about the underlying plant mechanisms is limited, results from Saccharomyces cerevisiae suggest the cell wall integrity maintenance mechanism represents an excellent example to illustrate how the molecular mechanisms responsible for stimulus perception, signal transduction and integration can function. Here I will review the available knowledge about the yeast cell wall integrity maintenance system for illustration purposes, summarize the limited knowledge available about the corresponding plant mechanism and discuss the relevance of the plant cell wall integrity maintenance mechanism in biotic stress responses. PMID:25446233

  3. Oral Cyclosporin A Inhibits CD4 T cell P-glycoprotein Activity in HIV-Infected Adults Initiating Treatment with Nucleoside Reverse Transcriptase Inhibitors

    PubMed Central

    Hulgan, Todd; Donahue, John P.; Smeaton, Laura; Pu, Minya; Wang, Hongying; Lederman, Michael M.; Smith, Kimberly; Valdez, Hernan; Pilcher, Christopher; Haas, David W.

    2010-01-01

    Purpose P-glycoprotein limits tissue penetration of many antiretroviral drugs. We characterized effects of the P-glycoprotein substrate cyclosporin A on T cell P-glycoprotein activity in HIV-infected AIDS Clinical Trials Group study A5138 participants. Methods We studied P-glycoprotein activity on CD4 and CD8 T cells in 16 participants randomized to receive oral cyclosporin A (n=9) or not (n=7) during initiation antiretroviral therapy (ART) that did not include protease or non-nucleoside reverse transcriptase inhibitors. Results CD4 T cell P-glycoprotein activity decreased by a median of 8 percentage points with cyclosporin A/ART (difference between cyclosporin A/ART versus ART only P=0.001). Plasma trough cyclosporin A concentrations correlated with change in P-glycoprotein activity in several T cell subsets. Conclusions Oral cyclosporin A can inhibit peripheral blood CD4 T cell P-glycoprotein activity. Targeted P-glycoprotein inhibition might enhance delivery of ART to T cells. PMID:19779705

  4. Cell wall structure and biogenesis in Aspergillus species.

    PubMed

    Yoshimi, Akira; Miyazawa, Ken; Abe, Keietsu

    2016-09-01

    Aspergillus species are among the most important filamentous fungi from the viewpoints of industry, pathogenesis, and mycotoxin production. Fungal cells are exposed to a variety of environmental stimuli, including changes in osmolality, temperature, and pH, which create stresses that primarily act on fungal cell walls. In addition, fungal cell walls are the first interactions with host cells in either human or plants. Thus, understanding cell wall structure and the mechanism of their biogenesis is important for the industrial, medical, and agricultural fields. Here, we provide a systematic review of fungal cell wall structure and recent findings regarding the cell wall integrity signaling pathways in aspergilli. This accumulated knowledge will be useful for understanding and improving the use of industrial aspergilli fermentation processes as well as treatments for some fungal infections. PMID:27140698

  5. Visualization of cellulose synthases in Arabidopsis secondary cell walls.

    PubMed

    Watanabe, Y; Meents, M J; McDonnell, L M; Barkwill, S; Sampathkumar, A; Cartwright, H N; Demura, T; Ehrhardt, D W; Samuels, A L; Mansfield, S D

    2015-10-01

    Cellulose biosynthesis in plant secondary cell walls forms the basis of vascular development in land plants, with xylem tissues constituting the vast majority of terrestrial biomass. We used plant lines that contained an inducible master transcription factor controlling xylem cell fate to quantitatively image fluorescently tagged cellulose synthase enzymes during cellulose deposition in living protoxylem cells. The formation of secondary cell wall thickenings was associated with a redistribution and enrichment of CESA7-containing cellulose synthase complexes (CSCs) into narrow membrane domains. The velocities of secondary cell wall-specific CSCs were faster than those of primary cell wall CSCs during abundant cellulose production. Dynamic intracellular of endomembranes, in combination with increased velocity and high density of CSCs, enables cellulose to be synthesized rapidly in secondary cell walls. PMID:26450210

  6. Increased Cell-Wall Extensibility in Elevated CO2 and O3 Indicates Modification of Leaf Cell-Wall Structure

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean leaf size is increased by growth in elevated [CO2] and decreased in elevated [O3]. The mechanism likely involves changes in cell biophysical properties. Cell growth rate is a function of cell-wall extensibility, a measure of how easily the wall expands in response to turgor. Modification of...

  7. Identification of Two Intracellular Mechanisms Leading to Reduced Expression of Oncoretrovirus Envelope Glycoproteins at the Cell Surface

    PubMed Central

    Grange, Marie-Pierre; Blot, Vincent; Delamarre, Lelia; Bouchaert, Isabelle; Rocca, Anna; Dautry-Varsat, Alice; Dokhélar, Marie-Christine

    2000-01-01

    All retrovirus glycoproteins have a cytoplasmic domain that plays several roles in virus replication. We have determined whether and how the cytoplasmic domains of oncoretrovirus glycoproteins modulate their intracellular trafficking, by using chimeric proteins that combined the α-chain of the interleukin-2 receptor with the glycoprotein cytoplasmic domains of five oncoretroviruses: human T-cell leukemia virus type 1 (HTLV-1), Rous sarcoma virus (RSV), bovine leukemia virus (BLV), murine leukemia virus (MuLV), and Mason-Pfizer monkey virus (MPMV). All of these proteins were synthesized and matured in the same way as a control protein with no retrovirus cytoplasmic domain. However, the amounts of all chimeric proteins at the cell surface were smaller than that of the control protein. The protein appearing at and leaving the cell surface and endocytosis were measured in stable transfectants expressing the chimera. We identified two groups of proteins which followed distinct intracellular pathways. Group 1 included chimeric proteins that reached the cell surface normally but were rapidly endocytosed afterwards. This group included the chimeric proteins with HTLV-1, RSV, and BLV cytoplasmic domains. Group 2 included chimeric proteins that were not detected at the cell surface, despite normal intracellular concentrations, and were accumulated in the Golgi complex. This group included the chimeric proteins with MuLV and MPMV cytoplasmic domains. Finally, we verified that the MuLV envelope glycoproteins behaved in the same way as the corresponding chimeras. These results indicate that retroviruses have evolved two distinct mechanisms to ensure a similar biological feature: low concentrations of their glycoproteins at the cell surface. PMID:11090173

  8. Inhibition of P-glycoprotein by psychotherapeutic drugs in a canine cell model.

    PubMed

    Schrickx, J A; Fink-Gremmels, J

    2014-10-01

    Drug-drug interactions related to long-term therapies are of increasing concern. Psychotherapeutic drugs, licensed for the use in dogs for the management of separation anxiety and other behavioural disorders, are examples of drugs used in long-term therapies. In an in vitro system with canine P-glycoprotein (P-gp) expressing cell lines, three psychotherapeutic drugs with a different mode of action were tested for their ability to inhibit the canine multidrug transporter P-gp. At 10 μm, the selective serotonin reuptake inhibitor fluoxetine and the tricyclic antidepressant clomipramine inhibited P-gp for 41% and 59%, respectively. In contrast, selegeline did not inhibit the function of the canine P-gp. PMID:24602126

  9. Immunological screening of a glycoprotein antigen expressed by Zajdela ascites hepatoma cells on normal rat tissues and tumour cells.

    PubMed

    Nato, F; Goulut, C; Mirshahi, M; Bourrillon, R

    1991-10-01

    Expression of the glycoprotein MII2 antigen originally identified in Zajdela ascites hepatoma cells was investigated in several normal rat tissues and in more or less differentiated tumours using biochemical and immunological approaches. SDS-polyacrylamide gel electrophoresis followed by fluorography or immunoblotting with an antiserum raised against the purified MII2 antigen revealed that this antigen was absent from normal liver cells. ELISA assays, indirect immunofluorescence and immunoprecipitation experiments using the same antiserum showed that this glycoprotein was not expressed in various normal tissues such as liver, spleen, lung, pancreas, intestine and stomach, but it was unexpectedly detected in kidney and thymic tissues. However, the molecular weight of the antigens immunoprecipitated from kidney and thymus was lower than the one of MII2 (Mr of 60,000 versus 110,000-160,000 for purified MII2). No staining was observed in embryonic rat liver at 10 and 20 days of development. Moreover, this antigen was present on the surface of Morris hepatoma 7777, another rapidly proliferating and poorly differentiated hepatocellular carcinoma. In contrast, this antigen was not detected on the surface of in vitro Zajdela hepatoma cells (ZHC) or of partially differentiated hepatomas (Faza) which have recovered some hepatic functions. In addition, the MII2 antigen was found on the human non-hepatic HT-29 tumour cell line, under its undifferentiated form (HT-29 G+ subline). The possible relationships between the expression of this antigen and both the malignant transformation process and the differentiation process are discussed. PMID:1656518

  10. Preparation of Cell Wall Antigens of Staphylococcus aureus

    PubMed Central

    Kowalski, J. J.; Tipper, Donald J.; Berman, David T.

    1970-01-01

    Cell walls were prepared from Staphylococcus aureus strains Copenhagen and 263 by high-speed mixing in the presence of glass beads followed by differential centrifugation. Insoluble peptidoglycan complexes were derived from cell walls by extraction of teichoic acid with 10% trichloroacetic acid. Intact teichoic acid was prepared from each strain by digestion of cell walls with lysostaphin and isolated by column chromatography. Soluble glycopeptide (peptidoglycan in which only the glycan has been fragmented) and the stable complex of teichoic acid with glycopeptide were prepared by digestion of cell walls with Chalaropsis B endo-N-acetylmuramidase and were separated by column chromatography. Amino acid and amino sugar contents of walls and subunits of walls were comparable to those reported by others. Images PMID:16557799

  11. Secretion of N- and O-linked Glycoproteins from 4T1 Murine Mammary Carcinoma Cells

    PubMed Central

    Phang, Wai-Mei; Tan, Aik-Aun; Gopinath, Subash C.B.; Hashim, Onn H.; Kiew, Lik Voon; Chen, Yeng

    2016-01-01

    Breast cancer is one of the most common cancers that affect women globally and accounts for ~23% of all cancers diagnosed in women. Breast cancer is also one of the leading causes of death primarily due to late stage diagnoses and a lack of effective treatments. Therefore, discovering protein expression biomarkers is mandatory for early detection and thus, critical for successful therapy. Two-dimensional electrophoresis (2D-E) coupled with lectin-based analysis followed by mass spectrometry were applied to identify potential biomarkers in the secretions of a murine mammary carcinoma cell line. Comparisons of the protein profiles of the murine 4T1 mammary carcinoma cell line and a normal murine MM3MG mammary cell line indicated that cadherin-1 (CDH), collagenase 3 (MMP-13), Viral envelope protein G7e (VEP), Gag protein (GAG) and Hypothetical protein LOC433182 (LOC) were uniquely expressed by the 4T1 cells, and pigment epithelium-derived factor (PEDF) was exclusively secreted by the MM3MG cells. Further analysis by a lectin-based study revealed that aberrant O-glycosylated CDH, N-glycosylated MMP-13 and LOC were present in the 4T1 medium. These differentially expressed N- and O-linked glycoprotein candidates, which were identified by combining lectin-based analysis with 2D-E, could serve as potential diagnostic and prognostic markers for breast cancer. PMID:27226773

  12. Secretion of N- and O-linked Glycoproteins from 4T1 Murine Mammary Carcinoma Cells.

    PubMed

    Phang, Wai-Mei; Tan, Aik-Aun; Gopinath, Subash C B; Hashim, Onn H; Kiew, Lik Voon; Chen, Yeng

    2016-01-01

    Breast cancer is one of the most common cancers that affect women globally and accounts for ~23% of all cancers diagnosed in women. Breast cancer is also one of the leading causes of death primarily due to late stage diagnoses and a lack of effective treatments. Therefore, discovering protein expression biomarkers is mandatory for early detection and thus, critical for successful therapy. Two-dimensional electrophoresis (2D-E) coupled with lectin-based analysis followed by mass spectrometry were applied to identify potential biomarkers in the secretions of a murine mammary carcinoma cell line. Comparisons of the protein profiles of the murine 4T1 mammary carcinoma cell line and a normal murine MM3MG mammary cell line indicated that cadherin-1 (CDH), collagenase 3 (MMP-13), Viral envelope protein G7e (VEP), Gag protein (GAG) and Hypothetical protein LOC433182 (LOC) were uniquely expressed by the 4T1 cells, and pigment epithelium-derived factor (PEDF) was exclusively secreted by the MM3MG cells. Further analysis by a lectin-based study revealed that aberrant O-glycosylated CDH, N-glycosylated MMP-13 and LOC were present in the 4T1 medium. These differentially expressed N- and O-linked glycoprotein candidates, which were identified by combining lectin-based analysis with 2D-E, could serve as potential diagnostic and prognostic markers for breast cancer. PMID:27226773

  13. Enterotoxigenic Escherichia coli TibA Glycoprotein Adheres to Human Intestine Epithelial Cells

    PubMed Central

    Lindenthal, Christoph; Elsinghorst, Eric A.

    2001-01-01

    Enterotoxigenic Escherichia coli (ETEC) is capable of invading epithelial cell lines derived from the human ileum and colon. Two separate invasion loci (tia and tib) that direct noninvasive E. coli strains to adhere to and invade cultured human intestine epithelial cells have previously been isolated from the classical ETEC strain H10407. The tib locus directs the synthesis of TibA, a 104-kDa outer membrane glycoprotein. Synthesis of TibA is directly correlated with the adherence and invasion phenotypes of the tib locus, suggesting that this protein is an adhesin and invasin. Here we report the purification of TibA and characterization of its biological activity. TibA was purified by continuous-elution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified TibA was biotin labeled and then shown to bind to HCT8 human ileocecal epithelial cells in a specific and saturable manner. Unlabeled TibA competed with biotin-labeled TibA, suggesting the presence of a specific TibA receptor in HCT8 cells. These results show that TibA acts as an adhesin. Polyclonal anti-TibA antiserum inhibited invasion of ETEC strain H10407 and of recombinant E. coli bearing tib locus clones, suggesting that TibA also acts as an invasin. The ability of TibA to direct epithelial cell adhesion suggests a role for this protein in ETEC pathogenesis. PMID:11119488

  14. Enterotoxigenic Escherichia coli TibA glycoprotein adheres to human intestine epithelial cells.

    PubMed

    Lindenthal, C; Elsinghorst, E A

    2001-01-01

    Enterotoxigenic Escherichia coli (ETEC) is capable of invading epithelial cell lines derived from the human ileum and colon. Two separate invasion loci (tia and tib) that direct noninvasive E. coli strains to adhere to and invade cultured human intestine epithelial cells have previously been isolated from the classical ETEC strain H10407. The tib locus directs the synthesis of TibA, a 104-kDa outer membrane glycoprotein. Synthesis of TibA is directly correlated with the adherence and invasion phenotypes of the tib locus, suggesting that this protein is an adhesin and invasin. Here we report the purification of TibA and characterization of its biological activity. TibA was purified by continuous-elution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified TibA was biotin labeled and then shown to bind to HCT8 human ileocecal epithelial cells in a specific and saturable manner. Unlabeled TibA competed with biotin-labeled TibA, suggesting the presence of a specific TibA receptor in HCT8 cells. These results show that TibA acts as an adhesin. Polyclonal anti-TibA antiserum inhibited invasion of ETEC strain H10407 and of recombinant E. coli bearing tib locus clones, suggesting that TibA also acts as an invasin. The ability of TibA to direct epithelial cell adhesion suggests a role for this protein in ETEC pathogenesis. PMID:11119488

  15. Assembly and enlargement of the primary cell wall in plants

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1997-01-01

    Growing plant cells are shaped by an extensible wall that is a complex amalgam of cellulose microfibrils bonded noncovalently to a matrix of hemicelluloses, pectins, and structural proteins. Cellulose is synthesized by complexes in the plasma membrane and is extruded as a self-assembling microfibril, whereas the matrix polymers are secreted by the Golgi apparatus and become integrated into the wall network by poorly understood mechanisms. The growing wall is under high tensile stress from cell turgor and is able to enlarge by a combination of stress relaxation and polymer creep. A pH-dependent mechanism of wall loosening, known as acid growth, is characteristic of growing walls and is mediated by a group of unusual wall proteins called expansins. Expansins appear to disrupt the noncovalent bonding of matrix hemicelluloses to the microfibril, thereby allowing the wall to yield to the mechanical forces generated by cell turgor. Other wall enzymes, such as (1-->4) beta-glucanases and pectinases, may make the wall more responsive to expansin-mediated wall creep whereas pectin methylesterases and peroxidases may alter the wall so as to make it resistant to expansin-mediated creep.

  16. Structural Studies of Complex Carbohydrates of Plant Cell Walls

    SciTech Connect

    Darvill, Alan; Hahn, Michael G.; O'Neill, Malcolm A.; York, William S.

    2015-02-17

    Most of the solar energy captured by land plants is converted into the polysaccharides (cellulose, hemicellulose, and pectin) that are the predominant components of the cell wall. These walls, which account for the bulk of plant biomass, have numerous roles in the growth and development of plants. Moreover, these walls have a major impact on human life as they are a renewable source of biomass, a source of diverse commercially useful polymers, a major component of wood, and a source of nutrition for humans and livestock. Thus, understanding the molecular mechanisms that lead to wall assembly and how cell walls and their component polysaccharides contribute to plant growth and development is essential to improve and extend the productivity and value of plant materials. The proposed research will develop and apply advanced analytical and immunological techniques to study specific changes in the structures and interactions of the hemicellulosic and pectic polysaccharides that occur during differentiation and in response to genetic modification and chemical treatments that affect wall biosynthesis. These new techniques will make it possible to accurately characterize minute amounts of cell wall polysaccharides so that subtle changes in structure that occur in individual cell types can be identified and correlated to the physiological or developmental state of the plant. Successful implementation of this research will reveal fundamental relationships between polysaccharide structure, cell wall architecture, and cell wall functions.

  17. Characterization of a plasma membrane glycoprotein common to myoblasts, skeletal muscle satellite cells, and glia.

    PubMed

    Wakshull, E; Bayne, E K; Chiquet, M; Fambrough, D M

    1983-12-01

    A plasma membrane glycoprotein common to embryonic chick myoblasts and adult chicken skeletal muscle satellite cells is the antigen recognized by monoclonal antibody C3/1. Although traces of the same antigen are present on some muscle-derived fibroblasts, the density of antigenic sites on myoblasts and satellite cells is so high that these cell types can be identified in tissues by immunocytochemical techniques. The antigen is exposed on the surfaces of myogenic cells growing in tissue culture and can be solubilized with detergent. This and other criteria establish that the antigen is a plasma membrane protein. The antigen, purified by affinity techniques, consists of a single type of polypeptide chain which migrates as a relatively broad band of apparent molecular weight 38,000 Da in SDS-polyacrylamide gel electrophoresis. It has a very small sedimentation constant, suggesting that the solubilized form is either monomeric or dimeric. The concentration of antigenic sites increases during myogenesis in vitro; but during maturation the antigenic sites are lost from muscle fibers. Electron microscopic autoradiographic study of adult muscle labeled with iodinated monoclonal antibody demonstrated unequivocally that the antigenic sites in adult muscle are concentrated in the satellite cells. Although selective for myoblasts, immature myotubes and satellite cells in the myogenic lineage, the monoclonal antibody also binds at rather high levels to peripheral Schwann cells and teloglia, to some nonneuronal cells in cultures derived from embryonic spinal cord, to some glial elements of adult chicken brain, and to several cell types in the early embryo. PMID:6360753

  18. The use of surface immobilization of P-selectin glycoprotein ligand-1 on mesenchymal stem cells to facilitate selectin mediated cell tethering and rolling

    PubMed Central

    Lo, Chi Y.; Antonopoulos, Aristotelis; Dell, Anne; Haslam, Stuart M.; Lee, Techung; Neelamegham, Sriram

    2013-01-01

    Mesenchymal stem/stromal cells (MSCs) are an important candidate for cell-based therapy since they can be easily isolated and expanded, secrete beneficial paracrine factors, and differentiate into multiple lineages. Since the endothelium at sites of injury and inflammation often express adhesion molecules belonging to the selectin family, methods to endow MSCs with selectin-ligands can enhance the efficacy of cell delivery and tissue engraftment. Here, we describe a construct 19Fc[FUT7+], where the first 19 amino acids of the pan-selectin ligand PSGL-1 (P-selectin glycoprotein ligand-1) was fused to a human IgG1. When expressed in HEK293T cells over-expressing the α(1,3)fucosyltransferase FUT7, 19Fc[FUT7+] is decorated by a core-2 sialyl Lewis-X sialofucosylated O-glycan. The non-covalent coupling of this protein onto MSC surface using palmitated protein G (PPG) enhanced cell binding to E- and P-selectin under hydrodynamic shear, without altering MSC multipotency. MSCs functionalized with 19Fc[FUT7+] were captured/tethered onto stimulated endothelial cell monolayers at wall shear stresses up to 4 dyn/cm2. Once captured, the cells rolled robustly up to the highest shear stress tested, 10 dyn/cm2. Unlike previous work where MSCs could only be captured onto selectin-bearing substrates at low or no-flow conditions, the current work presents a ‘glycan engineering’ strategy to enable leukocyte-like capture and rolling. PMID:23891082

  19. Retargeting of Coronavirus by Substitution of the Spike Glycoprotein Ectodomain: Crossing the Host Cell Species Barrier

    PubMed Central

    Kuo, Lili; Godeke, Gert-Jan; Raamsman, Martin J. B.; Masters, Paul S.; Rottier, Peter J. M.

    2000-01-01

    Coronaviruses generally have a narrow host range, infecting one or just a few species. Using targeted RNA recombination, we constructed a mutant of the coronavirus mouse hepatitis virus (MHV) in which the ectodomain of the spike glycoprotein (S) was replaced with the highly divergent ectodomain of the S protein of feline infectious peritonitis virus. The resulting chimeric virus, designated fMHV, acquired the ability to infect feline cells and simultaneously lost the ability to infect murine cells in tissue culture. This reciprocal switch of species specificity strongly supports the notion that coronavirus host cell range is determined primarily at the level of interactions between the S protein and the virus receptor. The isolation of fMHV allowed the localization of the region responsible for S protein incorporation into virions to the carboxy-terminal 64 of the 1,324 residues of this protein. This establishes a basis for further definition of elements involved in virion assembly. In addition, fMHV is potentially the ideal recipient virus for carrying out reverse genetics of MHV by targeted RNA recombination, since it presents the possibility of selecting recombinants, no matter how defective, that have regained the ability to replicate in murine cells. PMID:10627550

  20. Reversion of p-glycoprotein-mediated multidrug resistance in human leukemic cell line by diallyl trisulfide.

    PubMed

    Xia, Qing; Wang, Zhi-Yong; Li, Hui-Qing; Diao, Yu-Tao; Li, Xiao-Li; Cui, Jia; Chen, Xue-Liang; Li, Hao

    2012-01-01

    Multidrug resistance (MDR) is the major obstacle in chemotherapy, which involves multiple signaling pathways. Diallyl trisulfide (DATS) is the main sulfuric compound in garlic. In the present study, we aimed to explore whether DATS could overcome P-glycoprotein-(P-gp-)mediated MDR in K562/A02 cells, and to investigate whether NF-κB suppression is involved in DATS-induced reversal of MDR. MTT assay revealed that cotreatment with DATS increased the response of K562/A02 cells to adriamycin (the resistance reversal fold was 3.79) without toxic side effects. DATS could enhance the intracellular concentration of adriamycin by inhibiting the function and expression of P-gp, as shown by flow cytometry, RT-PCR, and western blot. In addition, DATS resulted in more K562/A02 cell apoptosis, accompanied by increased expression of caspase-3. The expression of NF-κB/p65 (downregulation) was significantly linked to the drug-resistance mechanism of DATS, whereas the expression of IκBα was not affected by DATS. Our findings demonstrated that DATS can serve as a novel, nontoxic modulator of MDR, and can reverse the MDR of K562/A02 cells in vitro by increasing intracellular adriamycin concentration and inducing apoptosis. More importantly, we proved for the first time that the suppression of NF-κB possibly involves the molecular mechanism in the course of reversion by DATS. PMID:22919419

  1. Unique glycoprotein antigen defined by monoclonal antibody on human neurobiastoma cells

    SciTech Connect

    Mujoo, K.; Spiro, R.C.; Reisfeld, R.A.

    1986-05-01

    The authors have characterized a new target antigen on the surface of human neuroblastoma cells and defined it with a monoclonal antibody (Mab) 5G3. This antibody is of IgG2a type and has an association constant of 8 x 10/sup 9/ M/sup -1/. In ELISA assays, Mab 5G3 reacted with human neuroblastoma as well as melanoma, squamous lung, skin carcinoma, and osteogenic sarcoma. Immunocytochemical analysis of frozen tissue sections revealed strong reactivity with all neuroblastoma tissues and marginal reactivity with melanoma and glioma tissues. There was no reactivity with fetal or normal tissues with the exception of cerebellum. The antigen recognized by Mab 5G3 is a glycoprotein of 200 and 215 kDa expressed on the SK-N-AS neuroblastoma cells. The antigen appears to contain N-linked carbohydrates based on treatment of human neuroblastoma cells with tunicamycin before and after intrinsic radiolabeling followed by indirect immunoprecipitation. The pulse-chase biosynthetic studies followed by indirect immunoprecipitation and SDS-PAGE indicated the precursor/product relationship between 200 and 215 kDa molecules. The 200 kDa component is endoglycosidase H-sensitive, whereas 215 kDa molecule is Endo-H resistant. The 215 kDa component is also sulfated, sialylated, and phosphorylated at serine residues. Preliminary data suggests that Mab, aside from identifying a unique target antigen on human neuroblastoma cells, may be suited as a targeting device for chemotherapeutic drugs.

  2. Host Cell P-glycoprotein Is Essential for Cholesterol Uptake and Replication of Toxoplasma gondii*

    PubMed Central

    Bottova, Iveta; Hehl, Adrian B.; Štefanić, Saša; Fabriàs, Gemma; Casas, Josefina; Schraner, Elisabeth; Pieters, Jean; Sonda, Sabrina

    2009-01-01

    P-glycoprotein (P-gp) is a membrane-bound efflux pump that actively exports a wide range of compounds from the cell and is associated with the phenomenon of multidrug resistance. However, the role of P-gp in normal physiological processes remains elusive. Using P-gp-deficient fibroblasts, we showed that P-gp was critical for the replication of the intracellular parasite Toxoplasma gondii but was not involved in invasion of host cells by the parasite. Importantly, we found that the protein participated in the transport of host-derived cholesterol to the intracellular parasite. T. gondii replication in P-gp-deficient host cells not only resulted in reduced cholesterol content in the parasite but also altered its sphingolipid metabolism. In addition, we found that different levels of P-gp expression modified the cholesterol metabolism in uninfected fibroblasts. Collectively our findings reveal a key and previously undocumented role of P-gp in host-parasite interaction and suggest a physiological role for P-gp in cholesterol trafficking in mammalian cells. PMID:19389707

  3. Host cell P-glycoprotein is essential for cholesterol uptake and replication of Toxoplasma gondii.

    PubMed

    Bottova, Iveta; Hehl, Adrian B; Stefanić, Sasa; Fabriàs, Gemma; Casas, Josefina; Schraner, Elisabeth; Pieters, Jean; Sonda, Sabrina

    2009-06-26

    P-glycoprotein (P-gp) is a membrane-bound efflux pump that actively exports a wide range of compounds from the cell and is associated with the phenomenon of multidrug resistance. However, the role of P-gp in normal physiological processes remains elusive. Using P-gp-deficient fibroblasts, we showed that P-gp was critical for the replication of the intracellular parasite Toxoplasma gondii but was not involved in invasion of host cells by the parasite. Importantly, we found that the protein participated in the transport of host-derived cholesterol to the intracellular parasite. T. gondii replication in P-gp-deficient host cells not only resulted in reduced cholesterol content in the parasite but also altered its sphingolipid metabolism. In addition, we found that different levels of P-gp expression modified the cholesterol metabolism in uninfected fibroblasts. Collectively our findings reveal a key and previously undocumented role of P-gp in host-parasite interaction and suggest a physiological role for P-gp in cholesterol trafficking in mammalian cells. PMID:19389707

  4. Anti-metastatic effects of antrodan, the Antrodia cinnamomea mycelia glycoprotein, in lung carcinoma cells.

    PubMed

    Fa, Kuan-Ning; Yang, Chih-Min; Chen, Pei-Chun; Lee, Yin-Ying; Chyau, Charng-Cherng; Hu, Miao-Lin

    2015-03-01

    This study investigated the anti-metastatic effects of antrodan, the glycoprotein from Antrodia cinnamomea (AC) mycelia, through direct actions and indirect immunomodulatory effects in Lewis lung carcinoma (LLC). Antrodan was isolated from AC mycelia by alkali extraction, acid precipitation, and purification using sepharose CL-6B column chromatography. In the direct anti-metastatic action, antrodan (30-70 μg/mL) was found to significantly inhibit invasion and migration of LLC cells, and these effects involved up-regulation of tissue inhibitor of matrix metalloproteinase (TIMP)-1, TIMP-2, and nm23-H1 protein expression leading to decreased activities and protein expression of MMP-2 and MMP-9. For testing the indirect immunomodulatory effect, antrodan was incubated for 3d with mononuclear cells (MNCs) isolated from human peripheral blood to obtain the condition medium (CM). Antrodan significantly increased interleukin (IL)-12 and IL-1β levels, but decreased TNF-α, IL-6 and IL-8 levels in the MMC-CM, which also significantly inhibited invasion, migration, and the activities and protein expression of MMP-2 and MMP-9, but significantly increased protein expression of TIMP-1, TIMP-2, and nm23-H1 in LLC cells. The indirect immunomodulatory effect of antrodan was stronger than the direct anti-metastatic effect at the same concentrations (50 and 60 μg/mL). Overall, the results suggest the anti-metastatic potential of antrodan in LLC cells. PMID:25583024

  5. Engineering the Oryza sativa cell wall with rice NAC transcription factors regulating secondary wall formation

    PubMed Central

    Yoshida, Kouki; Sakamoto, Shingo; Kawai, Tetsushi; Kobayashi, Yoshinori; Sato, Kazuhito; Ichinose, Yasunori; Yaoi, Katsuro; Akiyoshi-Endo, Miho; Sato, Hiroko; Takamizo, Tadashi; Ohme-Takagi, Masaru; Mitsuda, Nobutaka

    2013-01-01

    Plant tissues that require structural rigidity synthesize a thick, strong secondary cell wall of lignin, cellulose and hemicelluloses in a complicated bridged structure. Master regulators of secondary wall synthesis were identified in dicots, and orthologs of these regulators have been identified in monocots, but regulation of secondary cell wall formation in monocots has not been extensively studied. Here we demonstrate that the rice transcription factors SECONDARY WALL NAC DOMAIN PROTEINs (SWNs) can regulate secondary wall formation in rice (Oryza sativa) and are potentially useful for engineering the monocot cell wall. The OsSWN1 promoter is highly active in sclerenchymatous cells of the leaf blade and less active in xylem cells. By contrast, the OsSWN2 promoter is highly active in xylem cells and less active in sclerenchymatous cells. OsSWN2 splicing variants encode two proteins; the shorter protein (OsSWN2S) has very low transcriptional activation ability, but the longer protein (OsSWN2L) and OsSWN1 have strong transcriptional activation ability. In rice, expression of an OsSWN2S chimeric repressor, driven by the OsSWN2 promoter, resulted in stunted growth and para-wilting (leaf rolling and browning under normal water conditions) due to impaired vascular vessels. The same OsSWN2S chimeric repressor, driven by the OsSWN1 promoter, caused a reduction of cell wall thickening in sclerenchymatous cells, a drooping leaf phenotype, reduced lignin and xylose contents and increased digestibility as forage. These data suggest that OsSWNs regulate secondary wall formation in rice and manipulation of OsSWNs may enable improvements in monocotyledonous crops for forage or biofuel applications. PMID:24098302

  6. Engineering the Oryza sativa cell wall with rice NAC transcription factors regulating secondary wall formation.

    PubMed

    Yoshida, Kouki; Sakamoto, Shingo; Kawai, Tetsushi; Kobayashi, Yoshinori; Sato, Kazuhito; Ichinose, Yasunori; Yaoi, Katsuro; Akiyoshi-Endo, Miho; Sato, Hiroko; Takamizo, Tadashi; Ohme-Takagi, Masaru; Mitsuda, Nobutaka

    2013-01-01

    Plant tissues that require structural rigidity synthesize a thick, strong secondary cell wall of lignin, cellulose and hemicelluloses in a complicated bridged structure. Master regulators of secondary wall synthesis were identified in dicots, and orthologs of these regulators have been identified in monocots, but regulation of secondary cell wall formation in monocots has not been extensively studied. Here we demonstrate that the rice transcription factors SECONDARY WALL NAC DOMAIN PROTEINs (SWNs) can regulate secondary wall formation in rice (Oryza sativa) and are potentially useful for engineering the monocot cell wall. The OsSWN1 promoter is highly active in sclerenchymatous cells of the leaf blade and less active in xylem cells. By contrast, the OsSWN2 promoter is highly active in xylem cells and less active in sclerenchymatous cells. OsSWN2 splicing variants encode two proteins; the shorter protein (OsSWN2S) has very low transcriptional activation ability, but the longer protein (OsSWN2L) and OsSWN1 have strong transcriptional activation ability. In rice, expression of an OsSWN2S chimeric repressor, driven by the OsSWN2 promoter, resulted in stunted growth and para-wilting (leaf rolling and browning under normal water conditions) due to impaired vascular vessels. The same OsSWN2S chimeric repressor, driven by the OsSWN1 promoter, caused a reduction of cell wall thickening in sclerenchymatous cells, a drooping leaf phenotype, reduced lignin and xylose contents and increased digestibility as forage. These data suggest that OsSWNs regulate secondary wall formation in rice and manipulation of OsSWNs may enable improvements in monocotyledonous crops for forage or biofuel applications. PMID:24098302

  7. Methods for degrading or converting plant cell wall polysaccharides

    DOEpatents

    Berka, Randy; Cherry, Joel

    2008-08-19

    The present invention relates to methods for converting plant cell wall polysaccharides into one or more products, comprising: treating the plant cell wall polysaccharides with an effective amount of a spent whole fermentation broth of a recombinant microorganism, wherein the recombinant microorganism expresses one or more heterologous genes encoding enzymes which degrade or convert the plant cell wall polysaccharides into the one or more products. The present invention also relates to methods for producing an organic substance, comprising: (a) saccharifying plant cell wall polysaccharides with an effective amount of a spent whole fermentation broth of a recombinant microorganism, wherein the recombinant microorganism expresses one or more heterologous genes encoding enzymes which degrade or convert the plant cell wall polysaccharides into saccharified material; (b) fermenting the saccharified material of step (a) with one or more fermenting microoganisms; and (c) recovering the organic substance from the fermentation.

  8. Tissue-specific cell wall hydration in sugarcane stalks.

    PubMed

    Maziero, Priscila; Jong, Jennifer; Mendes, Fernanda M; Gonçalves, Adilson R; Eder, Michaela; Driemeier, Carlos

    2013-06-19

    Plant cell walls contain water, especially under biological and wet processing conditions. The present work characterizes this water in tissues of sugarcane stalks. Environmental scanning electron microscopy shows tissue deformation upon drying. Dynamic vapor sorption determines the equilibrium and kinetics of moisture uptake. Thermoporometry by differential scanning calorimetry quantifies water in nanoscale pores. Results show that cell walls from top internodes of stalks are more deformable, slightly more sorptive to moisture, and substantially more porous. These differences of top internode are attributed to less lignified walls, which is confirmed by lower infrared spectral signal from aromatics. Furthermore, cell wall nanoscale porosity, an architectural and not directly compositional characteristic, is shown to be tissue-specific. Nanoscale porosities are ranked as follows: pith parenchyma > pith vascular bundles > rind. This ranking coincides with wall reactivity and digestibility in grasses, suggesting that nanoscale porosity is a major determinant of wall recalcitrance. PMID:23738592

  9. The role of wall calcium in the extension of cell walls of soybean hypocotyls

    NASA Technical Reports Server (NTRS)

    Virk, S. S.; Cleland, R. E.

    1990-01-01

    Calcium crosslinks are load-bearing bonds in soybean (Glycine max (L.) Merr.) hypocotyl cell walls, but they are not the same load-bearing bonds that are broken during acid-mediated cell elongation. This conclusion is reached by studying the relationship between wall calcium, pH and the facilitated creep of frozen-thawed soybean hypocotyl sections. Supporting data include the following observations: 1) 2-[(2-bis-[carboxymethyl]amino-5-methylphenoxy)methyl]-6-methoxy-8-bis[car boxymethyl]aminoquinoline (Quin 2) and ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) caused only limited facilitated creep as compared with acid, despite removal of comparable or larger amounts of wall calcium; 2) the pH-response curves for calcium removal and acid-facilitated creep were different; 3) reversible acid-extension occurred even after removal of almost all wall calcium with Quin 2; and 4) growth of abraded sections did not involve a proportional loss of wall calcium. Removal of wall calcium, however, increased the capacity of the walls to undergo acid-facilitated creep. These data indicate that breakage of calcium crosslinks is not a major mechanism of cell-wall loosening in soybean hypocotyl tissues.

  10. Collenchyma: a versatile mechanical tissue with dynamic cell walls

    PubMed Central

    Leroux, Olivier

    2012-01-01

    Background Collenchyma has remained in the shadow of commercially exploited mechanical tissues such as wood and fibres, and therefore has received little attention since it was first described. However, collenchyma is highly dynamic, especially compared with sclerenchyma. It is the main supporting tissue of growing organs with walls thickening during and after elongation. In older organs, collenchyma may become more rigid due to changes in cell wall composition or may undergo sclerification through lignification of newly deposited cell wall material. While much is known about the systematic and organographic distribution of collenchyma, there is rather less information regarding the molecular architecture and properties of its cell walls. Scope and conclusions This review summarizes several aspects that have not previously been extensively discussed including the origin of the term ‘collenchyma’ and the history of its typology. As the cell walls of collenchyma largely determine the dynamic characteristics of this tissue, I summarize the current state of knowledge regarding their structure and molecular composition. Unfortunately, to date, detailed studies specifically focusing on collenchyma cell walls have not been undertaken. However, generating a more detailed understanding of the structural and compositional modifications associated with the transition from plastic to elastic collenchyma cell wall properties is likely to provide significant insights into how specific configurations of cell wall polymers result in specific functional properties. This approach, focusing on architecture and functional properties, is likely to provide improved clarity on the controversial definition of collenchyma. PMID:22933416

  11. The Interplay between Cell Wall Mechanical Properties and the Cell Cycle in Staphylococcus aureus

    PubMed Central

    Bailey, Richard G.; Turner, Robert D.; Mullin, Nic; Clarke, Nigel; Foster, Simon J.; Hobbs, Jamie K.

    2014-01-01

    The nanoscale mechanical properties of live Staphylococcus aureus cells during different phases of growth were studied by atomic force microscopy. Indentation to different depths provided access to both local cell wall mechanical properties and whole-cell properties, including a component related to cell turgor pressure. Local cell wall properties were found to change in a characteristic manner throughout the division cycle. Splitting of the cell into two daughter cells followed a local softening of the cell wall along the division circumference, with the cell wall on either side of the division circumference becoming stiffer. Once exposed, the newly formed septum was found to be stiffer than the surrounding, older cell wall. Deeper indentations, which were affected by cell turgor pressure, did not show a change in stiffness throughout the division cycle, implying that enzymatic cell wall remodeling and local variations in wall properties are responsible for the evolution of cell shape through division. PMID:25468333

  12. Characterisation of Eubacterium cell wall: peptidoglycan structure determines arthritogenicity

    PubMed Central

    Zhang, X; Rimpilainen, M; Toivanen, P

    2001-01-01

    OBJECTIVE—To elucidate factors involved in the arthritogenicity of bacterial cell walls.
METHODS—For characterisation of an arthritogenic Eubacterium aerofaciens cell wall, peptidoglycan-polysaccharide (PG-PS) polymers were isolated by removing cell wall associated proteins (CWPs), PG and PS moieties were separated, and an attempt was made to de-O-acetylate PG-PS. The cell wall of E limosum was used as a non-arthritogenic control. The chemical composition of these cell wall preparations was analysed by gas chromatography-mass spectrometry. Also, their ability to resist lysozyme degradation and to sustain experimental chronic arthritis was tested.
RESULTS—The observations made with the cell wall of E aerofaciens, an anaerobic habitant of the human intestine, were compared with those reported from a pathogenic Streptococcus, showing that in both strains a complex consisting of PG-PS is required for the induction of chronic arthritis. The PS moiety most probably protects PG from enzyme degradation, allowing prolonged tissue persistence and leading to the chronic synovial inflammation. CWPs attached to PG-PS are not necessary for this function. O-Acetylation of PG, which is required for arthritogenicity of the streptococcal cell wall, seems not to be present in the arthritogenic E aerofaciens PG or only occurs to a small degree; attempts to de-O-acylate the E aerofaciens cell wall did not affect its arthritogenicity or lysozyme resistance.
CONCLUSION—The results obtained indicate that the source of bacterial cell wall plays no part in the chemical or structural requirements for PG to induce chronic cell wall arthritis in the rats; the chemical structure of the PG moiety is decisive.

 PMID:11171690

  13. Glucuronoarabinoxylan structure in the walls of Aechmea leaf chlorenchyma cells is related to wall strength.

    PubMed

    Ceusters, Johan; Londers, Elsje; Brijs, Kristof; Delcour, Jan A; De Proft, Maurice P

    2008-09-01

    In CAM-plants rising levels of malic acid in the early morning cause elevated turgor pressures in leaf chlorenchyma cells. Under specific conditions this process is lethal for sensitive plants resulting in chlorenchyma cell burst while other species can cope with these high pressures and do not show cell burst under comparable conditions. The non-cellulosic polysaccharide composition of chlorenchyma cell walls was investigated and compared in three cultivars of Aechmea with high sensitivity for chlorenchyma cell burst and three cultivars with low sensitivity. Chlorenchyma layers were cut from the leaf and the non-cellulosic carbohydrate fraction of the cell wall fraction was analyzed by gas-liquid chromatography. Glucuronoarabinoxylans (GAXs) were the major non-cellulosic polysaccharides in Aechmea. The fine structure of these GAXs was strongly related to chlorenchyma wall strength. Chlorenchyma cell walls from cultivars with low sensitivity to cell burst were characterized by an A/X ratio of ca. 0.13 while those from cultivars with high sensitivity showed an A/X ratio of ca. 0.23. Xylose chains from cultivars with high cell burst sensitivity were ca. 40% more substituted with arabinose compared to cultivars with low sensitivity for cell burst. The results indicate a relationship in vivo between glucuronoarabinoxylan fine structure and chlorenchyma cell wall strength in Aechmea. The evidence obtained supports the hypothesis that GAXs with low degrees of substitution cross-link cellulose microfibrils, while GAXs with high degrees of substitution do not. A lower degree of arabinose substitution on the xylose backbone implies stronger cell walls and the possibility of withstanding higher internal turgor pressures without cell bursting. PMID:18632122

  14. Structure of plant cell walls: XIX. Isolation and characterization of wall polysaccharides from suspension-cultured Douglas fir cells

    SciTech Connect

    Thomas, J.R.; McNeil, M.; Darvill, A.G.; Albersheim, P.

    1987-03-01

    The partial purification and characterization of cell wall polysaccharides isolated from suspension-cultured Douglas fir (Pseudotsuga menziesii) cells are described. Extraction of isolated cell walls from 1.0 M LiCl solubilized pectic polysaccharides with glycosyl-linkage compositions similar to those of rhamnogalacturonans I and II, pectic polysaccharides isolated from walls of suspension-cultured sycamore cells. Treatment of LiCl-extracted Douglas fir walls with an endo-..cap alpha..-1,4-polygalacturonase released only small, additional amounts of pectic polysaccharide, which had a glycosyl-linkage composition similar to that of rhamnogalacturonan I. Xyloglucan oligosaccharides were released from the endo-..cap alpha..-1,4-polygalacturonase-treated walls by treatment with an endo-..beta..-1,4-glucanase. These oligosaccharides included hepta- and nonasaccharides similar or identical to those released from sycamore cell walls by the same enzyme, and structurally related octa- and decasaccharides similar to those isolated from various angiosperms. Finally, additional xyloglucan and small amounts of xylan were extracted from the endo-..beta..-1,4-glucanase-treated walls by 0.5 N NaOH. The xylan resembled that extracted by NaOH from dicot cell walls in that it contained 2,4- but not 3,4-linked xylosyl residues. In this study, a total of 15% of the cell wall was isolated as pectic material, 10% as xyloglucan, and less than 1% as xylan. The noncellulosic polysaccharides accounted for 25% of the cell walls, cellulose for 23%, protein for 34%, and ash for 5%, for a total of 88% of the cell wall.

  15. Glycoprotein J of infectious laryngotracheitis virus is required for efficient egress of infectious virions from cells.

    PubMed

    Mundt, Alice; Mundt, Egbert; Hogan, Robert J; García, Maricarmen

    2011-11-01

    Glycoprotein J (gJ) of infectious laryngotracheitis virus (ILTV) represents a major viral antigen and is dispensable for replication in cell culture and chickens. We generated gJ deletion mutants derived from the United States Department of Agriculture standard challenge strain (USDA-ch), a GFP-expressing mutant GΔgJ, a gJ deletion mutant void of any foreign DNA insertion (BΔgJ) and a gJ rescue mutant gJR with US5 restored. GΔgJ, BΔgJ and gJR were characterized in cell culture and embryonated eggs. Entry kinetic assays showed that the gJ deletion mutants did not differ in their entry kinetics from gJR. Replication kinetics strongly indicated that gJ plays an important role during egress of the virus. Differences in the abilities of the mutants to replicate in chorioallantoic membranes of chicken embryos and to release infectious virus into the allantoic fluid supported a function of gJ during the egress of ILTV from infected cells. PMID:21752963

  16. The secreted glycoprotein lubricin protects cartilage surfaces and inhibits synovial cell overgrowth

    PubMed Central

    Rhee, David K.; Marcelino, Jose; Baker, MacArthur; Gong, Yaoqin; Smits, Patrick; Lefebvre, Véronique; Jay, Gregory D.; Stewart, Matthew; Wang, Hongwei; Warman, Matthew L.; Carpten, John D.

    2005-01-01

    The long-term integrity of an articulating joint is dependent upon the nourishment of its cartilage component and the protection of the cartilage surface from friction-induced wear. Loss-of-function mutations in lubricin (a secreted glycoprotein encoded by the gene PRG4) cause the human autosomal recessive disorder camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP). A major feature of CACP is precocious joint failure. In order to delineate the mechanism by which lubricin protects joints, we studied the expression of Prg4 mRNA during mouse joint development, and we created lubricin-mutant mice. Prg4 began to be expressed in surface chondrocytes and synoviocytes after joint cavitation had occurred and remained strongly expressed by these cells postnatally. Mice lacking lubricin were viable and fertile. In the newborn period, their joints appeared normal. As the mice aged, we observed abnormal protein deposits on the cartilage surface and disappearance of underlying superficial zone chondrocytes. In addition to cartilage surface changes and subsequent cartilage deterioration, intimal cells in the synovium surrounding the joint space became hyperplastic, which further contributed to joint failure. Purified or recombinant lubricin inhibited the growth of these synoviocytes in vitro. Tendon and tendon sheath involvement was present in the ankle joints, where morphologic changes and abnormal calcification of these structures were observed. We conclude that lubricin has multiple functions in articulating joints and tendons that include the protection of surfaces and the control of synovial cell growth. PMID:15719068

  17. Envelope Glycoprotein Internalization Protects Human and Simian Immunodeficiency Virus-Infected Cells from Antibody-Dependent Cell-Mediated Cytotoxicity

    PubMed Central

    von Bredow, Benjamin; Arias, Juan F.; Heyer, Lisa N.; Gardner, Matthew R.; Farzan, Michael; Rakasz, Eva G.

    2015-01-01

    ABSTRACT The cytoplasmic tails of human and simian immunodeficiency virus (HIV and SIV, respectively) envelope glycoproteins contain a highly conserved, membrane-proximal endocytosis motif that prevents the accumulation of Env on the surface of infected cells prior to virus assembly. Using an assay designed to measure the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC), we show that substitutions in this motif increase the susceptibility of HIV-1- and SIV-infected cells to ADCC in a manner that directly correlates with elevated Env levels on the surface of virus-infected cells. In the case of HIV-1, this effect is additive with a deletion in vpu recently shown to enhance the susceptibility of HIV-1-infected cells to ADCC as a result of tetherin-mediated retention of budding virions on the cell surface. These results reveal a previously unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from antibody responses by regulating the amount of Env present on the cell surface. IMPORTANCE This study reveals an unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from elimination by Env-specific antibodies. Thus, strategies designed to interfere with this mechanism of Env internalization may improve the efficacy of antibody-based vaccines and antiretroviral therapies designed to enhance the immunological control of HIV-1 replication in chronically infected individuals. PMID:26269175

  18. High-throughput microarray mapping of cell wall polymers in roots and tubers during the viscosity-reducing process.

    PubMed

    Huang, Yuhong; Willats, William G; Lange, Lene; Jin, Yanling; Fang, Yang; Salmeán, Armando A; Pedersen, Henriette L; Busk, Peter Kamp; Zhao, Hai

    2016-03-01

    Viscosity reduction has a great impact on the efficiency of ethanol production when using roots and tubers as feedstock. Plant cell wall-degrading enzymes have been successfully applied to overcome the challenges posed by high viscosity. However, the changes in cell wall polymers during the viscosity-reducing process are poorly characterized. Comprehensive microarray polymer profiling, which is a high-throughput microarray, was used for the first time to map changes in the cell wall polymers of sweet potato (Ipomoea batatas), cassava (Manihot esculenta), and Canna edulis Ker. over the entire viscosity-reducing process. The results indicated that the composition of cell wall polymers among these three roots and tubers was markedly different. The gel-like matrix and glycoprotein network in the C. edulis Ker. cell wall caused difficulty in viscosity reduction. The obvious viscosity reduction of the sweet potato and the cassava was attributed to the degradation of homogalacturonan and the released 1,4-β-d-galactan and 1,5-α-l-arabinan. PMID:25757626

  19. Murine cell surface glycoproteins. Characterization of a major component of 80,000 daltons as a polymorphic differentiation antigen of mesenchymal cells.

    PubMed

    Hughes, E N; Mengod, G; August, J T

    1981-07-10

    Monoclonal antibodies reactive with NIH/3T3 cell surface antigens were obtained from hybridomas of murine myeloma cells fused to spleen cells of rats immunized with NIH/3T3 cell plasma membranes. Four of the antibodies, of forty that have been studied, appeared to react with allospecific antigenic determinants: they bound to NIH/3T3 cells but not to BALB/ 3T3 cells. Each of these four antibodies immunoprecipitated a glycoprotein of about 80,000 daltons that migrated to an isoelectric point of about pH 5.0. Polypeptides of identical molecular weight and isoelectric points, and yielding the same proteolytic cleavage fragments, were present in BALB/3T3 cells, but were not antigenically reactive. The 80,000-dalton glycoprotein was a major constituent of the plasma membrane. It was a predominant lactoperoxidase iodinated component of intact NIH/3T3 cells, and saturation binding of 125I-labeled antibody indicated that there were about 10(6) antigenic sites/cell. Studies of the distribution of the immunoreactive glycoprotein among different strains of mice confirmed the polymorphic expression of the determinant: Spleen cells of BALB/c, DBA/1, DBA/2, and CBA mice did not bind anti-80,000-dalton glycoprotein monoclonal antibodies, whereas spleen cells of a large number of other strains of mice were positive for antibody-binding. The antigenic reactivity varied markedly among different cell lines and was greatest with the NIH/3T3 mouse embryo fibroblast, G8-1 Swiss Webster myoblast, and IC-21 SV40-transformed C57BL/6 mouse peritoneal macrophage. The properties of the 80,000-dalton glycoprotein characterized this molecule as a new cell surface differentiation alloantigen of murine mesenchymal cells. PMID:6787057

  20. Modulation of P-glycoprotein function and multidrug resistance in cancer cells by Thai plant extracts.

    PubMed

    Takano, M; Kakizoe, S; Kawami, M; Nagai, J; Patanasethnont, D; Sripanidkulchai, B; Yumoto, R

    2014-11-01

    The effects of ethanol extracts from Thai plants belonging to the families of Annonaceae, Rutaceae, and Zingiberaceae on P-glycoprotein (P-gp) function and multidrug resistance were examined in paclitaxel-resistant HepG2 (PR-HepG2) cells. All the extracts tested, significantly increased the accumulation of [3H]paclitaxel, a P-gp substrate, in the cells. Among nine extracts, Z01 and Z02, extracts from Curcuma comosa and Kaempferia marginata (Zingiberaceae family), respectively, potently increased the accumulation. In addition, Z01 and Z02 increased the accumulation of other P-gp substrates, rhodamine 123 and doxorubicin, in PR-HepG2 cells in a concentration-dependent manner. Increased accumulation of rhodamine 123 and doxorubicin by Z01 and Z02 was also confirmed by confocal laser scanning microscopy. The effect of Z01 and Z02 pretreatment on the expression of MDR1 mRNA was also examined. The expression of MDR1 mRNA was not affected by the treatment of PR-HepG2 cells with these extracts for 48 hours. Cytotoxicity of paclitaxel was examined by XTT and protein assays in the absence and presence of Z02. Z02 potentiated the cytotoxicity of paclitaxel in PR-HepG2 cells. These results suggest that Curcuma comosa and Kaempferia marginata belonging to Zingiberaceae are useful sources to search for new P-gp modulator(s) that can be used to overcome multidrug resistance of cancer cells. PMID:25985578

  1. Characterization of the soluble glycoprotein released from vesicular stomatitis virus-infected cells.

    PubMed Central

    Chatis, P A; Morrison, T G

    1983-01-01

    Vesicular stomatitis virus-infected Chinese hamster ovary cells release into the extracellular medium a soluble form of the vesicular stomatitis virus glycoprotein (G protein) termed Gs (Kang and Prevec, Virology 46:678-680, 1971). The properties of this molecule and the cellular site at which it is generated were characterized. By comparing the sizes and the peptide maps of the unglycosylated forms of G and Gs, we found that between 5,000 and 6,000 daltons of the carboxy-terminal end of the G protein is cleaved to generate the Gs molecule. This truncated molecule contains no fatty acid. Gs released from cells grown at 39 degrees C migrated on polyacrylamide gels slightly slower than Gs released at 30 degrees C. The unglycosylated form of Gs also showed this size difference. Furthermore, unglycosylated Gs was resolved into two species upon isoelectric focusing: the relative amounts of the two species depended upon the temperature at which infected cells were incubated. Full-sized unglycosylated virus-associated G also was resolved into two species, but the more basic form predominated at both 30 and 39 degrees C. The appearance of Gs in the extracellular medium depended upon the presence of stable, full-sized G at the cell surface. The amount of Gs released was quantitated in seven different situations in which the migration of G to the cell surface was inhibited. In all cases, the amount of Gs released was also decreased. In addition, incubation of cells surface labeled with 125I resulted in the release of 125I-labeled Gs protein, as well as full-sized G protein. These results suggest that Gs is generated primarily by proteolytic cleavage of plasma membrane-associated G at a site in the molecule just amino terminal to the membrane-spanning region of the molecule. Images PMID:6296461

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  4. The inhibitory and combinative mechanism of HZ08 with P-glycoprotein expressed on the membrane of Caco-2 cell line

    SciTech Connect

    Zhang, Yanyan; Hu, Yahui; Feng, Yidong; Kodithuwakku, Nandani Darshika; Fang, Weirong; Li, Yunman; Huang, Wenlong

    2014-01-15

    Recently, the research and development of agents to reverse the phenomenon of multidrug resistance has been an attractive goal as well as a key approach to elevating the clinical survival of cancer patients. Although three generations of P-glycoprotein modulators have been identified, poor clearance and metabolism render these agents too toxic to be used in clinical application. HZ08, which has been under investigation for several years, shows a dramatic reversal effect with low cytotoxicity. For the first time, we aimed to describe the interaction between HZ08 and P-glycoprotein in Caco-2 cell line in which P-glycoprotein is overexpressed naturally. Cytotoxicity and multidrug resistance reversal assays, together with flow cytometry, fluorescence microscopy and siRNA interference as well as Caco-2 monolayer transport model were employed in this study to evaluate the interaction between HZ08 and P-glycoprotein. This study revealed that HZ08 was capable of reversing adriamycin resistance mediated by P-glycoprotein as a result of intracellular enhancement of adriamycin accumulation, which was found to be superior to verapamil. In addition, we confirmed that HZ08 suppressed the transport of Rhodamine123 in the Caco-2 monolayer model but had little effect on P-glycoprotein expression. The transport of HZ08 was diminished by P-glycoprotein inhibitors (verapamil and LY335979) and its accumulation was increased via siRNA targeting MDR1 in Caco-2 cells. Furthermore, considering the binding site of P-glycoprotein, verapamil performed as a competitive inhibitor with HZ08. In conclusion, as a P-glycoprotein substrate, HZ08 inhibited P-glycoprotein activity and may share the same binding site of verapamil to P-glycoprotein. - Highlights: • The cytotoxicity and reversing effect of HZ08 was measured in Caco-2 cell line. • HZ08 inhibited the transport of Rhodamine123 across Caco-2 cell monolayer. • The efflux ratio of HZ08 was dropped when combined with P-glycoprotein

  5. Paramyxovirus mediated cell fusion requires co-expression of both the fusion and hemagglutinin-neuraminidase glycoproteins.

    PubMed

    Heminway, B R; Yu, Y; Galinski, M S

    1994-01-01

    Syncytia formation in either CV-1 or HeLa T4+ cells required recombinant expression of both fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins from the human parainfluenza virus type 3 (HPIV3), human parainfluenza virus type 2 (HPIV2), and simian virus 5 (SV5). In this system, recombinant T7 transcription vectors (pT7-5 or pGEM) containing F or HN, were transfected individually or in combination into cells previously infected with a recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3). While both proteins were processed and expressed at the cell surface, syncytia formation occurred only when both glycoproteins were co-expressed. The function of HN in the fusion process could not be replaced using lectins or by co-expression of heterologous F and HN proteins. Further, cell fusion was not observed when experiments were performed using individually expressed F and HN proteins in adjacent cells. The data presented in this report support the notion that a specific interaction between both paramyxoviral glycoproteins is required for the formation of syncytia in tissue culture monolayers. PMID:8165862

  6. How cell wall complexity influences saccharification efficiency in Miscanthus sinensis.

    PubMed

    De Souza, Amanda P; Alvim Kamei, Claire L; Torres, Andres F; Pattathil, Sivakumar; Hahn, Michael G; Trindade, Luisa M; Buckeridge, Marcos S

    2015-07-01

    The production of bioenergy from grasses has been developing quickly during the last decade, with Miscanthus being among the most important choices for production of bioethanol. However, one of the key barriers to producing bioethanol is the lack of information about cell wall structure. Cell walls are thought to display compositional differences that lead to emergence of a very high level of complexity, resulting in great diversity in cell wall architectures. In this work, a set of different techniques was used to access the complexity of cell walls of different genotypes of Miscanthus sinensis in order to understand how they interfere with saccharification efficiency. Three genotypes of M. sinensis displaying different patterns of correlation between lignin content and saccharification efficiency were subjected to cell wall analysis by quantitative/qualitative analytical techniques such as monosaccharide composition, oligosaccharide profiling, and glycome profiling. When saccharification efficiency was correlated negatively with lignin, the structural features of arabinoxylan and xyloglucan were found to contribute positively to hydrolysis. In the absence of such correlation, different types of pectins, and some mannans contributed to saccharification efficiency. Different genotypes of M. sinensis were shown to display distinct interactions among their cell wall components, which seem to influence cell wall hydrolysis. PMID:25908240

  7. How cell wall complexity influences saccharification efficiency in Miscanthus sinensis

    PubMed Central

    De Souza, Amanda P.; Kamei, Claire L. Alvim; Torres, Andres F.; Pattathil, Sivakumar; Hahn, Michael G.; Trindade, Luisa M.; Buckeridge, Marcos S.

    2015-01-01

    The production of bioenergy from grasses has been developing quickly during the last decade, with Miscanthus being among the most important choices for production of bioethanol. However, one of the key barriers to producing bioethanol is the lack of information about cell wall structure. Cell walls are thought to display compositional differences that lead to emergence of a very high level of complexity, resulting in great diversity in cell wall architectures. In this work, a set of different techniques was used to access the complexity of cell walls of different genotypes of Miscanthus sinensis in order to understand how they interfere with saccharification efficiency. Three genotypes of M. sinensis displaying different patterns of correlation between lignin content and saccharification efficiency were subjected to cell wall analysis by quantitative/qualitative analytical techniques such as monosaccharide composition, oligosaccharide profiling, and glycome profiling. When saccharification efficiency was correlated negatively with lignin, the structural features of arabinoxylan and xyloglucan were found to contribute positively to hydrolysis. In the absence of such correlation, different types of pectins, and some mannans contributed to saccharification efficiency. Different genotypes of M. sinensis were shown to display distinct interactions among their cell wall components, which seem to influence cell wall hydrolysis. PMID:25908240

  8. Cell Surface Glycoprotein of Reactive Stromal Fibroblasts as a Potential Antibody Target in Human Epithelial Cancers

    NASA Astrophysics Data System (ADS)

    Garin-Chesa, Pilar; Old, Lloyd J.; Rettig, Wolfgang J.

    1990-09-01

    The F19 antigen is a cell surface glycoprotein (M_r, 95,000) of human sarcomas and proliferating, cultured fibroblasts that is absent from resting fibroblasts in normal adult tissues. Normal and malignant epithelial cells are also F19^-. The present immunohistochemical study describes induction of F19 in the reactive mesenchyme of epithelial tumors. F19^+ fibroblasts were found in primary and metastatic carcinomas, including colorectal (18 of 18 cases studied), breast (14/14), ovarian (21/21), bladder (9/10), and lung carcinomas (13/13). In contrast, the stroma of benign colorectal adenomas, fibrocystic disease and fibroadenomas of breast, benign prostate hyperplasia, in situ bladder carcinomas, and benign ovarian tumors showed no or only moderate numbers of F19^+ fibroblasts. Analysis of dermal incision wounds revealed that F19 is strongly induced during scar formation. Comparison of F19 with the extracellular matrix protein tenascin, a putative marker of tumor mesenchyme, showed a cellular staining pattern for F19 vs. the extracellular matrix pattern for tenascin and widespread expression of tenascin in F19^- normal tissues and benign tumors. Our results suggest that the F19^+ phenotype correlates with specialized fibroblast functions in wound healing and malignant tumor growth. Because of its abundance in tumor mesenchyme, F19 may serve as a target for antibodies labeled with radioisotopes or toxic agents, or inflammatogenic antibodies, in carcinoma patients.

  9. Modulation of P-glycoprotein activity by cannabinoid molecules in HK-2 renal cells

    PubMed Central

    Nieri, Paola; Romiti, Nadia; Adinolfi, Barbara; Chicca, Andrea; Massarelli, Ilaria; Chieli, Elisabetta

    2006-01-01

    Endogenous and synthetic cannabinoid molecules have been investigated as possible MDR-1/P-glycoprotein (P-gp) modulators in HK-2-immortalized renal cells, using calcein acetoxymethylester (calcein-AM) as a P-gp substrate. Among the endocannabinoid molecules tested, anandamide (AEA), but not 2-arachidonoyl-glycerol (2-AG) or palmitoyl-ethanolamide (PEA), increased the intracellular fluorescence emitted by calcein, a metabolic derivative of the P-gp substrate calcein-AM, indicative of a reduction in transport capacity. All the three synthetic cannabimimetics tested, that is, R-(+)-methanandamide (R(+)-MET), AM 251 and CP55,940 significantly increased calcein accumulation in the cytosol. RT–PCR demonstrated that HK-2 cells do not express CB1 or CB2 cannabinoid receptors. R(+)-MET, AM251 and CP55,940 were also evaluated as modulators of P-gp expression, by Western blot analysis. Only AM251 weakly enhanced the protein levels (by 1.2-fold) after a 4-day-long incubation with the noncytotoxic drug concentration 2 μM. The present data provide the first evidence that the endocannabinoid AEA and different synthetic cannabinoids may inhibit the P-gp activity in vitro via a cannabinoid receptor-independent mechanism. PMID:16715117

  10. P-glycoprotein-dependent resistance of cancer cells toward the extrinsic TRAIL apoptosis signaling pathway

    PubMed Central

    Galski, Hanan; Oved-Gelber, Tamar; Simanovsky, Masha; Lazarovici, Philip; Gottesman, Michael M.; Nagler, Arnon

    2014-01-01

    The TNF-related apoptosis-inducing ligand (TRAIL or Apo2L) preferentially cause apoptosis of malignant cells in vitro and in vivo without severe toxicity. Therefore, TRAIL or agonist antibodies to the TRAIL DR4 and DR5 receptors are used in cancer therapy. However, many malignant cells are intrinsically resistant or acquire resistance to TRAIL. It has been previously proposed that the multidrug transporter P-glycoprotein (Pgp) might play a role in resistance of cells to intrinsic apoptotic pathways by interfering with components of ceramide metabolism or by modulating the electrochemical gradient across the plasma membrane. In this study we investigated whether Pgp also confers resistance toward extrinsic death ligands of the TNF family. To this end we focused our study on HeLa cells carrying a tetracycline-repressible plasmid system which shuts down Pgp expression in the presence of tetracycline. Our findings demonstrate that expression of Pgp is a significant factor conferring resistance to TRAIL administration, but not to other death ligands such as TNF-α and Fas ligand. Moreover, blocking Pgp transport activity sensitizes the malignant cells toward TRAIL. Therefore, Pgp transport function is required to confer resistance to TRAIL. Although the resistance to TRAIL-induced apoptosis is Pgp specific, TRAIL itself is not a direct substrate of Pgp. Pgp expression has no effect on the level of the TRAIL receptors DR4 and DR5. These findings might have clinical implications since the combination of TRAIL therapy with administration of Pgp modulators might sensitize TRAIL resistant tumors. PMID:23774624

  11. P-glycoprotein-dependent resistance of cancer cells toward the extrinsic TRAIL apoptosis signaling pathway.

    PubMed

    Galski, Hanan; Oved-Gelber, Tamar; Simanovsky, Masha; Lazarovici, Philip; Gottesman, Michael M; Nagler, Arnon

    2013-09-01

    The TNF-related apoptosis-inducing ligand (TRAIL or Apo2L) preferentially cause apoptosis of malignant cells in vitro and in vivo without severe toxicity. Therefore, TRAIL or agonist antibodies to the TRAIL DR4 and DR5 receptors are used in cancer therapy. However, many malignant cells are intrinsically resistant or acquire resistance to TRAIL. It has been previously proposed that the multidrug transporter P-glycoprotein (Pgp) might play a role in resistance of cells to intrinsic apoptotic pathways by interfering with components of ceramide metabolism or by modulating the electrochemical gradient across the plasma membrane. In this study we investigated whether Pgp also confers resistance toward extrinsic death ligands of the TNF family. To this end we focused our study on HeLa cells carrying a tetracycline-repressible plasmid system which shuts down Pgp expression in the presence of tetracycline. Our findings demonstrate that expression of Pgp is a significant factor conferring resistance to TRAIL administration, but not to other death ligands such as TNF-α and Fas ligand. Moreover, blocking Pgp transport activity sensitizes the malignant cells toward TRAIL. Therefore, Pgp transport function is required to confer resistance to TRAIL. Although the resistance to TRAIL-induced apoptosis is Pgp specific, TRAIL itself is not a direct substrate of Pgp. Pgp expression has no effect on the level of the TRAIL receptors DR4 and DR5. These findings might have clinical implications since the combination of TRAIL therapy with administration of Pgp modulators might sensitize TRAIL resistant tumors. PMID:23774624

  12. Varicella-zoster virus glycoprotein expression differentially induces the unfolded protein response in infected cells.

    PubMed

    Carpenter, John E; Grose, Charles

    2014-01-01

    Varicella-zoster virus (VZV) is a human herpesvirus that spreads to children as varicella or chicken pox. The virus then establishes latency in the nervous system and re-emerges, typically decades later, as zoster or shingles. We have reported previously that VZV induces autophagy in infected cells as well as exhibiting evidence of the Unfolded Protein Response (UPR): XBP1 splicing, a greatly expanded Endoplasmic Reticulum (ER) and CHOP expression. Herein we report the results of a UPR specific PCR array that measures the levels of mRNA of 84 different components of the UPR in VZV infected cells as compared to tunicamycin treated cells as a positive control and uninfected, untreated cells as a negative control. Tunicamycin is a mixture of chemicals that inhibits N-linked glycosylation in the ER with resultant protein misfolding and the UPR. We found that VZV differentially induces the UPR when compared to tunicamycin treatment. For example, tunicamycin treatment moderately increased (8-fold) roughly half of the array elements while downregulating only three (one ERAD and two FOLD components). VZV infection on the other hand upregulated 33 components including a little described stress sensor CREB-H (64-fold) as well as ER membrane components INSIG and gp78, which modulate cholesterol synthesis while downregulating over 20 components mostly associated with ERAD and FOLD. We hypothesize that this expression pattern is associated with an expanding ER with downregulation of active degradation by ERAD and apoptosis as the cell attempts to handle abundant viral glycoprotein synthesis. PMID:25071735

  13. Cell wall remodeling in mycorrhizal symbiosis: a way towards biotrophism

    PubMed Central

    Balestrini, Raffaella; Bonfante, Paola

    2014-01-01

    Cell walls are deeply involved in the molecular talk between partners during plant and microbe interactions, and their role in mycorrhizae, i.e., the widespread symbiotic associations established between plant roots and soil fungi, has been investigated extensively. All mycorrhizal interactions achieve full symbiotic functionality through the development of an extensive contact surface between the plant and fungal cells, where signals and nutrients are exchanged. The exchange of molecules between the fungal and the plant cytoplasm takes place both through their plasma membranes and their cell walls; a functional compartment, known as the symbiotic interface, is thus defined. Among all the symbiotic interfaces, the complex intracellular interface of arbuscular mycorrhizal (AM) symbiosis has received a great deal of attention since its first description. Here, in fact, the host plasma membrane invaginates and proliferates around all the developing intracellular fungal structures, and cell wall material is laid down between this membrane and the fungal cell surface. By contrast, in ectomycorrhizae (ECM), where the fungus grows outside and between the root cells, plant and fungal cell walls are always in direct contact and form the interface between the two partners. The organization and composition of cell walls within the interface compartment is a topic that has attracted widespread attention, both in ecto- and endomycorrhizae. The aim of this review is to provide a general overview of the current knowledge on this topic by integrating morphological observations, which have illustrated cell wall features during mycorrhizal interactions, with the current data produced by genomic and transcriptomic approaches. PMID:24926297

  14. A proteomic and genetic analysis of the Neurospora crassa conidia cell wall proteins identifies two glycosyl hydrolases involved in cell wall remodeling.

    PubMed

    Ao, Jie; Aldabbous, Mash'el; Notaro, Marysa J; Lojacono, Mark; Free, Stephen J

    2016-09-01

    A proteomic analysis of the conidial cell wall identified 35 cell wall proteins. A comparison with the proteome of the vegetative hyphae showed that 16 cell wall proteins were shared, and that these shared cell wall proteins were cell wall biosynthetic proteins or cell wall structural proteins. Deletion mutants for 34 of the genes were analyzed for phenotypes indicative of conidial cell wall defects. Mutants for two cell wall glycosyl hydrolases, the CGL-1 β-1,3-glucanase (NCU07523) and the NAG-1 exochitinase (NCU10852), were found to have a conidial separation phenotype. These two enzymes function in remodeling the cell wall between adjacent conidia to facilitate conidia formation and dissemination. Using promoter::RFP and promoter::GFP constructs, we demonstrated that the promoters for 15 of the conidia-specific cell wall genes, including cgl-1 and nag-1, provided for conidia-specific gene expression or for a significant increase in their expression during conidiation. PMID:27381444

  15. Plant expansins: diversity and interactions with plant cell walls

    PubMed Central

    Cosgrove, Daniel J.

    2015-01-01

    Expansins were discovered two decades ago as cell wall proteins that mediate acid-induced growth by catalyzing loosening of plant cell walls without lysis of wall polymers. In the interim our understanding of expansins has gotten more complex through bioinformatic analysis of expansin distribution and evolution, as well as through expression analysis, dissection of the upstream transcription factors regulating expression, and identification of additional classes of expansin by sequence and structural similarities. Molecular analyses of expansins from bacteria have identified residues essential for wall loosening activity and clarified the bifunctional nature of expansin binding to complex cell walls. Transgenic modulation of expansin expression modifies growth and stress physiology of plants, but not always in predictable and even understandable ways. PMID:26057089

  16. Measurement of pectin methylation in plant cell walls

    SciTech Connect

    McFeeters, R.F.; Armstrong, S.A.

    1984-01-01

    A procedure was developed to measure the degree of pectin methylation in small samples of isolated cell walls from nonlignified plant tissues or pectin solutions. Galacturonic acid was determined colorimetrically with the 3,5-dimethylphenol reagent. Methylation was measured by base hydrolysis of galacturonic acid methyl esters, followed by gas chromatographic determination of released methanol. Estimates of the precision of analysis of pectin and cell wall samples were made. The coefficient of variation for estimates of the pectin esterification in cell walls isolated from 10-g samples of cucumber tissue ranged from 7.7 to 13.2%.

  17. On the growth of walled cells: From shells to vesicles.

    NASA Astrophysics Data System (ADS)

    Boudaoud, Arezki

    2003-03-01

    The growth of isolated walled cells is investigated. Examples of such cells range from bacteria to giant algae, and include cochlear hair, plant root hair, fungi and yeast cells. They are modeled as elastic shells inflated by a liquid. Cell growth is driven by fluid pressure and is similar to a plastic deformation of the wall. The requirement of mechanical equilibrium leads to two new scaling laws for cell size that are in quantitative agreement with the compiled biological data. Given these results, possible shapes for growing cells are computed by analogy with those of vesicle membranes.

  18. Growth of Walled Cells: From Shells to Vesicles

    NASA Astrophysics Data System (ADS)

    Boudaoud, Arezki

    2003-07-01

    The growth of isolated walled cells is investigated. Examples of such cells range from bacteria to giant algae, and include cochlear hair, plant root hair, fungi, and yeast cells. They are modeled as elastic shells containing a liquid. Cell growth is driven by fluid pressure and is is similar to a plastic deformation of the wall. The requirement of mechanical equilibrium leads to two new scaling laws for cell size that are in quantitative agreement with the compiled biological data. Given these results, possible shapes for growing cells are computed by analogy with those of vesicle membranes.

  19. An Arabidopsis Gene Regulatory Network for Secondary Cell Wall Synthesis

    PubMed Central

    Taylor-Teeples, M; Lin, L; de Lucas, M; Turco, G; Toal, TW; Gaudinier, A; Young, NF; Trabucco, GM; Veling, MT; Lamothe, R; Handakumbura, PP; Xiong, G; Wang, C; Corwin, J; Tsoukalas, A; Zhang, L; Ware, D; Pauly, M; Kliebenstein, DJ; Dehesh, K; Tagkopoulos, I; Breton, G; Pruneda-Paz, JL; Ahnert, SE; Kay, SA; Hazen, SP; Brady, SM

    2014-01-01

    Summary The plant cell wall is an important factor for determining cell shape, function and response to the environment. Secondary cell walls, such as those found in xylem, are composed of cellulose, hemicelluloses and lignin and account for the bulk of plant biomass. The coordination between transcriptional regulation of synthesis for each polymer is complex and vital to cell function. A regulatory hierarchy of developmental switches has been proposed, although the full complement of regulators remains unknown. Here, we present a protein-DNA network between Arabidopsis transcription factors and secondary cell wall metabolic genes with gene expression regulated by a series of feed-forward loops. This model allowed us to develop and validate new hypotheses about secondary wall gene regulation under abiotic stress. Distinct stresses are able to perturb targeted genes to potentially promote functional adaptation. These interactions will serve as a foundation for understanding the regulation of a complex, integral plant component. PMID:25533953

  20. Regulation of Meristem Morphogenesis by Cell Wall Synthases in Arabidopsis.

    PubMed

    Yang, Weibing; Schuster, Christoph; Beahan, Cherie T; Charoensawan, Varodom; Peaucelle, Alexis; Bacic, Antony; Doblin, Monika S; Wightman, Raymond; Meyerowitz, Elliot M

    2016-06-01

    The cell walls of the shoot apical meristem (SAM), containing the stem cell niche that gives rise to the above-ground tissues, are crucially involved in regulating differentiation. It is currently unknown how these walls are built and refined or their role, if any, in influencing meristem developmental dynamics. We have combined polysaccharide linkage analysis, immuno-labeling, and transcriptome profiling of the SAM to provide a spatiotemporal plan of the walls of this dynamic structure. We find that meristematic cells express only a core subset of 152 genes encoding cell wall glycosyltransferases (GTs). Systemic localization of all these GT mRNAs by in situ hybridization reveals members with either enrichment in or specificity to apical subdomains such as emerging flower primordia, and a large class with high expression in dividing cells. The highly localized and coordinated expression of GTs in the SAM suggests distinct wall properties of meristematic cells and specific differences between newly forming walls and their mature descendants. Functional analysis demonstrates that a subset of CSLD genes is essential for proper meristem maintenance, confirming the key role of walls in developmental pathways. PMID:27212401

  1. Cell Wall Metabolism in Response to Abiotic Stress

    PubMed Central

    Gall, Hyacinthe Le; Philippe, Florian; Domon, Jean-Marc; Gillet, Françoise; Pelloux, Jérôme; Rayon, Catherine

    2015-01-01

    This review focuses on the responses of the plant cell wall to several abiotic stresses including drought, flooding, heat, cold, salt, heavy metals, light, and air pollutants. The effects of stress on cell wall metabolism are discussed at the physiological (morphogenic), transcriptomic, proteomic and biochemical levels. The analysis of a large set of data shows that the plant response is highly complex. The overall effects of most abiotic stress are often dependent on the plant species, the genotype, the age of the plant, the timing of the stress application, and the intensity of this stress. This shows the difficulty of identifying a common pattern of stress response in cell wall architecture that could enable adaptation and/or resistance to abiotic stress. However, in most cases, two main mechanisms can be highlighted: (i) an increased level in xyloglucan endotransglucosylase/hydrolase (XTH) and expansin proteins, associated with an increase in the degree of rhamnogalacturonan I branching that maintains cell wall plasticity and (ii) an increased cell wall thickening by reinforcement of the secondary wall with hemicellulose and lignin deposition. Taken together, these results show the need to undertake large-scale analyses, using multidisciplinary approaches, to unravel the consequences of stress on the cell wall. This will help identify the key components that could be targeted to improve biomass production under stress conditions. PMID:27135320

  2. Cell Wall Metabolism in Response to Abiotic Stress.

    PubMed

    Le Gall, Hyacinthe; Philippe, Florian; Domon, Jean-Marc; Gillet, Françoise; Pelloux, Jérôme; Rayon, Catherine

    2015-01-01

    This review focuses on the responses of the plant cell wall to several abiotic stresses including drought, flooding, heat, cold, salt, heavy metals, light, and air pollutants. The effects of stress on cell wall metabolism are discussed at the physiological (morphogenic), transcriptomic, proteomic and biochemical levels. The analysis of a large set of data shows that the plant response is highly complex. The overall effects of most abiotic stress are often dependent on the plant species, the genotype, the age of the plant, the timing of the stress application, and the intensity of this stress. This shows the difficulty of identifying a common pattern of stress response in cell wall architecture that could enable adaptation and/or resistance to abiotic stress. However, in most cases, two main mechanisms can be highlighted: (i) an increased level in xyloglucan endotransglucosylase/hydrolase (XTH) and expansin proteins, associated with an increase in the degree of rhamnogalacturonan I branching that maintains cell wall plasticity and (ii) an increased cell wall thickening by reinforcement of the secondary wall with hemicellulose and lignin deposition. Taken together, these results show the need to undertake large-scale analyses, using multidisciplinary approaches, to unravel the consequences of stress on the cell wall. This will help identify the key components that could be targeted to improve biomass production under stress conditions. PMID:27135320

  3. Arabinogalactan protein-rich cell walls, paramural deposits and ergastic globules define the hyaline bodies of rhinanthoid Orobanchaceae haustoria

    PubMed Central

    Pielach, Anna; Leroux, Olivier; Domozych, David S.; Knox, J. Paul; Popper, Zoë A.

    2014-01-01

    Background and Aims Parasitic plants obtain nutrients from their hosts through organs called haustoria. The hyaline body is a specialized parenchymatous tissue occupying the central parts of haustoria in many Orobanchaceae species. The structure and functions of hyaline bodies are poorly understood despite their apparent necessity for the proper functioning of haustoria. Reported here is a cell wall-focused immunohistochemical study of the hyaline bodies of three species from the ecologically important clade of rhinanthoid Orobanchaceae. Methods Haustoria collected from laboratory-grown and field-collected plants of Rhinanthus minor, Odontites vernus and Melampyrum pratense attached to various hosts were immunolabelled for cell wall matrix glycans and glycoproteins using specific monoclonal antibodies (mAbs). Key Results Hyaline body cell wall architecture differed from that of the surrounding parenchyma in all species investigated. Enrichment in arabinogalactan protein (AGP) epitopes labelled with mAbs LM2, JIM8, JIM13, JIM14 and CCRC-M7 was prominent and coincided with reduced labelling of de-esterified homogalacturonan with mAbs JIM5, LM18 and LM19. Furthermore, paramural bodies, intercellular deposits and globular ergastic bodies composed of pectins, xyloglucans, extensins and AGPs were common. In Rhinanthus they were particularly abundant in pairings with legume hosts. Hyaline body cells were not in direct contact with haustorial xylem, which was surrounded by a single layer of paratracheal parenchyma with thickened cell walls abutting the xylem. Conclusions The distinctive anatomy and cell wall architecture indicate hyaline body specialization. Altered proportions of AGPs and pectins may affect the mechanical properties of hyaline body cell walls. This and the association with a transfer-like type of paratracheal parenchyma suggest a role in nutrient translocation. Organelle-rich protoplasts and the presence of exceptionally profuse intra- and intercellular

  4. Biosynthesis of ascites sialoglycoprotein-1, the major O-linked glycoprotein of 13762 rat mammary adenocarcinoma ascites cells

    SciTech Connect

    Spielman, J.

    1987-01-01

    The present studies were undertaken to determine the timing of the major events in biosynthesis, and to characterize the contributions of chain initiation and elongation in maturation of the glycoprotein. Initiation of the earliest O-linked chains was detected by analysis of conversion of {sup 3}H-thr to {sup 3}H 2-aminobutyrate following mild alkaline borohydride elimination of O-linked sugars from peanut lectin-precipitated ASGP-1. Initiation was detected within 5 min of translation; amino sugar analysis of GlcNH{sub 2}-labeled, trypsinized cells also showed that GalNAc was added as late as 5 min prior to arrival of ASGP-1 at the cell surface. Thus initiation occurs throughout biosynthesis. Maturation of the glycoprotein from a lightly-glycosylated immature form to the heavily-glycosylated mature from involved both continued initiation of new chains and chain elongation, and occurred with a half-time of about 30 min. Analysis of labeled ASGP-1 released from the cell surface by trypsinization showed that although some newly-synthesized ASGP-1 reached the cell surface within 70-80 min of protein synthesis, the half-time for appearance of mature glycoprotein was in excess of 4 hr, indicating that most molecules reside in an intracellular compartment(s) for a considerable time.

  5. Human plasmacytoid dendritic cells efficiently capture HIV-1 envelope glycoproteins via CD4 for antigen presentation.

    PubMed

    Sandgren, Kerrie J; Smed-Sörensen, Anna; Forsell, Mattias N; Soldemo, Martina; Adams, William C; Liang, Frank; Perbeck, Leif; Koup, Richard A; Wyatt, Richard T; Karlsson Hedestam, Gunilla B; Loré, Karin

    2013-07-01

    Advances in HIV-1 vaccine clinical trials and preclinical research indicate that the virus envelope glycoproteins (Env) are likely to be an essential component of a prophylactic vaccine. Efficient Ag uptake and presentation by dendritic cells (DCs) is important for strong CD4(+) Th cell responses and the development of effective humoral immune responses. In this study, we examined the capacity of distinct primary human DC subsets to internalize and present recombinant Env to CD4(+) T cells. Consistent with their specific receptor expression, skin DCs bound and internalized Env via C-type lectin receptors, whereas blood DC subsets, including CD1c(+) myeloid DCs, CD123(+) plasmacytoid DCs (PDCs), and CD141(+) DCs exhibited a restricted repertoire of C-type lectin receptors and relied on CD4 for uptake of Env. Despite a generally poor capacity for Ag uptake compared with myeloid DCs, the high expression of CD4 on PDCs allowed them to bind and internalize Env very efficiently. CD4-mediated uptake delivered Env to EEA1(+) endosomes that progressed to Lamp1(+) and MHC class II(+) lysosomes where internalized Env was degraded rapidly. Finally, all three blood DC subsets were able to internalize an Env-CMV pp65 fusion protein via CD4 and stimulate pp65-specific CD4(+) T cells. Thus, in the in vitro systems described in this paper, CD4-mediated uptake of Env is a functional pathway leading to Ag presentation, and this may therefore be a mechanism used by blood DCs, including PDCs, for generating immune responses to Env-based vaccines. PMID:23729440

  6. Cell Wall Composition, Biosynthesis and Remodeling during Pollen Tube Growth

    PubMed Central

    Mollet, Jean-Claude; Leroux, Christelle; Dardelle, Flavien; Lehner, Arnaud

    2013-01-01

    The pollen tube is a fast tip-growing cell carrying the two sperm cells to the ovule allowing the double fertilization process and seed setting. To succeed in this process, the spatial and temporal controls of pollen tube growth within the female organ are critical. It requires a massive cell wall deposition to promote fast pollen tube elongation and a tight control of the cell wall remodeling to modify the mechanical properties. In addition, during its journey, the pollen tube interacts with the pistil, which plays key roles in pollen tube nutrition, guidance and in the rejection of the self-incompatible pollen. This review focuses on our current knowledge in the biochemistry and localization of the main cell wall polymers including pectin, hemicellulose, cellulose and callose from several pollen tube species. Moreover, based on transcriptomic data and functional genomic studies, the possible enzymes involved in the cell wall remodeling during pollen tube growth and their impact on the cell wall mechanics are also described. Finally, mutant analyses have permitted to gain insight in the function of several genes involved in the pollen tube cell wall biosynthesis and their roles in pollen tube growth are further discussed. PMID:27137369

  7. MP1 encodes an abundant and highly antigenic cell wall mannoprotein in the pathogenic fungus Penicillium marneffei.

    PubMed

    Cao, L; Chan, C M; Lee, C; Wong, S S; Yuen, K Y

    1998-03-01

    We cloned the MP1 gene, which encodes an abundant antigenic cell wall mannoprotein from the dimorphic pathogenic fungus Penicillium marneffei. MP1 is a unique gene without homologs in sequence databases. It codes for a protein, Mp1p, of 462 amino acid residues, with a few sequence features that are present in several cell wall proteins of Saccharomyces cerevisiae and Candida albicans. It contains two putative N glycosylation sites, a serine- and threonine-rich region for O glycosylation, a signal peptide, and a putative glycosylphosphatidylinositol attachment signal sequence. Specific anti-Mp1p antibody was generated with recombinant Mp1p protein purified from Escherichia coli to allow further characterization of Mp1p. Western blot analysis with anti-Mp1p antibody revealed that Mp1p has predominant bands with molecular masses of 58 and 90 kDa and that it belongs to a group of cell wall proteins that can be readily removed from yeast cell surfaces by glucanase digestion. In addition, Mp1p is an abundant yeast glycoprotein and has high affinity for concanavalin A, a characteristic indicative of a mannoprotein. Furthermore, ultrastructural analysis with immunogold staining indicated that Mp1p is present in the cell walls of the yeast, hyphae, and conidia of P. marneffei. Finally, it was observed that infected patients develop a specific antibody response against Mp1p, suggesting that this protein represents a good cell surface target for host humoral immunity. PMID:9488383

  8. neo-Clerodane diterpenoids from Scutellaria barbata mediated inhibition of P-glycoprotein in MCF-7/ADR cells.

    PubMed

    Xue, Gui-Min; Xia, Yuan-Zheng; Wang, Zhi-Min; Li, Ling-Nan; Luo, Jian-Guang; Kong, Ling-Yi

    2016-10-01

    Ten new (1-10) and seventeen known (11-27) neo-clerodane diterpenoids substituted with nicotinoyloxyl were isolated from the plant Scutellaria barbata and their structures were established by extensive spectroscopic analysis. Chemoreversal effects of these neo-clerodane diterpenoids on multidrug resistance were evaluated in breast cancer multidrug-resistant MCF-7/ADR cells that overexpress P-glycoprotein. Four compounds (11, 14, 16, and 18) exhibited better chemoreversal abilities than the classical P-gp inhibitor verapamil and the most potent compound 11 reduced IC50 value of adriamycin in MCF-7/ADR cells from 58.8 μM to 1.3 μM. Mechanistic investigations showed that compound 11 reversed multidrug resistance through suppressing the activity of P-gp and restraining the expression of P-glycoprotein. In the present study, the structure-activity relationships of neo-clerodane diterpenoids were also discussed. PMID:27240278

  9. A formin-nucleated actin aster concentrates cell wall hydrolases for cell fusion in fission yeast

    PubMed Central

    Dudin, Omaya; Bendezú, Felipe O.; Groux, Raphael; Laroche, Thierry; Seitz, Arne

    2015-01-01

    Cell–cell fusion is essential for fertilization. For fusion of walled cells, the cell wall must be degraded at a precise location but maintained in surrounding regions to protect against lysis. In fission yeast cells, the formin Fus1, which nucleates linear actin filaments, is essential for this process. In this paper, we show that this formin organizes a specific actin structure—the actin fusion focus. Structured illumination microscopy and live-cell imaging of Fus1, actin, and type V myosins revealed an aster of actin filaments whose barbed ends are focalized near the plasma membrane. Focalization requires Fus1 and type V myosins and happens asynchronously always in the M cell first. Type V myosins are essential for fusion and concentrate cell wall hydrolases, but not cell wall synthases, at the fusion focus. Thus, the fusion focus focalizes cell wall dissolution within a broader cell wall synthesis zone to shift from cell growth to cell fusion. PMID:25825517

  10. Re-evaluation of the role of P-glycoprotein in in vitro drug permeability studies with the bovine brain microvessel endothelial cells.

    PubMed

    Hakkarainen, Jenni J; Rilla, Kirsi; Suhonen, Marjukka; Ruponen, Marika; Forsberg, Markus M

    2014-03-01

    1.  Currently available in vitro blood-brain barrier models all have recognized restrictions. In addition to leakiness, inconsistent data about P-glycoprotein mediated efflux limit the attractiveness of the primary bovine brain microvessel endothelial cells (BBMECs). Therefore, we re-evaluated the role of P-glycoprotein mediated efflux with two culture conditions in BBMECs for prediction of drug permeability of potential P-glycoprotein substrates. 2.  BBMECs were monocultured on filters on petri dishes and on filter inserts, and expression and localization of P-glycoprotein were compared by using western blot and confocal microscopy, respectively. The functionality of P-glycoprotein was assessed by using cellular uptake, calcein-AM and bidirectional transport assays. 3.  P-glycoprotein expression was higher in BBMECs cultured on filter inserts decreasing the permeability of digoxin and paclitaxel, but not the permeability of vinblastine. However, the monocultured BBMECs were not able to demonstrate efflux in the bidirectional transport assays. Under certain culture conditions, occludin may not be correctly located, perhaps explaining in part the leakiness of BBMECs. 4.  In conclusion, BBMECs, despite possessing a functional P-glycoprotein, under certain culture conditions may not be a suitable in vitro model for the bidirectional transport assays and for predicting the permeability of drugs and xenobiotics that are potential P-glycoprotein substrates. PMID:23924297

  11. Plant cell wall characterization using scanning probe microscopy techniques

    PubMed Central

    Yarbrough, John M; Himmel, Michael E; Ding, Shi-You

    2009-01-01

    Lignocellulosic biomass is today considered a promising renewable resource for bioenergy production. A combined chemical and biological process is currently under consideration for the conversion of polysaccharides from plant cell wall materials, mainly cellulose and hemicelluloses, to simple sugars that can be fermented to biofuels. Native plant cellulose forms nanometer-scale microfibrils that are embedded in a polymeric network of hemicelluloses, pectins, and lignins; this explains, in part, the recalcitrance of biomass to deconstruction. The chemical and structural characteristics of these plant cell wall constituents remain largely unknown today. Scanning probe microscopy techniques, particularly atomic force microscopy and its application in characterizing plant cell wall structure, are reviewed here. We also further discuss future developments based on scanning probe microscopy techniques that combine linear and nonlinear optical techniques to characterize plant cell wall nanometer-scale structures, specifically apertureless near-field scanning optical microscopy and coherent anti-Stokes Raman scattering microscopy. PMID:19703302

  12. Modification of cell wall polysaccharides during retting of cassava roots.

    PubMed

    Ngolong Ngea, Guillaume Legrand; Guillon, Fabienne; Essia Ngang, Jean Justin; Bonnin, Estelle; Bouchet, Brigitte; Saulnier, Luc

    2016-12-15

    Retting is an important step in traditional cassava processing that involves tissue softening of the roots to transform the cassava into flour and various food products. The tissue softening that occurs during retting was attributed to the degradation of cell wall pectins through the action of pectin-methylesterase and pectate-lyase that possibly originated from a microbial source or the cassava plant itself. Changes in cell wall composition were investigated during retting using chemical analysis, specific glycanase degradation and immuno-labelling of cell wall polysaccharides. Pectic 1,4-β-d-galactan was the main cell wall polysaccharide affected during the retting of cassava roots. This result suggested that better control of pectic galactan degradation and a better understanding of the degradation mechanism by endogenous endo-galactanase and/or exogenous microbial enzymes might contribute to improve the texture properties of cassava products. PMID:27451197

  13. Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation.

    PubMed

    Shah, Punit; Wang, Xiangchun; Yang, Weiming; Toghi Eshghi, Shadi; Sun, Shisheng; Hoti, Naseruddin; Chen, Lijun; Yang, Shuang; Pasay, Jered; Rubin, Abby; Zhang, Hui

    2015-10-01

    Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two- dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in

  14. Evolution and development of cell walls in cereal grains

    PubMed Central

    Burton, Rachel A.; Fincher, Geoffrey B.

    2014-01-01

    The composition of cell walls in cereal grains and other grass species differs markedly from walls in seeds of other plants. In the maternal tissues that surround the embryo and endosperm of the grain, walls contain higher levels of cellulose and in many cases are heavily lignified. This may be contrasted with walls of the endosperm, where the amount of cellulose is relatively low, and the walls are generally not lignified. The low cellulose and lignin contents are possible because the walls of the endosperm perform no load-bearing function in the mature grain and indeed the low levels of these relatively intractable wall components are necessary because they allow rapid degradation of the walls following germination of the grain. The major non-cellulosic components of endosperm walls are usually heteroxylans and (1,3;1,4)-β-glucans, with lower levels of xyloglucans, glucomannans, and pectic polysaccharides. Pectic polysaccharides and xyloglucans are the major non-cellulosic wall constituents in most dicot species, in which (1,3;1,4)-β-glucans are usually absent and heteroxylans are found at relatively low levels. Thus, the “core” non-cellulosic wall polysaccharides in grain of the cereals and other grasses are the heteroxylans and, more specifically, arabinoxylans. The (1,3;1,4)-β-glucans appear in the endosperm of some grass species but are essentially absent from others; they may constitute from zero to more than 45% of the cell walls of the endosperm, depending on the species. It is clear that in some cases these (1,3;1,4)-β-glucans function as a major store of metabolizable glucose in the grain. Cereal grains and their constituent cell wall polysaccharides are centrally important as a source of dietary fiber in human societies and breeders have started to select for high levels of non-cellulosic wall polysaccharides in grain. To meet end-user requirements, it is important that we understand cell wall biology in the grain both during development and

  15. An improved protocol to study the plant cell wall proteome

    PubMed Central

    Printz, Bruno; Dos Santos Morais, Raphaël; Wienkoop, Stefanie; Sergeant, Kjell; Lutts, Stanley; Hausman, Jean-Francois; Renaut, Jenny

    2015-01-01

    Cell wall proteins were extracted from alfalfa stems according to a three-steps extraction procedure using sequentially CaCl2, EGTA, and LiCl-complemented buffers. The efficiency of this protocol for extracting cell wall proteins was compared with the two previously published methods optimized for alfalfa stem cell wall protein analysis. Following LC-MS/MS analysis the three-steps extraction procedure resulted in the identification of the highest number of cell wall proteins (242 NCBInr identifiers) and gave the lowest percentage of non-cell wall proteins (about 30%). However, the three protocols are rather complementary than substitutive since 43% of the identified proteins were specific to one protocol. This three-step protocol was therefore selected for a more detailed proteomic characterization using 2D-gel electrophoresis. With this technique, 75% of the identified proteins were shown to be fraction-specific and 72.7% were predicted as belonging to the cell wall compartment. Although, being less sensitive than LC-MS/MS approaches in detecting and identifying low-abundant proteins, gel-based approaches are valuable tools for the differentiation and relative quantification of protein isoforms and/or modified proteins. In particular isoforms, having variations in their amino-acid sequence and/or carrying different N-linked glycan chains were detected and characterized. This study highlights how the extracting protocols as well as the analytical techniques devoted to the study of the plant cell wall proteome are complementary and how they may be combined to elucidate the dynamism of the plant cell wall proteome in biological studies. Data are available via ProteomeXchange with identifier PXD001927. PMID:25914713

  16. Construction, molecular modeling, and simulation of Mycobacterium tuberculosis cell walls.

    PubMed

    Hong, Xuan; Hopfinger, A J

    2004-01-01

    The mycobacterial cell wall is extraordinarily thick and tight consisting mainly of (1). long chain fatty acids, the mycolic acids, and (2). a unique polysaccharide, arabinogalactan (AG). These two chemical constituents are covalently linked through ester bonds. Minnikin (The Biology of the Mycobacteria; Academic: London, 1982) proposed that the mycobacterial cell wall is composed of an asymmetric lipid bilayer. The inner leaflet of the cell wall contains mycolic acids covalently linked to AG. This inner leaflet is believed to have the lowest permeability to organic compounds of the overall cell wall. Conformational search and molecular dynamics simulation were used to explore the conformational profile of AG and the conformations and structural organization of the mycolic acid-AG complex, and overall, an inner leaflet molecular model of the cell wall was constructed. The terminal arabinose residues of AG that serve as linkers between AG and mycolic acids were found to exist in four major chemical configurations. The mycolate hydrocarbon chains were determined to be tightly packed and perpendicular to the "plane" formed by the oxygen atoms of the 5-hydroxyl groups of the terminal arabinose residues. For Mycobacterium tuberculosis, the average packing distance between mycolic acids is estimated to be approximately 7.3 A. Thus, Minnikin's model is supported by this computational study. Overall, this modeling and simulation approach provides a way to probe the mechanism of low permeability of the cell wall and the intrinsic drug resistance of M. tuberculosis. In addition, monolayer models were built for both dipalmitoylphosphatidylethanolamine and dimyristoylphosphatidylcholine, two common phospholipids in bacterial and animal membranes, respectively. Structural comparisons of these cell wall phospholipid membrane models were made to the M. tuberculosis cell wall model. PMID:15132700

  17. Cell wall polysaccharides from fern leaves: evidence for a mannan-rich Type III cell wall in Adiantum raddianum.

    PubMed

    Silva, Giovanna B; Ionashiro, Mari; Carrara, Thalita B; Crivellari, Augusto C; Tiné, Marco A S; Prado, Jefferson; Carpita, Nicholas C; Buckeridge, Marcos S

    2011-12-01

    Primary cell walls from plants are composites of cellulose tethered by cross-linking glycans and embedded in a matrix of pectins. Cell wall composition varies between plant species, reflecting in some instances the evolutionary distance between them. In this work the monosaccharide compositions of isolated primary cell walls of nine fern species and one lycophyte were characterized and compared with those from Equisetum and an angiosperm dicot. The relatively high abundance of mannose in these plants suggests that mannans may constitute the major cross-linking glycan in the primary walls of pteridophytes and lycophytes. Pectin-related polysaccharides contained mostly rhamnose and uronic acids, indicating the presence of rhamnogalacturonan I highly substituted with galactose and arabinose. Structural and fine-structural analyses of the hemicellulose fraction of leaves of Adiantum raddianum confirmed this hypothesis. Linkage analysis showed that the mannan contains mostly 4-Man with very little 4,6-Man, indicating a low percentage of branching with galactose. Treatment of the mannan-rich fractions with endo-β-mannanase produced characteristic mannan oligosaccharides. Minor amounts of xyloglucan and xylans were also detected. These data and those of others suggest that all vascular plants contain xyloglucans, arabinoxylans, and (gluco)mannans, but in different proportions that define cell wall types. Whereas xyloglucan and pectin-rich walls define Type I walls of dicots and many monocots, arabinoxylans and lower proportion of pectin define the Type II walls of commelinoid monocots. The mannan-rich primary walls with low pectins of many ferns and a lycopod indicate a fundamentally different wall type among land plants, the Type III wall. PMID:21955619

  18. Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment

    PubMed Central

    Bender, Ruben R.; Muth, Anke; Schneider, Irene C.; Friedel, Thorsten; Hartmann, Jessica; Plückthun, Andreas; Maisner, Andrea; Buchholz, Christian J.

    2016-01-01

    Receptor-targeted lentiviral vectors (LVs) can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV) glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance). Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV) glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4) was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs. PMID:27281338

  19. Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment.

    PubMed

    Bender, Ruben R; Muth, Anke; Schneider, Irene C; Friedel, Thorsten; Hartmann, Jessica; Plückthun, Andreas; Maisner, Andrea; Buchholz, Christian J

    2016-06-01

    Receptor-targeted lentiviral vectors (LVs) can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV) glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance). Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV) glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4) was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs. PMID:27281338

  20. Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor

    SciTech Connect

    Pytela, R.; Pierschbacher, M.D.; Ruoslahti, E.

    1985-01-01

    Affinity chromatography was used to identify a putative cell surface receptor for fibronectin. A large cell-attachment-promoting fibronectin fragment was used as the affinity matrix, and specific elution was effected by using synthetic peptides containing the sequence Arg-Gly-Asp, which is derived from the cell recognition sequence in the fibronectin cell attachment site. A 140 kd protein was bound by the affinity matrix from octylglucoside extracts of MG-63 human osteosarcoma cells and specifically eluted with the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro. The 140 kd protein was labeled by cell surface specific radioiodination and became incorporated into liposomes at a high efficiency. Liposomes containing this protein showed specific affinity toward fibronectin-coated surfaces, and this binding could be selectively inhibited by the synthetic cell-attachment peptide but not by inactive peptides. Affinity chromatography on wheat germ agglutinin-Sepharose showed that the 140 kd protein is a glycoprotein and, in combination with the fibronectin fragment chromatography, gave highly enriched preparations of the 140 kd protein. These properties suggest that the 140 kd glycoprotein is a membrane-embedded cell surface protein directly involved in the initial step of cell adhesion to fibronectin substrates.

  1. Characterization and Localization of Insoluble Organic Matrices Associated with Diatom Cell Walls: Insight into Their Roles during Cell Wall Formation

    PubMed Central

    Tesson, Benoit; Hildebrand, Mark

    2013-01-01

    Organic components associated with diatom cell wall silica are important for the formation, integrity, and function of the cell wall. Polysaccharides are associated with the silica, however their localization, structure, and function remain poorly understood. We used imaging and biochemical approaches to describe in detail characteristics of insoluble organic components associated with the cell wall in 5 different diatom species. Results show that an insoluble organic matrix enriched in mannose, likely the diatotepum, is localized on the proximal surface of the silica cell wall. We did not identify any organic matrix embedded within the silica. We also identified a distinct material consisting of glucose polymer with variable localization depending on the species. In some species this component was directly involved in the morphogenesis of silica structure while in others it appeared to be only a structural component of the cell wall. A novel glucose-rich structure located between daughter cells during division was also identified. This work for the first time correlates the structure, composition, and localization of insoluble organic matrices associated with diatom cell walls. Additionally we identified a novel glucose polymer and characterized its role during silica structure formation. PMID:23626714

  2. Live cell imaging of the cytoskeleton and cell wall enzymes in plant cells.

    PubMed

    Sampathkumar, Arun; Wightman, Raymond

    2015-01-01

    The use of live imaging techniques to visualize the dynamic changes and interactions within plant cells has given us detailed information on the function and organization of the cytoskeleton and cell wall associated proteins. This information has grown with the constant improvement in imaging hardware and molecular tools. In this chapter, we describe the procedure for the preparation and live visualization of fluorescent protein fusions associated with the cytoskeleton and the cell wall in Arabidopsis. PMID:25408450

  3. Cell-mediated Immunity to Human Tamm-Horsfall Glycoprotein in Autoimmune Liver Disease with Renal Tubular Acidosis

    PubMed Central

    Tsantoulas, D. C.; McFarlane, I. G.; Portmann, B.; Eddleston, A. L. W. F.; Williams, Roger

    1974-01-01

    Cell-mediated immune responses to Tamm-Horsfall glycoprotein isolated from human urine were investigated using the leucocyte migration test. Abnormal responses were found in 91% of patients with active chronic hepatitis or primary biliary cirrhosis with an associated renal tubular acidosis (R.T.A.) but in only 19% of those without R.T.A. In nearly all of a group of patients without autoimmune liver disease and in a control group of normal subjects results were within normal limits. In addition, using an immunofluorescent technique with rabbit antibody to human Tamm-Horsfall glycoprotein, it was possible to show the presence in human liver cell membrane of material reacting immunologically as Tamm-Horsfall. These findings suggest that the development of an immune response to this glycoprotein, initiated by release of cross-reacting antigens from damaged hepatocytes, could be the mechanism underlying the occurrence of R.T.A. in some patients with autoimmune liver disease. ImagesFIG. 3 PMID:4611578

  4. An optimized approach for enrichment of glycoproteins from cell culture lysates using native multi-lectin affinity chromatography.

    PubMed

    Lee, Ling Y; Hincapie, Marina; Packer, Nicolle; Baker, Mark S; Hancock, William S; Fanayan, Susan

    2012-09-01

    Lectins are capable of recognizing specific glycan structures and serve as invaluable tools for the separation of glycosylated proteins from nonglycosylated proteins in biological samples. We report on the optimization of native multi-lectin affinity chromatography, combining three lectins, namely, concanavalin A, jacalin, and wheat germ agglutinin for fractionation of cellular glycoproteins from MCF-7 breast cancer lysate. We evaluated several conditions for optimum recovery of total proteins and glycoproteins such as low pH and saccharide elution buffers, and the inclusion of detergents in binding and elution buffers. Optimum recovery was observed with overnight incubation of cell lysate with lectins at 4°C, and inclusion of detergent in binding and saccharide elution buffers. Total protein and bound recoveries were 80 and 9%, respectively. Importantly, we found that high saccharide strength elution buffers were not necessary to release bound glycoproteins. This study demonstrates that multi-lectin affinity chromatography can be extended to total cell lysate to investigate the cellular glycoproteome. PMID:22997032

  5. Characterizing visible and invisible cell wall mutant phenotypes.

    PubMed

    Carpita, Nicholas C; McCann, Maureen C

    2015-07-01

    About 10% of a plant's genome is devoted to generating the protein machinery to synthesize, remodel, and deconstruct the cell wall. High-throughput genome sequencing technologies have enabled a reasonably complete inventory of wall-related genes that can be assembled into families of common evolutionary origin. Assigning function to each gene family member has been aided immensely by identification of mutants with visible phenotypes or by chemical and spectroscopic analysis of mutants with 'invisible' phenotypes of modified cell wall composition and architecture that do not otherwise affect plant growth or development. This review connects the inference of gene function on the basis of deviation from the wild type in genetic functional analyses to insights provided by modern analytical techniques that have brought us ever closer to elucidating the sequence structures of the major polysaccharide components of the plant cell wall. PMID:25873661

  6. Ultrastructure and Composition of the Nannochloropsis gaditana Cell Wall

    PubMed Central

    Scholz, Matthew J.; Weiss, Taylor L.; Jinkerson, Robert E.; Jing, Jia; Roth, Robyn; Goodenough, Ursula; Posewitz, Matthew C.

    2014-01-01

    Marine algae of the genus Nannochloropsis are promising producers of biofuel precursors and nutraceuticals and are also harvested commercially for aquaculture feed. We have used quick-freeze, deep-etch electron microscopy, Fourier transform infrared spectroscopy, and carbohydrate analyses to characterize the architecture of the Nannochloropsis gaditana (strain CCMP 526) cell wall, whose recalcitrance presents a significant barrier to biocommodity extraction. The data indicate a bilayer structure consisting of a cellulosic inner wall (∼75% of the mass balance) protected by an outer hydrophobic algaenan layer. Cellulase treatment of walls purified after cell lysis generates highly enriched algaenan preparations without using the harsh chemical treatments typically used in algaenan isolation and characterization. Nannochloropsis algaenan was determined to comprise long, straight-chain, saturated aliphatics with ether cross-links, which closely resembles the cutan of vascular plants. Chemical identification of >85% of the isolated cell wall mass is detailed, and genome analysis is used to identify candidate biosynthetic enzymes. PMID:25239976

  7. A Model for Cell Wall Dissolution in Mating Yeast Cells: Polarized Secretion and Restricted Diffusion of Cell Wall Remodeling Enzymes Induces Local Dissolution

    PubMed Central

    Huberman, Lori B.; Murray, Andrew W.

    2014-01-01

    Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells. PMID:25329559

  8. A model for cell wall dissolution in mating yeast cells: polarized secretion and restricted diffusion of cell wall remodeling enzymes induces local dissolution.

    PubMed

    Huberman, Lori B; Murray, Andrew W

    2014-01-01

    Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells. PMID:25329559

  9. Bending forces plastically deform growing bacterial cell walls.

    PubMed

    Amir, Ariel; Babaeipour, Farinaz; McIntosh, Dustin B; Nelson, David R; Jun, Suckjoon

    2014-04-22

    Cell walls define a cell's shape in bacteria. The walls are rigid to resist large internal pressures, but remarkably plastic to adapt to a wide range of external forces and geometric constraints. Currently, it is unknown how bacteria maintain their shape. In this paper, we develop experimental and theoretical approaches and show that mechanical stresses regulate bacterial cell wall growth. By applying a precisely controllable hydrodynamic force to growing rod-shaped Escherichia coli and Bacillus subtilis cells, we demonstrate that the cells can exhibit two fundamentally different modes of deformation. The cells behave like elastic rods when subjected to transient forces, but deform plastically when significant cell wall synthesis occurs while the force is applied. The deformed cells always recover their shape. The experimental results are in quantitative agreement with the predictions of the theory of dislocation-mediated growth. In particular, we find that a single dimensionless parameter, which depends on a combination of independently measured physical properties of the cell, can describe the cell's responses under various experimental conditions. These findings provide insight into how living cells robustly maintain their shape under varying physical environments. PMID:24711421

  10. Respiratory syncytial virus glycoproteins uptake occurs through clathrin-mediated endocytosis in a human epithelial cell line

    PubMed Central

    Gutiérrez-Ortega, Abel; Sánchez-Hernández, Carla; Gómez-García, Beatriz

    2008-01-01

    Cell-surface viral proteins most frequently enter the cell through clathrin or caveolae endocytosis. Respiratory syncytial virus antigen internalization by immune cells is via caveolin, however, uptake of paramyxovirus cell membrane proteins by non-immune cells is done through clathrin-coated pits. In this work, the uptake of respiratory syncytial virus cell surface glycoproteins by non-immune human epithelial cells was investigated through indirect immunofluorescence with polyclonal anti-RSV antibody and confocal lasser-scanner microscopy. Clathrin and caveolae internalization pathways were monitored through specific inhibitors monodansylcadaverine (MDC) and methyl-beta-cyclodextrin (MBCD), respectively. Internalization of RSV antigens was inhibited by MDC but not by MBCD, implying that clathrin-mediated endocytosis is the major uptake route of RSV antigens by an epithelial human cell line. PMID:18950517

  11. Production Model Press for the Preparation of Bacterial Cell Walls

    PubMed Central

    Perrine, T. D.; Ribi, E.; Maki, W.; Miller, B.; Oertli, E.

    1962-01-01

    A modification of the apparatus previously described permits the preparation of cell walls in quantity. This consists of a heavy duty, double-acting hydraulic press with motor-driven pump, and a superstrength alloy steel pressure cell which is corrosion resistant. Liquid cooling of the jet is substituted for the previously used gas cooling to minimize aerosol formation and to facilitate subsequent treatment of the products. The device produces cell walls of excellent quality in good yield. The pressure cell has been used satisfactorily up to about 60,000 psi. Design details are given. Images FIG. 1 FIG. 2 FIG. 6 PMID:14485524

  12. Motion of red blood cells near microvessel walls: effects of a porous wall layer

    PubMed Central

    HARIPRASAD, DANIEL S.; SECOMB, TIMOTHY W.

    2013-01-01

    A two-dimensional model is used to simulate the motion and deformation of a single mammalian red blood cell (RBC) flowing close to the wall of a microvessel, taking into account the effects of a porous endothelial surface layer (ESL) lining the vessel wall. Migration of RBCs away from the wall leads to the formation of a cell-depleted layer near the wall, which has a large effect on the resistance to blood flow in microvessels. The objective is to examine the mechanical factors causing this migration, including the effects of the ESL. The vessel is represented as a straight parallel-sided channel. The RBC is represented as a set of interconnected viscoelastic elements, suspended in plasma, a Newtonian fluid. The ESL is represented as a porous medium, and plasma flow in the layer is computed using the Brinkman approximation. It is shown that an initially circular cell positioned close to the ESL in a shear flow is deformed into an asymmetric shape. This breaking of symmetry leads to migration away from the wall. With increasing hydraulic resistivity of the layer, the rate of lateral migration increases. It is concluded that mechanical interactions of RBCs flowing in microvessels with a porous wall layer may reduce the rate of lateral migration and hence reduce the width of the cell-depleted zone external to the ESL, relative to the cell-depleted zone that would be formed if the interface between the ESL and free-flowing plasma were replaced by an impermeable boundary. PMID:23493820

  13. Podocalyxin Is a Glycoprotein Ligand of the Human Pluripotent Stem Cell-Specific Probe rBC2LCN

    PubMed Central

    Tateno, Hiroaki; Matsushima, Asako; Hiemori, Keiko; Onuma, Yasuko; Ito, Yuzuru; Hasehira, Kayo; Nishimura, Ken; Ohtaka, Manami; Takayasu, Satoko; Nakanishi, Mahito; Ikehara, Yuzuru; Nakanishi, Mio; Ohnuma, Kiyoshi; Chan, Techuan; Toyoda, Masashi; Akutsu, Hidenori; Umezawa, Akihiro; Asashima, Makoto

    2013-01-01

    In comprehensive glycome analysis with a high-density lectin microarray, we have previously shown that the recombinant N-terminal domain of the lectin BC2L-C from Burkholderia cenocepacia (rBC2LCN) binds exclusively to undifferentiated human induced pluripotent stem (iPS) cells and embryonic stem (ES) cells but not to differentiated somatic cells. Here we demonstrate that podocalyxin, a heavily glycosylated type 1 transmembrane protein, is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. When analyzed by DNA microarray, podocalyxin was found to be highly expressed in both iPS cells and ES cells. Western and lectin blotting revealed that rBC2LCN binds to podocalyxin with a high molecular weight of more than 240 kDa in undifferentiated iPS cells of six different origins and four ES cell lines, but no binding was observed in either differentiated mouse feeder cells or somatic cells. The specific binding of rBC2LCN to podocalyxin prepared from a large set of iPS cells (138 types) and ES cells (15 types) was also confirmed using a high-throughput antibody-overlay lectin microarray. Alkaline digestion greatly reduced the binding of rBC2LCN to podocalyxin, indicating that the major glycan ligands of rBC2LCN are presented on O-glycans. Furthermore, rBC2LCN was found to exhibit significant affinity to a branched O-glycan comprising an H type 3 structure (Ka, 2.5 × 104 M−1) prepared from human 201B7 iPS cells, indicating that H type 3 is a most probable potential pluripotency marker. We conclude that podocalyxin is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. PMID:23526252

  14. Candidate topical microbicides bind herpes simplex virus glycoprotein B and prevent viral entry and cell-to-cell spread.

    PubMed

    Cheshenko, Natalia; Keller, Marla J; MasCasullo, Veronica; Jarvis, Gary A; Cheng, Hui; John, Minnie; Li, Jin-Hua; Hogarty, Kathleen; Anderson, Robert A; Waller, Donald P; Zaneveld, Lourens J D; Profy, Albert T; Klotman, Mary E; Herold, Betsy C

    2004-06-01

    Topical microbicides designed to prevent acquisition of sexually transmitted infections are urgently needed. Nonoxynol-9, the only commercially available spermicide, damages epithelium and may enhance human immunodeficiency virus transmission. The observation that herpes simplex virus (HSV) and human immunodeficiency virus bind heparan sulfate provided the rationale for the development of sulfated or sulfonated polymers as topical agents. Although several of the polymers have advanced to clinical trials, the spectrum and mechanism of anti-HSV activity and the effects on soluble mediators of inflammation have not been evaluated. The present studies address these gaps. The results indicate that PRO 2000, polystyrene sulfonate, cellulose sulfate, and polymethylenehydroquinone sulfonate inhibit HSV infection 10,000-fold and are active against clinical isolates, including an acyclovir-resistant variant. The compounds formed stable complexes with glycoprotein B and inhibit viral binding, entry, and cell-to-cell spread. The effects may be long lasting due to the high affinity and stability of the sulfated compound-virus complex, as evidenced by surface plasmon resonance studies. The candidate microbicides retained their antiviral activities in the presence of cervical secretions and over a broad pH range. There was little reduction in cell viability following repeated exposure of human endocervical cells to these compounds, although a reduction in secretory leukocyte protease inhibitor levels was observed. These studies support further development and rigorous evaluation of these candidate microbicides. PMID:15155195

  15. Interaction of Cryptococcus neoformans Extracellular Vesicles with the Cell Wall

    PubMed Central

    Wolf, Julie M.; Espadas-Moreno, Javier; Luque-Garcia, Jose L.

    2014-01-01

    Cryptococcus neoformans produces extracellular vesicles containing a variety of cargo, including virulence factors. To become extracellular, these vesicles not only must be released from the plasma membrane but also must pass through the dense matrix of the cell wall. The greatest unknown in the area of fungal vesicles is the mechanism by which these vesicles are released to the extracellular space given the presence of the fungal cell wall. Here we used electron microscopy techniques to image the interactions of vesicles with the cell wall. Our goal was to define the ultrastructural morphology of the process to gain insights into the mechanisms involved. We describe single and multiple vesicle-leaving events, which we hypothesized were due to plasma membrane and multivesicular body vesicle origins, respectively. We further utilized melanized cells to “trap” vesicles and visualize those passing through the cell wall. Vesicle size differed depending on whether vesicles left the cytoplasm in single versus multiple release events. Furthermore, we analyzed different vesicle populations for vesicle dimensions and protein composition. Proteomic analysis tripled the number of proteins known to be associated with vesicles. Despite separation of vesicles into batches differing in size, we did not identify major differences in protein composition. In summary, our results indicate that vesicles are generated by more than one mechanism, that vesicles exit the cell by traversing the cell wall, and that vesicle populations exist as a continuum with regard to size and protein composition. PMID:24906412

  16. Another Brick in the Cell Wall: Biosynthesis Dependent Growth Model

    PubMed Central

    Barbacci, Adelin; Lahaye, Marc; Magnenet, Vincent

    2013-01-01

    Expansive growth of plant cell is conditioned by the cell wall ability to extend irreversibly. This process is possible if (i) a tensile stress is developed in the cell wall due to the coupling effect between turgor pressure and the modulation of its mechanical properties through enzymatic and physicochemical reactions and if (ii) new cell wall elements can be synthesized and assembled to the existing wall. In other words, expansive growth is the result of coupling effects between mechanical, thermal and chemical energy. To have a better understanding of this process, models must describe the interplay between physical or mechanical variable with biological events. In this paper we propose a general unified and theoretical framework to model growth in function of energy forms and their coupling. This framework is based on irreversible thermodynamics. It is then applied to model growth of the internodal cell of Chara corallina modulated by changes in pressure and temperature. The results describe accurately cell growth in term of length increment but also in term of cell pectate biosynthesis and incorporation to the expanding wall. Moreover, the classical growth model based on Lockhart's equation such as the one proposed by Ortega, appears as a particular and restrictive case of the more general growth equation developed in this paper. PMID:24066142

  17. Another brick in the cell wall: biosynthesis dependent growth model.

    PubMed

    Barbacci, Adelin; Lahaye, Marc; Magnenet, Vincent

    2013-01-01

    Expansive growth of plant cell is conditioned by the cell wall ability to extend irreversibly. This process is possible if (i) a tensile stress is developed in the cell wall due to the coupling effect between turgor pressure and the modulation of its mechanical properties through enzymatic and physicochemical reactions and if (ii) new cell wall elements can be synthesized and assembled to the existing wall. In other words, expansive growth is the result of coupling effects between mechanical, thermal and chemical energy. To have a better understanding of this process, models must describe the interplay between physical or mechanical variable with biological events. In this paper we propose a general unified and theoretical framework to model growth in function of energy forms and their coupling. This framework is based on irreversible thermodynamics. It is then applied to model growth of the internodal cell of Chara corallina modulated by changes in pressure and temperature. The results describe accurately cell growth in term of length increment but also in term of cell pectate biosynthesis and incorporation to the expanding wall. Moreover, the classical growth model based on Lockhart's equation such as the one proposed by Ortega, appears as a particular and restrictive case of the more general growth equation developed in this paper. PMID:24066142

  18. Molecular basis of preferential resistance to colchicine in multidrug-resistant human cells conferred by Gly-185 yields Val-185 substitution in P-glycoprotein

    SciTech Connect

    Safa, A.R.; Stern, R.K.; Choi, Kyunghee; Agresti, M.; Tamai, Ikumi; Mehta, N.D.; Roninson, I.B. )

    1990-09-01

    Expression of P-glycoprotein, encoded by the human MDR1 gene, results in cross-resistance to many lipophilic cytotoxic drugs (multidrug resistance). P-glycoprotein is believed to function as an energy-dependent efflux pump that is responsible for decreased drug accumulation in multidrug-resistant cells. Previous work showed that preferential resistance to colchicine in a colchicine-selected multidrug-resistant cell line was caused by spontaneous mutations in the MDR1 gene that resulted in a Gly-185 {yields} Val-185 substitution in P-glycoprotein. The authors have now compared transfectant cell lines expressing the wild-type Gly-185 or the mutant Val-185 P-glycoprotein with regard to their levels of resistance to and accumulation and binding of different drugs. In cells expressing the mutant protein, increased resistance to colchicine and decreased resistance to vinblastine correlated with a decreased accumulation of colchicine and increased accumulation of vinblastine. Expression of the mutant P-glycoprotein also resulted in significantly increased resistance to epipodophyllotoxin and decreased resistance to vincristine and actinomycin D; smaller changes in resistance were observed for several other drugs. Unexpectedly, the mutant P-glycoprotein showed increased binding of photoactive analogs of vinblastine and verapamil and the photoactive compound azidopine and decreased binding of a photoactive colchicine analog. These results suggest that the Gly-185 {yields} Val-185 substitution affects not the initial drug-binding site of P-glycoprotein but another site, associated with the release of P-glycoprotein-bound drugs to the outside of the cell.

  19. Grifola frondosa Glycoprotein GFG-3a Arrests S phase, Alters Proteome, and Induces Apoptosis in Human Gastric Cancer Cells.

    PubMed

    Cui, Fengjie; Zan, Xinyi; Li, Yunhong; Sun, Wenjing; Yang, Yan; Ping, Lifeng

    2016-01-01

    GFG-3a is a novel glycoprotein previously purified from the fermented mycelia of Grifola frondosa with novel sugar compositions and protein sequencing. The present study aims to investigate its effects on the cell cycle, differential proteins expression, and apoptosis of human gastric cancer SGC-7901 cells. Our findings revealed that GFG-3a induced the cell apoptosis and arrested cell cycle at S phase. GFG-3a treatment resulted in the differential expression of 21 proteins in SGC-7901 cells by upregulating 10 proteins including RBBP4 associated with cell cycle arrest and downregulating 11 proteins including RUVBL1, NPM, HSP90AB1, and GRP78 involved in apoptosis and stress response. qRT-PCR and Western blot analysis also suggested that GFG-3a could increase the expressions of Caspase-8/-3, p53, Bax, and Bad while decrease the expressions of Bcl2, Bcl-xl, PI3K, and Akt1. These results indicated that the stress response, p53-dependent mitochondrial-mediated, Caspase-8/-3-dependent, and PI3k/Akt pathways were involved in the GFG-3a-induced apoptosis process in SGC-7901 cells. These findings might provide a basis to prevent or treat human gastric cancer with GFG-3a and understand the tumor-inhibitory molecular mechanisms of mushroom glycoproteins. PMID:27040446

  20. Progesterone interacts with P-glycoprotein in multidrug-resistant cells and in the endometrium of gravid uterus.

    PubMed

    Yang, C P; DePinho, S G; Greenberger, L M; Arceci, R J; Horwitz, S B

    1989-01-15

    P-Glycoprotein (P-GP) plays a pivotal role in maintaining the multidrug-resistant (MDR) phenotype. This membrane glycoprotein is overproduced in MDR cells and the endometrium of the mouse gravid uterus (Arceci, R.J., Croop, J.M., Horwitz, S.B., and Housman, D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4350-4354). This latter observation and an interest in endogenous substrates for P-GP led to a study of the interaction of steroids with P-GP found in the endometrium of the mouse gravid uterus and in MDR cells derived from the murine macrophage-like cell J774.2. [3H]Azidopine labeling of P-GP from these two sources was inhibited by various steroids, particularly progesterone. Progesterone also markedly inhibited [3H]vinblastine binding to membrane vesicles prepared from MDR cells, enhanced vinblastine accumulation in MDR cells, and increased the sensitivity of MDR cells to vinblastine. In addition, we have demonstrated that the hydrophobicity of a steroid is important in determining its effect on inhibition of drug binding to P-GP. It is concluded that progesterone, a relatively nontoxic endogenous steroid, interacts with P-GP and is capable of reversing drug resistance in MDR cells. PMID:2562956

  1. Inhibitory effects of neochamaejasmin B on P-glycoprotein in MDCK-hMDR1 cells and molecular docking of NCB binding in P-glycoprotein.

    PubMed

    Pan, Lanying; Hu, Haihong; Wang, Xiangjun; Yu, Lushan; Jiang, Huidi; Chen, Jianzhong; Lou, Yan; Zeng, Su

    2015-01-01

    Stellera chamaejasme L. (Thymelaeaceae) is widely distributed in Mongolia, Tibet and the northern parts of China. Its roots are commonly used as "Langdu", which is embodied in the Pharmacopoeia of the P.R. China (2010) as a toxic Traditional Chinese Medicine. It is claimed to have antivirus, antitumor and antibacterial properties in China and other Asian countries. Studies were carried out to characterize the inhibition of neochamaejasmin B (NCB) on P-glycoprotein (P-gp, ABCB1, MDR1). Rhodamine-123 (R-123) transport and accumulation studies were performed in MDCK-hMDR1 cells. ABCB1 (MDR1) mRNA gene expression and P-gp protein expression were analyzed. Binding selectivity studies based on molecular docking were explored. R-123 transport and accumulation studies in MDCK-hMDR1 cells indicated that NCB inhibited the P-gp-mediated efflux in a concentration-dependent manner. RT-PCR and Western blot demonstrated that the P-gp expression was suppressed by NCB. To investigate the inhibition type of NCB on P-gp, Ki and Ki' values were determined by double-reciprocal plots in R-123 accumulation studies. Since Ki was greater than Ki', the inhibition of NCB on P-gp was likely a mixed type of competitive and non-competitive inhibition. The results were confirmed by molecular docking in our current work. The docking data indicated that NCB had higher affinity to P-gp than to Lig1 ((S)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one). PMID:25679052

  2. Relationship between P-glycoprotein expression and cyclosporin A in kidney. An immunohistological and cell culture study.

    PubMed Central

    García del Moral, R.; O'Valle, F.; Andújar, M.; Aguilar, M.; Lucena, M. A.; López-Hidalgo, J.; Ramírez, C.; Medina-Cano, M. T.; Aguilar, D.; Gómez-Morales, M.

    1995-01-01

    P-glycoprotein (P-gp), encoded in humans by the mdr-1 gene, acts physiologically as an efflux pump to expel hydrophobic substances from cells. This glycoprotein is closely related to multidrug resistance in tumor cells and can be modulated by cyclosporin A (CsA). We investigated the relationship between CsA and P-gp in 52 renal allograft biopsies and in cultures of Madin-Darby canine kidney (MDCK) renal tubule cells to determine whether the intrarenal accumulation of CsA or chronic stimulation with the drug modified the expression of P-gp. Expression of P-gp and CsA was analyzed by immunohistochemistry. Immunostaining was evaluated semiquantitatively. Modulation of P-gp in MDCK cells after chronic stimulation with CsA for 7, 30, and 60 days was analyzed by flow cytometry. P-gp and CsA immunostaining in renal post-transplant biopsies showed considerable overlap in all cases (Spearman's test, r = 0.577, P < 0.001). After 7 days in vitro, the number of cells expressing P-gp increased progressively; a further increase in mean fluorescence was found after 60 days (P < 0.001, Student's t-test). Our findings suggest that in non-neoplastic cells, CsA may stimulate P-gp as a mechanism of detoxification. Individual differences in the adaptive responses to glycoprotein may be responsible for the appearance of nephrotoxicity or a CsA-resistant rejection reaction in cases of overexpression on lymphocytes and macrophages. Images Figure 1 PMID:7856751

  3. Glycan Profiling of Plant Cell Wall Polymers using Microarrays

    PubMed Central

    Moller, Isabel E.; Pettolino, Filomena A.; Hart, Charlie; Lampugnani, Edwin R.; Willats, William G.T.; Bacic, Antony

    2012-01-01

    Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging. Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4. In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification. Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments

  4. Cell wall proteomics of the green alga Haematococcus pluvialis (Chlorophyceae).

    PubMed

    Wang, Sheng-Bing; Hu, Qiang; Sommerfeld, Milton; Chen, Feng

    2004-03-01

    The green microalga Haematococcus pluvialis can synthesize and accumulate large amounts of the ketocarotenoid astaxanthin, and undergo profound changes in cell wall composition and architecture during the cell cycle and in response to environmental stresses. In this study, cell wall proteins (CWPs) of H. pluvialis were systematically analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with peptide mass fingerprinting (PMF) and sequence-database analysis. In total, 163 protein bands were analyzed, which resulted in positive identification of 81 protein orthologues. The highly complex and dynamic composition of CWPs is manifested by the fact that the majority of identified CWPs are differentially expressed at specific stages of the cell cycle along with a number of common wall-associated 'housekeeping' proteins. The detection of cellulose synthase orthologue in the vegetative cells suggested that the biosynthesis of cellulose occurred during primary wall formation, in contrast to earlier observations that cellulose was exclusively present in the secondary wall of the organism. A transient accumulation of a putative cytokinin oxidase at the early stage of encystment pointed to a possible role in cytokinin degradation while facilitating secondary wall formation and/or assisting in cell expansion. This work represents the first attempt to use a proteomic approach to investigate CWPs of microalgae. The reference protein map constructed and the specific protein markers obtained from this study provide a framework for future characterization of the expression and physiological functions of the proteins involved in the biogenesis and modifications in the cell wall of Haematococcus and related organisms. PMID:14997492

  5. The composition of the cell wall of Aspergillus niger

    PubMed Central

    Johnston, I. R.

    1965-01-01

    1. The cell-wall composition of Aspergillus niger has been investigated. Analysis shows the presence of six sugars, glucose, galactose, mannose, arabinose, glucosamine and galactosamine, all in the d-configuration, except that a small amount of l-galactose may be present. Sixteen common amino acids are also present. 2. The wall consists chiefly of neutral carbohydrate (73–83%) and hexosamine (9–13%), with smaller amounts of lipid (2–7%), protein (0·5–2·5%) and phosphorus (less than 0·1%). The acetyl content (3·0–3·4%) corresponds to 1·0mole/mole of hexosamine nitrogen. 3. A fractionation of the cell-wall complex was achieved, with or without a preliminary phenol extraction, by using n-sodium hydroxide. Though this caused some degradation, 30–60% of the wall could be solubilized (depending on the preparation). Analyses on several fractions suggest that fractionation procedures bring about some separation of components although not in a clear-cut fashion. 4. Cell-wall preparations were shown to yield a fraction having [α]D approx. +240° (in n-sodium hydroxide) and consisting largely of glucose. This was separated into two subfractions, one of which had [α]D+281° (in n-sodium hydroxide) and had properties resembling the polysaccharide nigeran; the other had [α]D +231° (in n-sodium hydroxide). It is suggested that nigeran is a cell-wall component. PMID:5862404

  6. Control of cell wall extensibility during pollen tube growth.

    PubMed

    Hepler, Peter K; Rounds, Caleb M; Winship, Lawrence J

    2013-07-01

    In this review, we address the question of how the tip-growing pollen tube achieves its rapid rate of elongation while maintaining an intact cell wall. Although turgor is essential for growth to occur, the local expansion rate is controlled by local changes in the viscosity of the apical wall. We focus on several different structures and underlying processes that are thought to be major participants including exocytosis, the organization and activity of the actin cytoskeleton, calcium and proton physiology, and cellular energetics. We think that the actin cytoskeleton, in particular the apical cortical actin fringe, directs the flow of vesicles to the apical domain, where they fuse with the plasma membrane and contribute their contents to the expanding cell wall. While pH gradients, as generated by a proton-ATPase located on the plasma membrane along the side of the clear zone, may regulate rapid actin turnover and new polymerization in the fringe, the tip-focused calcium gradient biases secretion towards the polar axis. The recent data showing that exocytosis of new wall material precedes and predicts the process of cell elongation provide support for the idea that the intussusception of newly secreted pectin contributes to decreases in apical wall viscosity and to cell expansion. Other prime factors will be the localization and activity of the enzyme pectin methyl-esterase, and the chelation of calcium by pectic acids. Finally, we acknowledge a role for reactive oxygen species in the control of wall viscosity. PMID:23770837

  7. Control of Cell Wall Extensibility during Pollen Tube Growth

    PubMed Central

    Hepler, Peter K.

    2013-01-01

    In this review, we address the question of how the tip-growing pollen tube achieves its rapid rate of elongation while maintaining an intact cell wall. Although turgor is essential for growth to occur, the local expansion rate is controlled by local changes in the viscosity of the apical wall. We focus on several different structures and underlying processes that are thought to be major participants including exocytosis, the organization and activity of the actin cytoskeleton, calcium and proton physiology, and cellular energetics. We think that the actin cytoskeleton, in particular the apical cortical actin fringe, directs the flow of vesicles to the apical domain, where they fuse with the plasma membrane and contribute their contents to the expanding cell wall. While pH gradients, as generated by a proton-ATPase located on the plasma membrane along the side of the clear zone, may regulate rapid actin turnover and new polymerization in the fringe, the tip-focused calcium gradient biases secretion towards the polar axis. The recent data showing that exocytosis of new wall material precedes and predicts the process of cell elongation provide support for the idea that the intussusception of newly secreted pectin contributes to decreases in apical wall viscosity and to cell expansion. Other prime factors will be the localization and activity of the enzyme pectin methyl-esterase, and the chelation of calcium by pectic acids. Finally, we acknowledge a role for reactive oxygen species in the control of wall viscosity. PMID:23770837

  8. P-glycoprotein-mediated transport of oxytetracycline in the Caco-2 cell model.

    PubMed

    Schrickx, J; Fink-Gremmels, J

    2007-02-01

    ATP-dependent drug transporters such as P-glycoprotein (P-gp), multi-drug resistance associated protein (MRP2) and breast cancer resistant protein (BCRP) are expressed at the brush border membrane of enterocytes. These efflux transporters excrete their substrates, among other various classes of antibiotics, into the lumen thus reducing net absorption as indicated by a low bioavailability after oral administration. Oxytetracycline (OTC) has been used for decennia in veterinary medicine for its extensive spectrum of antimicrobial activity. A major limitation has been, and still remains, its low bioavailability following oral administration. The present study aimed to investigate to what extent this low bioavailability is attributable to the fact that OTC is a substrate for one or more efflux transporters. As an experimental model to study the transmembrane transport of OTC, differentiated Caco-2 cells grown as monolayers on permeable supports were used. With this model it was shown that the secretion of OTC is slightly higher than its absorption. PSC833, a potent inhibitor of P-gp, decreased the secretion of OTC without affecting its absorption, while the MRP-inhibitor MK571 did not exert any effect. These data indicate that OTC is a substrate for P-gp. The affinity of OTC to these transporters seems to be rather low, as suggested by the low efflux ratio of 1:1.3. In competition experiments, OTC decreased the effluxes of other P-gp substrates such as Rhodamine123 and ivermectin. These findings are of clinical relevance, as they clearly indicate potential drug-drug interactions at the level of P-gp-mediated drug transport. PMID:17217397

  9. Bending forces plastically deform growing bacterial cell walls

    PubMed Central

    Amir, Ariel; Babaeipour, Farinaz; McIntosh, Dustin B.; Nelson, David R.; Jun, Suckjoon

    2014-01-01

    Cell walls define a cell’s shape in bacteria. The walls are rigid to resist large internal pressures, but remarkably plastic to adapt to a wide range of external forces and geometric constraints. Currently, it is unknown how bacteria maintain their shape. In this paper, we develop experimental and theoretical approaches and show that mechanical stresses regulate bacterial cell wall growth. By applying a precisely controllable hydrodynamic force to growing rod-shaped Escherichia coli and Bacillus subtilis cells, we demonstrate that the cells can exhibit two fundamentally different modes of deformation. The cells behave like elastic rods when subjected to transient forces, but deform plastically when significant cell wall synthesis occurs while the force is applied. The deformed cells always recover their shape. The experimental results are in quantitative agreement with the predictions of the theory of dislocation-mediated growth. In particular, we find that a single dimensionless parameter, which depends on a combination of independently measured physical properties of the cell, can describe the cell’s responses under various experimental conditions. These findings provide insight into how living cells robustly maintain their shape under varying physical environments. PMID:24711421

  10. Microfabricated alkali vapor cell with anti-relaxation wall coating

    SciTech Connect

    Straessle, R.; Pétremand, Y.; Briand, D.; Rooij, N. F. de; Pellaton, M.; Affolderbach, C.; Mileti, G.

    2014-07-28

    We present a microfabricated alkali vapor cell equipped with an anti-relaxation wall coating. The anti-relaxation coating used is octadecyltrichlorosilane and the cell was sealed by thin-film indium-bonding at a low temperature of 140 °C. The cell body is made of silicon and Pyrex and features a double-chamber design. Depolarizing properties due to liquid Rb droplets are avoided by confining the Rb droplets to one chamber only. Optical and microwave spectroscopy performed on this wall-coated cell are used to evaluate the cell's relaxation properties and a potential gas contamination. Double-resonance signals obtained from the cell show an intrinsic linewidth that is significantly lower than the linewidth that would be expected in case the cell had no wall coating but only contained a buffer-gas contamination on the level measured by optical spectroscopy. Combined with further experimental evidence this proves the presence of a working anti-relaxation wall coating in the cell. Such cells are of interest for applications in miniature atomic clocks, magnetometers, and other quantum sensors.

  11. Inhibition of P-glycoprotein activity and chemosensitization of multidrug-resistant ovarian carcinoma 2780AD cells by hexanoylglucosylceramide.

    PubMed

    Veldman, R J; Sietsma, H; Klappe, K; Hoekstra, D; Kok, J W

    1999-12-20

    In the present study we show that neutral hexanoyl-(glyco)sphingolipids inhibit P-glycoprotein (Pgp) activity in human ovarian 2780AD cells. By contrast, hexanoylceramide and the gangliosides GM(3) and GM(2) had no effect on Pgp activity, whereas sphingosine had a stimulating effect. In the case of hexanoylglucosylceramide, inhibition of Pgp activity by was reflected by a regained doxorubicin sensitivity of cells, which were grown in medium supplemented with the lipid. Our results lead to the conclusion that a direct transmodulation of Pgp activity by glycolipids occurs, depending on lipid headgroup structure, which can result in reduced resistance to the chemotherapeutic agent doxorubicin. PMID:10600530

  12. Interaction of anthelmintic drugs with P-glycoprotein in recombinant LLC-PK1-mdr1a cells.

    PubMed

    Dupuy, Jacques; Alvinerie, Michel; Ménez, Cecile; Lespine, Anne

    2010-08-01

    Given the widespread use of formulations combining anthelmintics which are possible P-glycoprotein interfering agents, the understanding of drug interactions with efflux ABC transporters is of concern for improving anthelmintic control. We determined the ability of 14 anthelmintics from different classes to interact with abcb1a (mdr1a, P-glycoprotein, Pgp) by following the intracellular accumulation of rhodamine 123 (Rho 123), a fluorescent Pgp substrate, in LLC-PK1 cells overexpressing Pgp. The cytotoxicity of the compounds that are able to interfere with Pgp activity was evaluated in cells overexpressing Pgp and compared with parental cells using the MTS viability assay. Among all the anthelmintics used, ivermectin (IVM), triclabendazole (TCZ), triclabendazole sulfoxide (TCZ-SO), closantel (CLOS) and rafoxanide (RAF) increased the intracellular Rho 123 in Pgp overexpressing cells, while triclabendazole sulfone, albendazole, mebendazole, oxfendazole, thiabendazole, nitroxynil, levamisole, praziquantel and clorsulon failed to have any effect. The concentration needed to reach the maximal Rho 123 accumulation (E(max)) was obtained with 10 microM for IVM, 80 microM for CLOS, 40 microM for TCZ and TCZ-SO, and 80 microM for RAF. We showed that for these five drugs parental cell line was more sensitive to drug toxicity compared with Pgp recombinant cell line. Such in vitro approach constitutes a powerful tool to predict Pgp-drug interactions when formulations combining several anthelmintics are administered and may contribute to the required optimization of efficacy of anthelmintics. PMID:20513441

  13. Inhibitors targeting on cell wall biosynthesis pathway of MRSA.

    PubMed

    Hao, Haihong; Cheng, Guyue; Dai, Menghong; Wu, Qinghua; Yuan, Zonghui

    2012-11-01

    Methicillin resistant Staphylococcus aureus (MRSA), widely known as a type of new superbug, has aroused world-wide concern. Cell wall biosynthesis pathway is an old but good target for the development of antibacterial agents. Peptidoglycan and wall teichoic acids (WTAs) biosynthesis are two main processes of the cell wall biosynthesis pathway (CWBP). Other than penicillin-binding proteins (PBPs), some key factors (Mur enzymes, lipid I or II precursor, etc.) in CWBP are becoming attractive molecule targets for the discovery of anti-MRSA compounds. A number of new compounds, with higher affinity for PBPs or with inhibitory activity on such molecule targets in CWBP of MRSA, have been in the pipeline recently. This review concludes recent research achievements and provides a complete picture of CWBP of MRSA, including the peptidoglycan and wall teichoic acids synthesis pathway. The potential inhibitors targeting on CWBP are subsequently presented to improve development of novel therapeutic strategies for MRSA. PMID:22898792

  14. Co-delivery of cell-wall-forming enzymes in the same vesicle for coordinated fungal cell wall formation.

    PubMed

    Schuster, Martin; Martin-Urdiroz, Magdalena; Higuchi, Yujiro; Hacker, Christian; Kilaru, Sreedhar; Gurr, Sarah J; Steinberg, Gero

    2016-01-01

    Fungal cells are surrounded by an extracellular cell wall. This complex matrix of proteins and polysaccharides protects against adverse stresses and determines the shape of fungal cells. The polysaccharides of the fungal wall include 1,3-β-glucan and chitin, which are synthesized by membrane-bound synthases at the growing cell tip. A hallmark of filamentous fungi is the class V chitin synthase, which carries a myosin-motor domain. In the corn smut fungus Ustilago maydis, the myosin-chitin synthase Mcs1 moves to the plasma membrane in secretory vesicles, being delivered by kinesin-1 and myosin-5. The myosin domain of Mcs1 enhances polar secretion by tethering vesicles at the site of exocytosis. It remains elusive, however, how other cell-wall-forming enzymes are delivered and how their activity is coordinated post secretion. Here, we show that the U. maydis class VII chitin synthase and 1,3-β-glucan synthase travel in Mcs1-containing vesicles, and that their apical secretion depends on Mcs1. Once in the plasma membrane, anchorage requires enzyme activity, which suggests co-synthesis of chitin and 1,3-β-glucan polysaccharides at sites of exocytosis. Thus, delivery of cell-wall-forming enzymes in Mcs1 vesicles ensures local foci of fungal cell wall formation. PMID:27563844

  15. Characterization of rhamnogalacturonan I from cotton suspension culture cell walls

    SciTech Connect

    Not Available

    1991-01-01

    Progress has been made on the project of determining the structure of pectins. From recent progress, a covalent crosslink between rhamnogalacturonan I (RGI) and xyloglucan was hypothesized and a structure for RGI was proposed. The development of a method to determine the distribution of methyl esterification with pectins also progressed. The degree of methyl esterification of cotton cotyledon cell walls was compared to that of cotton suspension cultures. Cotyledon wall were found to have {approximately}55% of the galacturonic acid esterified whereas suspension culture wall were only about 14% methyl esterified. 10 refs. (SM)

  16. Simulated microgravity inhibits cell wall regeneration of Penicillium decumbens protoplasts

    NASA Astrophysics Data System (ADS)

    Zhao, C.; Sun, Y.; Yi, Z. C.; Rong, L.; Zhuang, F. Y.; Fan, Y. B.

    2010-09-01

    This work compares cell wall regeneration from protoplasts of the fungus Penicillium decumbens under rotary culture (simulated microgravity) and stationary cultures. Using an optimized lytic enzyme mixture, protoplasts were successfully released with a yield of 5.3 × 10 5 cells/mL. Under simulated microgravity conditions, the protoplast regeneration efficiency was 33.8%, lower than 44.9% under stationary conditions. Laser scanning confocal microscopy gave direct evidence for reduced formation of polysaccharides under simulated conditions. Scanning electron microscopy showed the delayed process of cell wall regeneration by simulated microgravity. The delayed regeneration of P. decumbens cell wall under simulated microgravity was likely caused by the inhibition of polysaccharide synthesis. This research contributes to the understanding of how gravitational loads affect morphological and physiological processes of fungi.

  17. Modification of glass cell walls by rubidium vapor

    NASA Astrophysics Data System (ADS)

    Ma, J.; Kishinevski, A.; Jau, Y.-Y.; Reuter, C.; Happer, W.

    2009-04-01

    It has long been known that the inner walls of freshly manufactured glass cells filled with a few droplets of alkali metal undergo a “curing” process, where the properties of the cell wall change over a period of days to weeks. We report quantitative studies of “curing” in Pyrex cells filled with rubidium metal. Our experiment shows that at 94°C , the surface of Pyrex glass adsorbs about 3×1015 rubidium atoms per cm2 , which is equivalent to 6-7 monolayers of liquid rubidium.

  18. Chromosome and cell wall segregation in Streptococcus faecium ATCC 9790

    SciTech Connect

    Higgins, M.L.; Glaser, D.; Dicker, D.T.; Zito, E.T.

    1989-01-01

    Segregation was studied by measuring the positions of autoradiographic grain clusters in chains formed from single cells containing on average less than one radiolabeled chromosome strand. The degree to which chromosomal and cell wall material cosegregated was quantified by using the methods of S. Cooper and M. Weinberger, dividing the number of chains labeled at the middle. This analysis indicated that in contrast to chromosomal segregation in Escherichia coli and, in some studies, to that in gram-positive rods, chromosomal segregation in Streptococcus faecium was slightly nonrandom and did not vary with growth rate. Results were not significantly affected by strand exchange. In contrast, labeled cell wall segregated predominantly nonrandomly.

  19. Molecular Rigidity in Dry and Hydrated Onion Cell Walls.

    PubMed Central

    Ha, M. A.; Apperley, D. C.; Jarvis, M. C.

    1997-01-01

    Solid-state nuclear magnetic resonance relaxation experiments can provide information on the rigidity of individual molecules within a complex structure such as a cell wall, and thus show how each polymer can potentially contribute to the rigidity of the whole structure. We measured the proton magnetic relaxation parameters T2 (spin-spin) and T1p (spin-lattice) through the 13C-nuclear magnetic resonance spectra of dry and hydrated cell walls from onion (Allium cepa L.) bulbs. Dry cell walls behaved as rigid solids. The form of their T2 decay curves varied on a continuum between Gaussian, as in crystalline solids, and exponential, as in more mobile materials. The degree of molecular mobility that could be inferred from the T2 and T1p decay patterns was consistent with a crystalline state for cellulose and a glassy state for dry pectins. The theory of composite materials may be applied to explain the rigidity of dry onion cell walls in terms of their components. Hydration made little difference to the rigidity of cellulose and most of the xyloglucan shared this rigidity, but the pectic fraction became much more mobile. Therefore, the cellulose/xyloglucan microfibrils behaved as solid rods, and the most significant physical distinction within the hydrated cell wall was between the microfibrils and the predominantly pectic matrix. A minor xyloglucan fraction was much more mobile than the microfibrils and probably corresponded to cross-links between them. Away from the microfibrils, pectins expanded upon hydration into a nonhomogeneous, but much softer, almost-liquid gel. These data are consistent with a model for the stress-bearing hydrated cell wall in which pectins provide limited stiffness across the thickness of the wall, whereas the cross-linked microfibril network provides much greater rigidity in other directions. PMID:12223827

  20. Molecular Rigidity in Dry and Hydrated Onion Cell Walls.

    PubMed

    Ha, M. A.; Apperley, D. C.; Jarvis, M. C.

    1997-10-01

    Solid-state nuclear magnetic resonance relaxation experiments can provide information on the rigidity of individual molecules within a complex structure such as a cell wall, and thus show how each polymer can potentially contribute to the rigidity of the whole structure. We measured the proton magnetic relaxation parameters T2 (spin-spin) and T1p (spin-lattice) through the 13C-nuclear magnetic resonance spectra of dry and hydrated cell walls from onion (Allium cepa L.) bulbs. Dry cell walls behaved as rigid solids. The form of their T2 decay curves varied on a continuum between Gaussian, as in crystalline solids, and exponential, as in more mobile materials. The degree of molecular mobility that could be inferred from the T2 and T1p decay patterns was consistent with a crystalline state for cellulose and a glassy state for dry pectins. The theory of composite materials may be applied to explain the rigidity of dry onion cell walls in terms of their components. Hydration made little difference to the rigidity of cellulose and most of the xyloglucan shared this rigidity, but the pectic fraction became much more mobile. Therefore, the cellulose/xyloglucan microfibrils behaved as solid rods, and the most significant physical distinction within the hydrated cell wall was between the microfibrils and the predominantly pectic matrix. A minor xyloglucan fraction was much more mobile than the microfibrils and probably corresponded to cross-links between them. Away from the microfibrils, pectins expanded upon hydration into a nonhomogeneous, but much softer, almost-liquid gel. These data are consistent with a model for the stress-bearing hydrated cell wall in which pectins provide limited stiffness across the thickness of the wall, whereas the cross-linked microfibril network provides much greater rigidity in other directions. PMID:12223827

  1. 15. View of interior, north wall of hot cell featuring ...

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

    15. View of interior, north wall of hot cell featuring radioactive materials containment box, facing east - Nevada Test Site, Reactor Maintenance & Disassembly Complex, Junior Hot Cell, Jackass Flats, Area 25, South of intersection of Roads F & G, Mercury, Nye County, NV

  2. 47. ARAI. Interior view of operating wall of hot cell ...

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

    47. ARA-I. Interior view of operating wall of hot cell in ARA-626. Note stands for operators at viewing windows. Manipulators with hand grips extend cables and other controls into hot cell through ducts above windows. Ineel photo no. 81-27. - Idaho National Engineering Laboratory, Army Reactors Experimental Area, Scoville, Butte County, ID

  3. Differential expression of sphingolipids in P-glycoprotein or multidrug resistance-related protein 1 expressing human neuroblastoma cell lines.

    PubMed

    Dijkhuis, Anne-Jan; Douwes, Jenny; Kamps, Willem; Sietsma, Hannie; Kok, Jan Willem

    2003-07-31

    The sphingolipid composition and multidrug resistance status of three human neuroblastoma cell lines were established. SK-N-FI cells displayed high expression and functional (efflux) activity of P-glycoprotein, while multidrug resistance-related protein 1 was relatively abundant and most active in SK-N-AS cells. These two cell lines exhibited higher sphingolipid levels, compared to SK-N-DZ, which had the lowest activity of either ATP-binding cassette transporter protein. SK-N-DZ cells also differed in ganglioside composition with predominant expression of b-series gangliosides. In conclusion, these three neuroblastoma cell lines offer a good model system to study sphingolipid metabolism in relation to ATP-binding cassette transporter protein function. PMID:12885402

  4. Alterations in auxin homeostasis suppress defects in cell wall function.

    PubMed

    Steinwand, Blaire J; Xu, Shouling; Polko, Joanna K; Doctor, Stephanie M; Westafer, Mike; Kieber, Joseph J

    2014-01-01

    The plant cell wall is a highly dynamic structure that changes in response to both environmental and developmental cues. It plays important roles throughout plant growth and development in determining the orientation and extent of cell expansion, providing structural support and acting as a barrier to pathogens. Despite the importance of the cell wall, the signaling pathways regulating its function are not well understood. Two partially redundant leucine-rich-repeat receptor-like kinases (LRR-RLKs), FEI1 and FEI2, regulate cell wall function in Arabidopsis thaliana roots; disruption of the FEIs results in short, swollen roots as a result of decreased cellulose synthesis. We screened for suppressors of this swollen root phenotype and identified two mutations in the putative mitochondrial pyruvate dehydrogenase E1α homolog, IAA-Alanine Resistant 4 (IAR4). Mutations in IAR4 were shown previously to disrupt auxin homeostasis and lead to reduced auxin function. We show that mutations in IAR4 suppress a subset of the fei1 fei2 phenotypes. Consistent with the hypothesis that the suppression of fei1 fei2 by iar4 is the result of reduced auxin function, disruption of the WEI8 and TAR2 genes, which decreases auxin biosynthesis, also suppresses fei1 fei2. In addition, iar4 suppresses the root swelling and accumulation of ectopic lignin phenotypes of other cell wall mutants, including procuste and cobra. Further, iar4 mutants display decreased sensitivity to the cellulose biosynthesis inhibitor isoxaben. These results establish a role for IAR4 in the regulation of cell wall function and provide evidence of crosstalk between the cell wall and auxin during cell expansion in the root. PMID:24859261

  5. The role of the cell wall in plant immunity

    PubMed Central

    Malinovsky, Frederikke G.; Fangel, Jonatan U.; Willats, William G. T.

    2014-01-01

    The battle between plants and microbes is evolutionarily ancient, highly complex, and often co-dependent. A primary challenge for microbes is to breach the physical barrier of host cell walls whilst avoiding detection by the plant’s immune receptors. While some receptors sense conserved microbial features, others monitor physical changes caused by an infection attempt. Detection of microbes leads to activation of appropriate defense responses that then challenge the attack. Plant cell walls are formidable and dynamic barriers. They are constructed primarily of complex carbohydrates joined by numerous distinct connection types, and are subject to extensive post-synthetic modification to suit prevailing local requirements. Multiple changes can be triggered in cell walls in response to microbial attack. Some of these are well described, but many remain obscure. The study of the myriad of subtle processes underlying cell wall modification poses special challenges for plant glycobiology. In this review we describe the major molecular and cellular mechanisms that underlie the roles of cell walls in plant defense against pathogen attack. In so doing, we also highlight some of the challenges inherent in studying these interactions, and briefly describe the analytical potential of molecular probes used in conjunction with carbohydrate microarray technology. PMID:24834069

  6. Effects of spaceflight on polysaccharides of Saccharomyces cerevisiae cell wall.

    PubMed

    Liu, Hong-Zhi; Wang, Qiang; Liu, Xiao-Yong; Tan, Sze-Sze

    2008-12-01

    Freeze-dried samples of four Saccharomyces cerevisiae strains, namely, FL01, FL03, 2.0016, and 2.1424, were subjected to spaceflight. After the satellite's landing on Earth, the samples were recovered and changes in yeast cell wall were analyzed. Spaceflight strains of all S. cerevisiae strains showed significant changes in cell wall thickness (P < 0.05). One mutant of S. cerevisiae 2.0016 with increased biomass, cell wall thickness, and cell wall glucan was isolated (P < 0.05). The spaceflight mutant of S. cerevisiae 2.0016 showed 46.7%, 62.6%, and 146.0% increment in biomass, cell wall thickness and beta-glucan content, respectively, when compared to the ground strain. Moreover, growth curve analysis showed spaceflight S. cerevisiae 2.0016 had a faster growth rate, shorter lag phase periods, higher final biomass, and higher content of beta-glucan. Genetic stability analysis showed that prolonged subculturing of spaceflight strain S. cerevisiae 2.0016 did not lead to the appearance of variants, indicating that the genetic stability of S. cerevisiae 2.0016 mutant could be sufficient for its exploitation of beta-glucan production. PMID:18797865

  7. Purification and characterization of a soybean cell wall protein

    SciTech Connect

    San Francisco, S.; Tierney, M.L. )

    1989-04-01

    Plant cell wall composition is thought to reflect cellular responses to developmental and environmental signals. We have purified a 33 kDa protein from cell wall extracts of soybean seedlings which is most abundant in extracts from the hook region of the hypocotyl and is rich in proline and hydroxypyroline. In vivo {sup 3}H-proline labelling of hypocotyl tissues indicates that the hook tissue is the predominant site for synthesis of this protein. In unwounded hook, label is incorporated into a 33 kDa protein, while in wounded hook this and additional proteins rich in proline are synthesized. Similarly treated cell wall extracts analyzed by Western blot analysis, using a polyclonal antibody raised against this 33kD protein, showed that the 33 kDa protein is most abundant in cell wall extracts from the hook region of unwounded seedlings and does not increase upon wounding. An immunologically related 35kD protein is also apparent in extracts from wounded hooks and appears to co-migrate with one of the labelled proteins extractable from this tissue. These data indicate that there are two related, proline-rich cell wall proteins in the hook region of soybean seedlings, one of which (33 kDa) is prominent during seedling development and another (35 kDa) which is wound inducible.

  8. A cell surface integral membrane glycoprotein of 85,000 mol wt (gp85) associated with triton X-100-insoluble cell skeleton.

    PubMed

    Tarone, G; Ferracini, R; Galetto, G; Comoglio, P

    1984-08-01

    The Triton X-100-insoluble skeleton of baby hamster kidney BHK cells consists of the nucleus, intermediate-size filaments, and actin fibers. By transmission electron microscopy, membrane fragments were found to be associated with these insoluble structures. When radioiodinated or [3H]glucosamine-labeled cells were extracted with 0.5% Triton, most plasma membrane glycoproteins were solubilized except for a glycoprotein with a molecular weight of 85,000 (gp85) that remained associated with the insoluble skeletons. Immunoprecipitation with a specific antiserum indicated that the gp85 is not a proteolytic degradation product of fibronectin, an extracellular matrix glycoprotein insoluble in detergent. A monoclonal antibody of BHK cells specific for gp85 was produced. Immunofluorescence analysis with this monoclonal antibody indicated that gp85 is not associated with the extracellular matrix, but is confined to the cell membrane. Both in fixed and unfixed intact cells, fluorescence was concentrated in dots preferentially aligned in streaks on the cell surface. Gp85 was found to behave as an integral membrane protein interacting with the hydrophobic core of the lipid bilayer since it was extracted from membrane preparations by ionic detergents such as SDS, but not by 0.1 N NaOH (pH 12) in the absence of detergents, a condition known to release peripheral molecules. Association of gp85 with the cell skeleton was unaffected by increasing the Triton concentration up to 5%, but it was affected when actin filaments were dissociated or when a protein-denaturing agent (6 M urea) was used in the presence of Triton, suggesting that protein-protein interactions are involved in the association of gp85 with the cell skeleton. We conclude that gp85 is an integral plasma membrane glycoprotein that might have a role in cell surface-cytoskeleton interaction. PMID:6378925

  9. Structure, function, and biosynthesis of plant cell walls: proceedings of the seventh annual symposium in botany

    SciTech Connect

    Dugger, W.M.; Bartnicki-Garcia, S.

    1984-01-01

    Papers in the following areas were included in these symposium proceedings: (1) cell wall chemistry and biosynthesis; (2) cell wall hydrolysis and associated physiology; (3) cellular events associated with cell wall biosynthesis; and (4) interactions of plant cell walls with pathogens and related responses. Papers have been individually abstracted for the data base. (ACR)

  10. Deletion of the α-(1,3)-Glucan Synthase Genes Induces a Restructuring of the Conidial Cell Wall Responsible for the Avirulence of Aspergillus fumigatus

    PubMed Central

    Beauvais, Anne; Bozza, Silvia; Kniemeyer, Olaf; Formosa, Céline; Balloy, Viviane; Henry, Christine; Roberson, Robert W.; Dague, Etienne; Chignard, Michel; Brakhage, Axel A.; Romani, Luigina; Latgé, Jean-Paul

    2013-01-01

    α-(1,3)-Glucan is a major component of the cell wall of Aspergillus fumigatus, an opportunistic human fungal pathogen. There are three genes (AGS1, AGS2 and AGS3) controlling the biosynthesis of α-(1,3)-glucan in this fungal species. Deletion of all the three AGS genes resulted in a triple mutant that was devoid of α-(1,3)-glucan in its cell wall; however, its growth and germination was identical to that of the parental strain in vitro. In the experimental murine aspergillosis model, this mutant was less pathogenic than the parental strain. The AGS deletion resulted in an extensive structural modification of the conidial cell wall, especially conidial surface where the rodlet layer was covered by an amorphous glycoprotein matrix. This surface modification was responsible for viability reduction of conidia in vivo, which explains decrease in the virulence of triple agsΔ mutant. PMID:24244155

  11. Ultrastructure of organic cell walls in Proterozoic microalgae

    NASA Astrophysics Data System (ADS)

    Moczydlowska-Vidal, M.

    2009-04-01

    The antiquity of life has been well appreciated since the discoveries of microfossils and confirmation of their authenticity, as well as the recognition of geochemical signs of biogenicity in the Archean successions. Resolving the biological affinities of early biota is essential for the unravelling the changes that led to modern biodiversity, but also for the detection of possible biogenic records outside of the terrestrial biosphere. Advanced techniques in microscopy, tomography and spectroscopy applied to examine individual microfossils at the highest attainable spatial resolution have provided unprecedented insights into micro- and nano-scale structure and composition of organic matter. Transmission and scanning electron microscopy studies of the wall ultrastructure of sphaeromorphic and ornamented acritarchs have revealed complex, single to multilayered walls, having a unique texture in sub-layers and an occasionally preserved trilaminar sheath structure (TLS) of the cell wall. A variety of optical characteristics, the electron density and texture of fabrics of discrete layers, and the properties of biopolymers may indicate the polyphyletic affiliations of such microfossils and/or the preservation of various stages (vegetative, resting) in their life cycle. I evaluate the morphological features of organic-walled unicellular microfossils in conjunction with their cell wall ultrastructure to infer their life cycle and to recognize various developmental stages represented among microfossils attributed to a single form-taxon. Several cases of fine wall ultrastructure in microfossils have been documented and have had a conclusive influence on understanding their affinities. Some Proterozoic and Cambrian leiosphaerids are of algal affinities. Certain specimens represent chlorophyceaens, having the multilayered composite wall with TLS structure known from vegetative and resting cells in modern genera of the Chlorococcales and Volvocales. The wall ultrastructure of

  12. Cellulose synthesis in two secondary cell wall processes in a single cell type

    PubMed Central

    Mendu, Venugopal; Stork, Jozsef; Harris, Darby; DeBolt, Seth

    2011-01-01

    Plant cells have a rigid cell wall that constrains internal turgor pressure yet extends in a regulated and organized manner to allow the cell to acquire shape. The primary load-bearing macromolecule of a plant cell wall is cellulose, which forms crystalline microfibrils that are organized with respect to a cell's function and shape requirements. A primary cell wall is deposited during expansion whereas secondary cell wall is synthesized post expansion during differentiation. A complex form of asymmetrical cellular differentiation occurs in Arabidopsis seed coat epidermal cells, where we have recently shown that two secondary cell wall processes occur that utilize different cellulose synthase (CESA) proteins. One process is to produce pectinaceous mucilage that expands upon hydration and the other is a radial wall thickening that reinforced the epidermal cell structure. Our data illustrate polarized specialization of CESA5 in facilitating mucilage attachment to the parent seed and CESA2, CESA5 and CESA9 in radial cell wall thickening and formation of the columella. Herein, we present a model for the complexity of cellulose biosynthesis in this highly differentiated cell type with further evidence supporting each cellulosic secondary cell wall process. PMID:22057330

  13. Cotton fiber: a powerful single-cell model for cell wall and cellulose research

    PubMed Central

    Haigler, Candace H.; Betancur, Lissete; Stiff, Michael R.; Tuttle, John R.

    2012-01-01

    Cotton fibers are single-celled extensions of the seed epidermis. They can be isolated in pure form as they undergo staged differentiation including primary cell wall synthesis during elongation and nearly pure cellulose synthesis during secondary wall thickening. This combination of features supports clear interpretation of data about cell walls and cellulose synthesis in the context of high throughput modern experimental technologies. Prior contributions of cotton fiber to building fundamental knowledge about cell walls will be summarized and the dynamic changes in cell wall polymers throughout cotton fiber differentiation will be described. Recent successes in using stable cotton transformation to alter cotton fiber cell wall properties as well as cotton fiber quality will be discussed. Futurec prospects to perform experiments more rapidly through altering cotton fiberwall properties via virus-induced gene silencing will be evaluated. PMID:22661979

  14. Cell wall integrity signalling in human pathogenic fungi.

    PubMed

    Dichtl, Karl; Samantaray, Sweta; Wagener, Johannes

    2016-09-01

    Fungi are surrounded by a rigid structure, the fungal cell wall. Its plasticity and composition depend on active regulation of the underlying biosynthesis and restructuring processes. This involves specialised signalling pathways that control gene expression and activities of biosynthetic enzymes. The cell wall integrity (CWI) pathway is the central signalling cascade required for the adaptation to a wide spectrum of cell wall perturbing conditions, including heat, oxidative stress and antifungals. In the recent years, great efforts were made to analyse the CWI pathway of diverse fungi. It turned out that the CWI signalling cascade is mostly conserved in the fungal kingdom. In this review, we summarise as well as compare the current knowledge on the canonical CWI pathway in the human pathogenic fungi Candida albicans, Candida glabrata, Aspergillus fumigatus and Cryptococcus neoformans. Understanding the differences and similarities in the stress responses of these organisms could become a key to improving existing or developing new antifungal therapies. PMID:27155139

  15. Fluorescent probes for exploring plant cell wall deconstruction: a review.

    PubMed

    Paës, Gabriel

    2014-01-01

    Plant biomass is a potential resource of chemicals, new materials and biofuels that could reduce our dependency on fossil carbon, thus decreasing the greenhouse effect. However, due to its chemical and structural complexity, plant biomass is recalcitrant to green biological transformation by enzymes, preventing the establishment of integrated bio-refineries. In order to gain more knowledge in the architecture of plant cell wall to facilitate their deconstruction, many fluorescent probes bearing various fluorophores have been devised and used successfully to reveal the changes in structural motifs during plant biomass deconstruction, and the molecular interactions between enzymes and plant cell wall polymers. Fluorescent probes are thus relevant tools to explore plant cell wall deconstruction. PMID:24995923

  16. A new method for extraction of pectin from cell walls

    SciTech Connect

    Maness, N.O.; Mort, A.J. )

    1991-05-01

    Pectin is often extracted from plant tissues using the Ca{sup ++} chelators ethylenediamine tetraacetate (EDTA) or cyclohexane-trans-1,2 diamine tetraacetate (CDTA). While these chelators are effective in solubilizing pectin, even after extensive dialysis against distilled water, EDTA or CDTA remains associated with the pectin. The authors have found that if 500 mM imidazole buffer, pH 7.0 is substituted for 50 mM CDTA, pH 6.5, and for equivalent extraction periods, an equivalent amount of pectin with the same sugar composition is extracted. But, the imidazole buffer can be dialyzed away completely into distilled water. Their alternative procedure for extraction of pectin from cell walls will be presented. Utilization of the procedure for extraction of whole cell walls or cell walls pretreated with liquid hydrogen fluoride is discussed.

  17. Human Adenovirus 52 Uses Sialic Acid-containing Glycoproteins and the Coxsackie and Adenovirus Receptor for Binding to Target Cells

    PubMed Central

    Lenman, Annasara; Liaci, A. Manuel; Liu, Yan; Årdahl, Carin; Rajan, Anandi; Nilsson, Emma; Bradford, Will; Kaeshammer, Lisa; Jones, Morris S.; Frängsmyr, Lars; Feizi, Ten; Stehle, Thilo; Arnberg, Niklas

    2015-01-01

    Most adenoviruses attach to host cells by means of the protruding fiber protein that binds to host cells via the coxsackievirus and adenovirus receptor (CAR) protein. Human adenovirus type 52 (HAdV-52) is one of only three gastroenteritis-causing HAdVs that are equipped with two different fiber proteins, one long and one short. Here we show, by means of virion-cell binding and infection experiments, that HAdV-52 can also attach to host cells via CAR, but most of the binding depends on sialylated glycoproteins. Glycan microarray, flow cytometry, surface plasmon resonance and ELISA analyses reveal that the terminal knob domain of the long fiber (52LFK) binds to CAR, and the knob domain of the short fiber (52SFK) binds to sialylated glycoproteins. X-ray crystallographic analysis of 52SFK in complex with 2-O-methylated sialic acid combined with functional studies of knob mutants revealed a new sialic acid binding site compared to other, known adenovirus:glycan interactions. Our findings shed light on adenovirus biology and may help to improve targeting of adenovirus-based vectors for gene therapy. PMID:25674795

  18. The role of eukaryotic subtilisin-like endoproteases for the activation of human immunodeficiency virus glycoproteins in natural host cells.

    PubMed Central

    Hallenberger, S; Moulard, M; Sordel, M; Klenk, H D; Garten, W

    1997-01-01

    Proteolytic activation of the precursor envelope glycoproteins gp160 of human immunodeficiency virus type 1 (HIV-1) and gp140 of HIV-2, a prerequisite for viral infection, results in the formation of gp120/gp41 and gp125/gp36, respectively. Cleavage is mediated by cellular proteases. Furin, a member of the eukaryotic subtilisin family, has been shown to be an activating protease for HIV. Here, we compared the presence of furin and other mammalian subtilisins in lymphatic cells and tissues. Northern blot analyses revealed that furin and the recently discovered protease LPC/PC7 were the only subtilisin-like enzymes transcribed in such cells. Furin was identified as an enzymatically active endoprotease present in different lymphocytic, as well as monocytic, cell lines. When expressed from vaccinia virus vectors, the proprotein convertases were correctly processed, transported, and secreted into the media and enzymatically active. Coexpression of different subtilisins with the HIV envelope precursors revealed that furin and LPC/PC7 are able to cleave HIV-1 gp160. Moreover, both enzymes proteolytically processed the envelope precursor of HIV-2. gp140 was also cleaved to some extent by PC1, which is not, however, present in lymphatic cells. Furin- and LPC/PC7-catalyzed cleavage of HIV-1 gp160 resulted in biologically active envelope protein. In conclusion, among the known members of the subtilisin family, only furin and LPC/PC7 fulfill the requirements of a protease responsible for in vivo activation of HIV envelope glycoproteins. PMID:8995623

  19. Fluorescent Protein-Tagged Sindbis Virus E2 Glycoprotein Allows Single Particle Analysis of Virus Budding from Live Cells

    PubMed Central

    Jose, Joyce; Tang, Jinghua; Taylor, Aaron B.; Baker, Timothy S.; Kuhn, Richard J.

    2015-01-01

    Sindbis virus (SINV) is an enveloped, mosquito-borne alphavirus. Here we generated and characterized a fluorescent protein-tagged (FP-tagged) SINV and found that the presence of the FP-tag (mCherry) affected glycoprotein transport to the plasma membrane whereas the specific infectivity of the virus was not affected. We examined the virions by transmission electron cryo-microscopy and determined the arrangement of the FP-tag on the surface of the virion. The fluorescent proteins are arranged icosahedrally on the virus surface in a stable manner that did not adversely affect receptor binding or fusion functions of E2 and E1, respectively. The delay in surface expression of the viral glycoproteins, as demonstrated by flow cytometry analysis, contributed to a 10-fold reduction in mCherry-E2 virus titer. There is a 1:1 ratio of mCherry to E2 incorporated into the virion, which leads to a strong fluorescence signal and thus facilitates single-particle tracking experiments. We used the FP-tagged virus for high-resolution live-cell imaging to study the spatial and temporal aspects of alphavirus assembly and budding from mammalian cells. These processes were further analyzed by thin section microscopy. The results demonstrate that SINV buds from the plasma membrane of infected cells and is dispersed into the surrounding media or spread to neighboring cells facilitated by its close association with filopodial extensions. PMID:26633461

  20. Modulation of Alternaria infectoria Cell Wall Chitin and Glucan Synthesis by Cell Wall Synthase Inhibitors

    PubMed Central

    Fernandes, Chantal; Anjos, Jorge; Walker, Louise A.; Silva, Branca M. A.; Cortes, Luísa; Mota, Marta; Munro, Carol A.; Gow, Neil A. R.

    2014-01-01

    The present work reports the effects of caspofungin, a β-1,3-glucan synthase inhibitor, and nikkomycin Z, an inhibitor of chitin synthases, on two strains of Alternaria infectoria, a melanized fungus involved in opportunistic human infections and respiratory allergies. One of the strains tested, IMF006, bore phenotypic traits that conferred advantages in resisting antifungal treatment. First, the resting cell wall chitin content was higher and in response to caspofungin, the chitin level remained constant. In the other strain, IMF001, the chitin content increased upon caspofungin treatment to values similar to basal IMF006 levels. Moreover, upon caspofungin treatment, the FKS1 gene was upregulated in IMF006 and downregulated in IMF001. In addition, the resting β-glucan content was also different in both strains, with higher levels in IMF001 than in IMF006. However, this did not provide any advantage with respect to echinocandin resistance. We identified eight different chitin synthase genes and studied relative gene expression when the fungus was exposed to the antifungals under study. In both strains, exposure to caspofungin and nikkomycin Z led to modulation of the expression of class V and VII chitin synthase genes, suggesting its importance in the robustness of A. infectoria. The pattern of A. infectoria phagocytosis and activation of murine macrophages by spores was not affected by caspofungin. Monotherapy with nikkomycin Z and caspofungin provided only fungistatic inhibition, while a combination of both led to fungal cell lysis, revealing a strong synergistic action between the chitin synthase inhibitor and the β-glucan synthase inhibitor against this fungus. PMID:24614372

  1. Merkel cell carcinoma of the abdominal wall

    PubMed Central

    Gaopande, Vandana L.; Joshi, Avinash R.; Khandeparkar, Siddhi G. S.; Deshmukh, Sanjay D.

    2015-01-01

    Merkel cell carcinoma also known as neuroendocrine carcinoma of the skin is a very rare skin tumor. It commonly presents in the old age and the common sites are head, neck and extremities. The diagnosis requires histopathological examination with immunohistochemical correlation. We report a case of Merkel cell carcinoma stage IIIB with bilateral inguinal lymphadenopathy that on FNAB showed metastatic deposits of the tumor. PMID:26225333

  2. Plant-originated glycoprotein (24 kDa) has an inhibitory effect on proliferation of BNL CL.2 cells in response to di(2-ethylhexyl)phthalate.

    PubMed

    Lee, Jin; Lim, Kye-Taek

    2011-08-01

    Di(2-ethylhexyl)phthalate (DEHP) is one of the many environmental chemicals that are widely used in polyvinyl chloride products, vinyl flooring, food packaging and infant toys. They cause cell proliferation or dysfunction of human liver. The purpose of this study is to investigate the inhibitory effect of a glycoprotein (24 kDa) isolated from Zanthoxylum piperitum DC (ZPDC) on proliferation of liver cell in the DEHP-induced BNL CL. 2 cells. [³H]-thymidine incorporation, intracellular reactive oxygen species (ROS), intracellular Ca²⁺ mobilization and activity of protein kinase C (PKC) were measured using radioactivity and fluorescence method respectively. The expression of mitogen-activated protein kinases [extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK)], activator protein (AP)-1 (c-Jun and c-Fos), proliferating cell nuclear antigen (PCNA) and cell cycle-related factors (cyclin D1/cyclin-dependent kinase [CDK] 4) were evaluated using Western blotting or electrophoretic mobility shift assay. The results in this study showed that the levels of [³H]-thymidine incorporation, intracellular ROS, intracellular Ca²⁺ mobilization and activity of PKCα were inhibited by ZPDC glycoprotein (100 µg/ml) in the DEHP-induced BNL CL. 2 cells. Also, activities of ERK, JNK and AP-1 were reduced by ZPDC glycoprotein (100 µg/ml). With regard to cell proliferation, activities of PCNA and cyclin D1/CDK4 were significantly suppressed at treatment with ZPDC glycoprotein (100 µg/ml) in the presence of DEHP. Taken together, these findings suggest that ZPDC glycoprotein significantly normalized activities of PCNA and cyclin D1/CDK4, which relate to cell proliferation factors. Thus, ZPDC glycoprotein appears to be one of the compounds derived from natural products that are able to inhibit cell proliferation in the phthalate-induced BNL CL. 2 cells. PMID:21721021

  3. A zoom into the nanoscale texture of secondary cell walls

    PubMed Central

    2014-01-01

    Background Besides classical utilization of wood and paper, lignocellulosic biomass has become increasingly important with regard to biorefinery, biofuel production and novel biomaterials. For these new applications the macromolecular assembly of cell walls is of utmost importance and therefore further insights into the arrangement of the molecules on the nanolevel have to be gained. Cell wall recalcitrance against enzymatic degradation is one of the key issues, since an efficient degradation of lignocellulosic plant material is probably the most crucial step in plant conversion to energy. A limiting factor for in-depth analysis is that high resolution characterization techniques provide structural but hardly chemical information (e.g. Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM)), while chemical characterization leads to a disassembly of the cell wall components or does not reach the required nanoscale resolution (Fourier Tranform Infrared Spectroscopy (FT-IR), Raman Spectroscopy). Results Here we use for the first time Scanning Near-Field Optical Microscopy (SNOM in reflection mode) on secondary plant cell walls and reveal a segmented circumferential nanostructure. This pattern in the 100 nm range was found in the secondary cell walls of a softwood (spruce), a hardwood (beech) and a grass (bamboo) and is thus concluded to be consistent among various plant species. As the nanostructural pattern is not visible in classical AFM height and phase images it is proven that the contrast is not due to changes in surfaces topography, but due to differences in the molecular structure. Conclusions Comparative analysis of model substances of casted cellulose nanocrystals and spin coated lignin indicate, that the SNOM signal is clearly influenced by changes in lignin distribution or composition. Therefore and based on the known interaction of lignin and visible light (e.g. fluorescence and resonance effects), we assume the elucidated nanoscale

  4. Distribution of cell wall components in Sphagnum hyaline cells and in liverwort and hornwort elaters.

    PubMed

    Kremer, Celeste; Pettolino, Filomena; Bacic, Antony; Drinnan, Andrew

    2004-10-01

    Spiral secondary walls are found in hyaline cells of Sphagnum, in the elaters of most liverworts, and in elaters of the hornwort Megaceros. Recent studies on these cells suggest that cytoskeletal and ultrastructural processes involved in cell differentiation and secondary wall formation are similar in bryophytes and vascular plant tracheary elements. To examine differences in wall structure, primary and secondary wall constituents of the hyaline cells of Sphagnum novo-zelandicum and elaters of the liverwort Radula buccinifera and the hornwort Megaceros gracilis were analyzed by immunohistochemical and chemical methods. Anti-arabinogalactan-protein antibodies, JIM8 and JIM13, labeled the central fibrillar secondary wall layer of Megaceros elaters and the walls of Sphagnum leaf cells, but did not label the walls of Radula elaters. The CCRC-M7 antibody, which detects an arabinosylated (1-->6)-linked beta-galactan epitope, exclusively labeled hyaline cells in Sphagnum leaves and the secondary walls of Radula elaters. Anti-pectin antibodies, LM5 and JIM5, labeled the primary wall in Megaceros elaters. LM5 also labeled the central layer of the secondary wall but only during formation. In Radula elaters, JIM5 and another anti-pectin antibody, JIM7, labeled the primary wall. The distribution of arabinogalactan-proteins and pectic polysaccharides restricted to specific wall types and stages of development provides evidence for the developmental and functional regulation of cell wall composition in bryophytes. Monosaccharide-linkage analysis of Sphagnum leaf cell walls suggests they contain polysaccharides similar to those of higher plants. The most abundant linkage was 4-Glc, typical of cellulose, but there was also evidence for xyloglucans, 4-linked mannans, 4-linked xylans and rhamnogalacturonan-type polysaccharides. PMID:15290291

  5. Pathogenic role of anti-ß2-glycoprotein I antibodies in antiphospholipid associated fetal loss: characterisation of ß2-glycoprotein I binding to trophoblast cells and functional effects of anti-ß2-glycoprotein I antibodies in vitro

    PubMed Central

    Di, S; Raschi, E; Testoni, C; Castellani, R; D'Asta, M; Shi, T; Krilis, S; Caruso, A; Meroni, P

    2005-01-01

    Background: Antiphospholipid antibodies reacting with ß2-glycoprotein I (ß2GPI) have been associated with recurrent fetal loss and pregnancy complications. Objective: To investigate whether specific mutations in the phospholipid binding site of ß2GPI might affect its binding to trophoblast and in turn the anti-ß2GPI antibody induced functional effects. Methods: ß2GPI adhesion to trophoblast was evaluated as human monoclonal IgM or polyclonal IgG anti-ß2GPI antibody binding to trophoblast monolayers cultured (1) in complete medium; (2) in serum-free medium; (3) after serum starvation in the presence of purified human ß2GPI; or (4) in the presence of ß2GPI with single or multiple mutations in the amino acid loop Cys281-Lys-Asn-Lys-Glu-Lys-Lys-Cys288. The effect of anti-ß2GPI binding to trophoblast was evaluated as chorionic gonadotropin (hCG) mRNA expression, and protein release by RT-PCR and radioimmunoassay, respectively. Results: ß2GPI adhesion to trophoblast and its consequent recognition by the specific antibodies were inversely proportional to the mutation number in the phospholipid binding site. Anti-ß2GPI antibodies reduced gonadotropin release, hormone dependent hCG mRNA expression, and protein synthesis in the presence of ß2GPI, while the addition of the mutants or the absence of ß2GPI had no effect. Conclusions: ß2GPI binds to trophoblast in vitro through its fifth domain, as reported for endothelial cells, and can be recognised by anti-ß2GPI antibodies; the antibody binding downregulates trophoblast hCG synthesis and secretion. Such a mechanism might contribute to defective placentation in women with fetal loss associated with the antiphospholipid syndrome. PMID:15256379

  6. Particle Trajectories in Rotating Wall Cell Culture Devices

    NASA Technical Reports Server (NTRS)

    Ramachandran N.; Downey, J. P.

    1999-01-01

    Cell cultures are extremely important to the medical community since such cultures provide an opportunity to perform research on human tissue without the concerns inherent in experiments on individual humans. Development of cells in cultures has been found to be greatly influenced by the conditions of the culture. Much work has focused on the effect of the motions of cells in the culture relative to the solution. Recently rotating wall vessels have been used with success in achieving improved cellular cultures. Speculation and limited research have focused on the low shear environment and the ability of rotating vessels to keep cells suspended in solution rather than floating or sedimenting as the primary reasons for the improved cellular cultures using these devices. It is widely believed that the cultures obtained using a rotating wall vessel simulates to some degree the effect of microgravity on cultures. It has also been speculated that the microgravity environment may provide the ideal acceleration environment for culturing of cellular tissues due to the nearly negligible levels of sedimentation and shear possible. This work predicts particle trajectories of cells in rotating wall vessels of cylindrical and annular design consistent with the estimated properties of typical cellular cultures. Estimates of the shear encountered by cells in solution and the interactions with walls are studied. Comparisons of potential experiments in ground and microgravity environments are performed.

  7. Glycoprotein isolated from Solanum nigrum L. modulates the apoptotic-related signals in 12-O-tetradecanoylphorbol 13-acetate-stimulated MCF-7 cells.

    PubMed

    Heo, Kyung-Sun; Lim, Kye-Taek

    2005-01-01

    Solanum nigrum L. (SNL) has been used in folk medicine for its anti-inflammatory activity. We isolated only the SNL glycoprotein from SNL and found that it was cytotoxic at low concentration. With respect to cytotoxicity, we investigated whether purified SNL glycoprotein is able to regulate protein kinase C (PKC) alpha activation and nuclear factor (NF)- kappaB activities in 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced tumor promotion, and whether it has an apoptosis-inducing effect in MCF-7 cells using western blot analysis. In addition, to elucidate the relationship between PKCalpha and NF-kappaB, inhibitory studies were performed with staurosporine (an inhibitor of phospholipid/calcium-dependent protein kinase) and pyrrolidine dithiocarbamate (an inhibitor of NF-kappaB activation). To verify induction of apoptosis by the SNL glycoprotein, we performed DNA fragmentation and nuclear staining assays using ethidium bromide and bisbenzamide H33342. The results in this study indicated that SNL glycoprotein induces apoptosis through modulation of PKCalpha and NF-kappaB activity in MCF-7 cells. In fact, SNL glycoprotein interfered with PKCalpha membrane translocation and inhibited NF-kappaB (p50) protein activity in MCF-7 cells stimulated with TPA (61.68 ng/mL, 100 nM) dose-dependently. Regarding the apoptotic-inducing effect, nucleosomal DNA fragmentation and nuclear staining by SNL glycoprotein in MCF-7 cells were shown. Collectively, the data demonstrate that SNL glycoprotein is a potential natural anticancer agent because of its ability to induce apoptosis in MCF-7 cells. PMID:15857213

  8. Interactions of Condensed Tannins with Saccharomyces cerevisiae Yeast Cells and Cell Walls: Tannin Location by Microscopy.

    PubMed

    Mekoue Nguela, Julie; Vernhet, Aude; Sieczkowski, Nathalie; Brillouet, Jean-Marc

    2015-09-01

    Interactions between grape tannins/red wine polyphenols and yeast cells/cell walls was previously studied within the framework of red wine aging and the use of yeast-derived products as an alternative to aging on lees. Results evidenced a quite different behavior between whole cells (biomass grown to elaborate yeast-derived products, inactivated yeast, and yeast inactivated after autolysis) and yeast cell walls (obtained from mechanical disruption of the biomass). Briefly, whole cells exhibited a high capacity to irreversibly adsorb grape and wine tannins, whereas only weak interactions were observed for cell walls. This last point was quite unexpected considering the literature and called into question the real role of cell walls in yeasts' ability to fix tannins. In the present work, tannin location after interactions between grape and wine tannins and yeast cells and cell walls was studied by means of transmission electron microscopy, light epifluorescence, and confocal microscopy. Microscopy observations evidenced that if tannins interact with cell walls, and especially cell wall mannoproteins, they also diffuse freely through the walls of dead cells to interact with their plasma membrane and cytoplasmic components. PMID:26223789

  9. Intercellular transfer of P-glycoprotein in human blood-brain barrier endothelial cells is increased by histone deacetylase inhibitors

    PubMed Central

    Noack, Andreas; Noack, Sandra; Buettner, Manuela; Naim, Hassan Y.; Löscher, Wolfgang

    2016-01-01

    The blood–brain barrier (BBB) controls the entry of compounds into the brain, thereby regulating brain homeostasis. Efflux transporters such as P-glycoprotein (Pgp) significantly contribute to BBB function. Multiple signaling pathways modulate the expression and activity of Pgp in response to xenobiotics and disease. A non-genetic way of intercellular transfer of Pgp occurs in cancer cells, but whether this also occurs in non-cancer cells such as endothelial cells that form the BBB is not known. A human brain endothelial cell line (hCMEC/D3) was used to study whether cell-to-cell Pgp transfer occurs during co-culturing with Pgp-EGFP expressing hCMEC/D3 cells. The Pgp-EGFP fusion protein was transferred from donor to recipient cells by cell-to-cell contact and Pgp-EGFP enriched vesicles, which were exocytosed by donor cells and endocytosed by adherent recipient cells. Flow cytometry experiments with the Pgp substrate eFLUXX-ID Gold demonstrated that the transferred Pgp is functional in the recipient cells. Exposure of the donor cells with inhibitors of histone deacetylases (HDACs) resulted in an enhanced intercellular Pgp transfer. Non-genetic transfer of a resistance phenotype and its regulation by HDACs is a novel mechanism of altering BBB functionality. This mechanism may have important implications for understanding drug-induced alterations in Pgp expression and activity. PMID:27375084

  10. Intercellular transfer of P-glycoprotein in human blood-brain barrier endothelial cells is increased by histone deacetylase inhibitors.

    PubMed

    Noack, Andreas; Noack, Sandra; Buettner, Manuela; Naim, Hassan Y; Löscher, Wolfgang

    2016-01-01

    The blood-brain barrier (BBB) controls the entry of compounds into the brain, thereby regulating brain homeostasis. Efflux transporters such as P-glycoprotein (Pgp) significantly contribute to BBB function. Multiple signaling pathways modulate the expression and activity of Pgp in response to xenobiotics and disease. A non-genetic way of intercellular transfer of Pgp occurs in cancer cells, but whether this also occurs in non-cancer cells such as endothelial cells that form the BBB is not known. A human brain endothelial cell line (hCMEC/D3) was used to study whether cell-to-cell Pgp transfer occurs during co-culturing with Pgp-EGFP expressing hCMEC/D3 cells. The Pgp-EGFP fusion protein was transferred from donor to recipient cells by cell-to-cell contact and Pgp-EGFP enriched vesicles, which were exocytosed by donor cells and endocytosed by adherent recipient cells. Flow cytometry experiments with the Pgp substrate eFLUXX-ID Gold demonstrated that the transferred Pgp is functional in the recipient cells. Exposure of the donor cells with inhibitors of histone deacetylases (HDACs) resulted in an enhanced intercellular Pgp transfer. Non-genetic transfer of a resistance phenotype and its regulation by HDACs is a novel mechanism of altering BBB functionality. This mechanism may have important implications for understanding drug-induced alterations in Pgp expression and activity. PMID:27375084

  11. The structure of secondary cell wall polymers: how Gram-positive bacteria stick their cell walls together.

    PubMed

    Schäffer, Christina; Messner, Paul

    2005-03-01

    The cell wall of Gram-positive bacteria has been a subject of detailed chemical study over the past five decades. Outside the cytoplasmic membrane of these organisms the fundamental polymer is peptidoglycan (PG), which is responsible for the maintenance of cell shape and osmotic stability. In addition, typical essential cell wall polymers such as teichoic or teichuronic acids are linked to some of the peptidoglycan chains. In this review these compounds are considered as 'classical' cell wall polymers. In the course of recent investigations of bacterial cell surface layers (S-layers) a different class of 'non-classical' secondary cell wall polymers (SCWPs) has been identified, which is involved in anchoring of S-layers to the bacterial cell surface. Comparative analyses have shown considerable differences in chemical composition, overall structure and charge behaviour of these SCWPs. This review discusses the progress that has been made in understanding the structural principles of SCWPs, which may have useful applications in S-layer-based 'supramolecular construction kits' in nanobiotechnology. PMID:15758211

  12. Chimeric antigen receptor (CAR)-engineered T cells redirected against hepatitis C virus (HCV) E2 glycoprotein

    PubMed Central

    Sautto, Giuseppe A; Wisskirchen, Karin; Clementi, Nicola; Castelli, Matteo; Diotti, Roberta A; Graf, Julia; Clementi, Massimo; Burioni, Roberto; Protzer, Ulrike; Mancini, Nicasio

    2016-01-01

    Objective The recent availability of novel antiviral drugs has raised new hope for a more effective treatment of hepatitis C virus (HCV) infection and its severe sequelae. However, in the case of non-responding or relapsing patients, alternative strategies are needed. To this end we have used chimeric antigen receptors (CARs), a very promising approach recently used in several clinical trials to redirect primary human T cells against different tumours. In particular, we designed the first CARs against HCV targeting the HCV/E2 glycoprotein (HCV/E2). Design Anti-HCV/E2 CARs were composed of single-chain variable fragments (scFvs) obtained from a broadly cross-reactive and cross-neutralising human monoclonal antibody (mAb), e137, fused to the intracellular signalling motif of the costimulatory CD28 molecule and the CD3ζ domain. Activity of CAR-grafted T cells was evaluated in vitro against HCV/E2-transfected cells as well as hepatocytes infected with cell culture-derived HCV (HCVcc). Results In this proof-of-concept study, retrovirus-transduced human T cells expressing anti-HCV/E2 CARs were endowed with specific antigen recognition accompanied by degranulation and secretion of proinflammatory and antiviral cytokines, such as interferon γ, interleukin 2 and tumour necrosis factor α. Moreover, CAR-grafted T cells were capable of lysing target cells of both hepatic and non-hepatic origin expressing on their surface the HCV/E2 glycoproteins of the most clinically relevant genotypes, including 1a, 1b, 2a, 3a, 4 and 5. Finally, and more importantly, they were capable of lysing HCVcc-infected hepatocytes. Conclusions Clearance of HCV-infected cells is a major therapeutic goal in chronic HCV infection, and adoptive transfer of anti-HCV/E2 CARs-grafted T cells represents a promising new therapeutic tool. PMID:25661083

  13. Effect of intercalated cell-specific Rh C glycoprotein deletion on basal and metabolic acidosis-stimulated renal ammonia excretion

    PubMed Central

    Lee, Hyun-Wook; Verlander, Jill W.; Bishop, Jesse M.; Nelson, Raoul D.; Handlogten, Mary E.

    2010-01-01

    Rh C glycoprotein (Rhcg) is an NH3-specific transporter expressed in both intercalated cells (IC) and principal cells (PC) in the renal collecting duct. Recent studies show that deletion of Rhcg from both intercalated and principal cells inhibits both basal and acidosis-stimulated renal ammonia excretion. The purpose of the current studies was to better understand the specific role of Rhcg expression in intercalated cells in basal and metabolic acidosis-stimulated renal ammonia excretion. We generated mice with intercalated cell-specific Rhcg deletion (IC-Rhcg-KO) using Cre-loxP techniques; control (C) mice were floxed Rhcg but Cre negative. Under basal conditions, IC-Rhcg-KO and C mice excreted urine with similar ammonia content and pH. Mice were then acid loaded by adding HCl to their diet. Ammonia excretion after acid loading increased similarly in IC-Rhcg-KO and C mice during the first 2 days of acid loading but on day 3 was significantly less in IC-Rhcg-KO than in C mice. During the first 2 days of acid loading, urine was significantly more acidic in IC-Rhcg-KO mice than in C mice; there was no difference on day 3. In IC-Rhcg-KO mice, acid loading increased principal cell Rhcg expression in both the cortex and outer medulla as well as expression of another ammonia transporter, Rh glycoprotein B (Rhbg), in principal cells in the outer medulla. We conclude that 1) Rhcg expression in intercalated cells is necessary for the normal renal response to metabolic acidosis; 2) principal cell Rhcg contributes to both basal and acidosis-stimulated ammonia excretion; and 3) adaptations in Rhbg expression occur in response to acid-loading. PMID:20462967

  14. Anammox Planctomycetes have a peptidoglycan cell wall

    PubMed Central

    van Teeseling, Muriel C.F.; Mesman, Rob J.; Kuru, Erkin; Espaillat, Akbar; Cava, Felipe; Brun, Yves V.; VanNieuwenhze, Michael S.; Kartal, Boran; van Niftrik, Laura

    2015-01-01

    Planctomycetes are intriguing microorganisms that apparently lack peptidoglycan, a structure that controls the shape and integrity of almost all bacterial cells. Therefore, the planctomycetal cell envelope is considered exceptional and their cell plan uniquely compartmentalized. Anaerobic ammonium-oxidizing (anammox) Planctomycetes play a key role in the global nitrogen cycle by releasing fixed nitrogen back to the atmosphere as N2. Here using a complementary array of state-of-the-art techniques including continuous culturing, cryo-transmission electron microscopy, peptidoglycan-specific probes and muropeptide analysis, we show that the anammox bacterium Kuenenia stuttgartiensis contains peptidoglycan. On the basis of the thickness, composition and location of peptidoglycan in K. stuttgartiensis, we propose to redefine Planctomycetes as Gram-negative bacteria. Our results demonstrate that Planctomycetes are not an exception to the universal presence of peptidoglycan in bacteria. PMID:25962786

  15. The Ca2(+)-binding glycoprotein SPARC modulates cell cycle progression in bovine aortic endothelial cells

    SciTech Connect

    Funk, S.E.; Sage, E.H. )

    1991-04-01

    SPARC (secreted protein, acidic and rich in cysteine) is an extracellular, Ca2(+)-binding protein associated with cellular populations undergoing migration, proliferation, and/or differentiation. Active preparations of SPARC bind to specific components of the extracellular matrix and cause mesenchymal cells to assume a rounded phenotype. In this study we show that SPARC modulates the progression of bovine aortic endothelial cells through the cell cycle. At a concentration of 20 micrograms/ml, SPARC inhibited the incorporation of (3H)thymidine into newly synthesized DNA by approximately 70%, as compared to control cultures within 24 hr after the release from G0 phase. The effect was dose-dependent and reached greater than 90% inhibition at 30 micrograms of SPARC per ml after 24 hr. A 20-residue synthetic peptide (termed 2.1) from a non-Ca2(+)-binding, disulfide-rich domain of SPARC also exhibited a dose-dependent inhibition of {sup 3}Hthymidine uptake in endothelial cells within 24 hr after release from G0 phase. An inhibition of 50% was seen with peptide 2.1 at a 0.4 mM concentration. Peptides from other regions of the SPARC protein did not produce this effect. Maximum inhibition of ({sup 3}H)thymidine uptake by SPARC and peptide 2.1 occurred during the early-to-middle G1 phase of the endothelial-cell cycle. From 0-12 hr after release from G0 phase, cells exhibited delayed entry into S phase, which normally occurred at 24 +/- 2 hr. These results were further corroborated by flow cytometry. In the presence of SPARC at 20 micrograms/ml, 72% fewer cells were in S phase after a 24-hr period; a similar, but less marked, reduction was seen with peptide 2.1. Peptide 2.1 did not cause cell rounding, whereas peptide 1.1, a highly efficient inhibitor of endothelial-cell spreading, exhibited essentially no activity with respect to cell-cycle progression.

  16. Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: I. Inhibition of glycoprotein migration in various rat cell types as shown by light microscope radioautography after injection of 3H-fucose

    SciTech Connect

    Bennett, G.; Parsons, S.; Carlet, E.

    1984-08-01

    Previous studies have shown that colchicine and vinblastine inhibit secretion in many cell types by interrupting the normal intracellular migration of secretory products. In the present work, radioautography has been used to study the effects of these drugs on migration of membrane and secretory glycoproteins in a variety of cell types. Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with 3H-fucose. Control rats received 3H-fucose only. All rats were sacrificed 90 min after 3H-fucose injection and their tissues processed for light microscope radioautography. Examination of secretory cell types such as ameloblasts and thyroid follicular cells in control animals revealed reactions of approximately equal intensity over the Golgi region and over extracellular secretion products, while in drug-treated rats most of the reaction was confined to the Golgi region. In a variety of other cell types, including endocrine cells (e.g., hepatocytes) and cells generally considered as nonsecretory (e.g., intestinal columnar cells), reaction in control animals occurred both over the Golgi region and over various portions of the cell surface. In drug-treated animals, a strong Golgi reaction was present, but reaction over the cell surface was weak or absent. These results indicate that in many cell types, colchicine and vinblastine inhibit migration out of the Golgi region not only of secretory glycoproteins, but also of membrane glycoproteins destined for the plasma membrane.

  17. The Cellulose System in the Cell Wall of Micrasterias

    PubMed

    Kim; Herth; Vuong; Chanzy

    1996-11-01

    The cellulose system of the cell wall of Micrasterias denticulata and Micrasterias rotata was analyzed by diffraction contrast transmission electron microscopy, electron diffraction, and X-ray analysis. The studies, achieved on disencrusted cell ghosts, confirmed that the cellulose microfibrils occurred in crisscrossed bands consisting of a number of parallel ribbon-like microfibrils. The individual microfibrils had thicknesses of 5 nm for a width of around 20 nm, but in some instances, two or three microfibrils merged into one another to yield larger monocrystalline domains reaching up to 60 nm in lateral size. The orientation of the cellulose of Micrasterias is very unusual, as it was found that in the cell wall, the equatorial crystallographic planes of cellulose having a d-spacing of 0.60 nm [(11;0) in the Ibeta cellulose unit cell defined by Sugiyama et al., 1991, Macromolecules 24, 4168-4175] were oriented perpendicular to the cell wall surface. Up to now, such orientation has been found only in Spirogyra, another member of the Zygnemataceae group. The unusual structure of the secondary wall cellulose of Micrasterias may be tentatively correlated with the unique organization of the terminal complexes, which in this alga occur as hexagonal arrays of rosettes. PMID:8986649

  18. Galectin-3 Induces Clustering of CD147 and Integrin-β1 Transmembrane Glycoprotein Receptors on the RPE Cell Surface

    PubMed Central

    Priglinger, Claudia S.; Szober, Christoph M.; Priglinger, Siegfried G.; Merl, Juliane; Euler, Kerstin N.; Kernt, Marcus; Gondi, Gabor; Behler, Jennifer; Geerlof, Arie; Kampik, Anselm; Ueffing, Marius; Hauck, Stefanie M.

    2013-01-01

    Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and

  19. Can mutational GC-pressure create new linear B-cell epitopes in herpes simplex virus type 1 glycoprotein B?

    PubMed

    Khrustalev, Vladislav Victorovich

    2009-01-01

    We showed that GC-content of nucleotide sequences coding for linear B-cell epitopes of herpes simplex virus type 1 (HSV1) glycoprotein B (gB) is higher than GC-content of sequences coding for epitope-free regions of this glycoprotein (G + C = 73 and 64%, respectively). Linear B-cell epitopes have been predicted in HSV1 gB by BepiPred algorithm ( www.cbs.dtu.dk/services/BepiPred ). Proline is an acrophilic amino acid residue (it is usually situated on the surface of protein globules, and so included in linear B-cell epitopes). Indeed, the level of proline is much higher in predicted epitopes of gB than in epitope-free regions (17.8% versus 1.8%). This amino acid is coded by GC-rich codons (CCX) that can be produced due to nucleotide substitutions caused by mutational GC-pressure. GC-pressure will also lead to disappearance of acrophobic phenylalanine, isoleucine, methionine and tyrosine coded by GC-poor codons. Results of our "in-silico directed mutagenesis" showed that single nonsynonymous substitutions in AT to GC direction in two long epitope-free regions of gB will cause formation of new linear epitopes or elongation of previously existing epitopes flanking these regions in 25% of 539 possible cases. The calculations of GC-content and amino acid content have been performed by CodonChanges algorithm ( www.barkovsky.hotmail.ru ). PMID:19811425

  20. A model of cell wall expansion based on thermodynamics of polymer networks

    NASA Technical Reports Server (NTRS)

    Veytsman, B. A.; Cosgrove, D. J.

    1998-01-01

    A theory of cell wall extension is proposed. It is shown that macroscopic properties of cell walls can be explained through the microscopic properties of interpenetrating networks of cellulose and hemicellulose. The qualitative conclusions of the theory agree with the existing experimental data. The dependence of the cell wall yield threshold on the secretion of the wall components is discussed.

  1. Molecular and immuno-characteristics of immunoglobulin-like glycoproteins in cancer cell-expressed biomarker, CA215.

    PubMed

    Lee, Gregory; Cheung, Anthony P; Li, Bo; Ge, Bixia; Chow, Po-Ming

    2012-01-01

    RP215 monoclonal antibody (Mab) was shown to recognize a specific carbohydrate-associated epitope found in cancer cell-expressed glycoproteins, known as CA215. The membrane-bound and soluble forms of CA215 were detected in almost all of the cancer cells in humans, but rarely found in normal tissues. Through MALDI-TOF MS analysis, it has been reported previously that as much as 40% of the detected tryptic peptides of CA215 showed high degrees of sequence homology to those found in immunoglobulin heavy chains. The cancer cell-derived immunoglobulins were further purified from CA215 by affinity column-linked with goat anti-human IgG for molecular characterizations. Semi-quantitative RT-PCR was used to determine the mRNA levels of various immunoglobulin genes expressed by cancer cells of single or multi-cell origins and compared with those found in normal human serum. The stability of CA215 was investigated under different experimental conditions. It was observed that the RP215-specific epitope in CA215 is stable at neutral pH, in human serum or in mice (half life of 5-18 days), but unstable at extreme pH's (pH ≤ 2.0; pH ≥ 12.0) or high temperatures. Enzyme immunoassays were performed with several secondary antibody probes related to human IgG. It was demonstrated that cancer cell-expressed immunoglobulins with RP215-specific epitope have much lower immunoactivity than that of normal human IgG (≤ 5%), despite the fact that both showed almost identical amino acid sequence in the respective Fc region reported previously. This could be the result of aberrant glycosylation of CA215 in cancer cells. Aberrant glycosylation of glycoproteins may have important biological implications on the proliferation of cancer cells in vitro or in vivo. PMID:22417288

  2. Alteration of the pH dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein.

    PubMed Central

    Gallagher, T M; Escarmis, C; Buchmeier, M J

    1991-01-01

    Infection of susceptible murine cells with the coronavirus mouse hepatitis virus type 4 (MHV4) results in extensive cell-cell fusion at pHs from 5.5 to 8.5. The endosomotropic weak bases chloroquine and ammonium chloride do not prevent MHV4 infection. In marked contrast, we have selected variants from a neural cell line persistently infected with MHV4 which are entirely dependent on acid pH to fuse host cells and are strongly inhibited by endosomotropic weak bases. Wild-type and variant viruses were compared at the level of the fusion-active surface (S) glycoprotein gene. Cloning and sequencing of each 4,131-base open reading frame predicted a total of eight amino acid differences which fell into three distinct clusters. Each S glycoprotein, when expressed from cDNA, was synthesized in equivalent amounts, and similar proportions were transported to the cell surface. Wild-type S induced cell-cell fusion at neutral pH, whereas variant S required prolonged exposure to acidic pH to induce fusion. Expression of hybrid S genes prepared by exchange of restriction fragments between wild-type and variant cDNAs revealed that elimination of neutral pH fusion was solely dependent on amino acid alterations at positions 1067 (Q to H), 1094 (Q to H), and 1114 (L to R). These changes lie within a predicted heptad repeat region of the transmembrane cleavage fragment of S (S2). These findings demonstrate that the pH dependence of coronavirus fusion is highly variable and that this variability can be determined by as few as three amino acid residues. Images PMID:1848311

  3. Purification of the Thy-1 molecule, a major cell-surface glycoprotein of rat thymocytes.

    PubMed Central

    Letarte-Muirhead, M; Barclay, A N; Williams, A F

    1975-01-01

    The Thy-1-molecule, which was identified by its antigenic activities, has been purified from rat thymocytes. The purification involved preparation of crude membranes and solubilization in deoxycholate, followed by gel filtration and affinity chromatography on antibody or lectin columns. In all cases the purified molecule was a glycoprotein that did not form higher polymers and was not associated with other polypeptide chains. The Thy-1 glycoprotein could be found in two forms, one binding to lentil lectin, the other not. Both forms had the same detectable antigens and were of a similar but not identical size. After sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the apparent molecular weight of Thy-1 binding to lentil lectin was 25 000, whereas that not binding to the lectin was 27 000, with heterogeneity towards forms of apparently higher molecular weight. Images PLATE 4 PLATE 1 PLATE 2 PLATE 3 PMID:56177

  4. The yin and yang of cell wall integrity control: brassinosteroid and FERONIA signaling.

    PubMed

    Höfte, Herman

    2015-02-01

    Understanding how developmental and environmental signals control plant cell expansion requires an intimate knowledge of the architecture of the primary cell wall and the chemo-rheological processes that underlie cell wall relaxation. In this review I discuss recent findings that reveal a more prominent role than previously suspected for covalent bonds and pectin cross-links in primary cell wall architecture. In addition, genetic studies have uncovered a role for receptor kinases in the control of cell wall homeostasis in growing cells. The emerging view is that, upon cell wall disruption, compensatory changes are induced in the cell wall through the interplay between the brassinosteroid signaling module, which positively regulates wall extensibility and receptor kinases of the CrRLKL1 family, which may act as negative regulators of cell wall stiffness. These findings lift the tip of the veil of a complex signaling network allowing normal homeostasis in walls of growing cells but also crisis management under stress conditions. PMID:25481004

  5. Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT, MMR, P-Glycoprotein and CD133 Expression

    PubMed Central

    Prados, Jose; Caba, Octavio; Cabeza, Laura; Berdasco, Maria; Gónzalez, Beatriz; Melguizo, Consolación

    2015-01-01

    Background The use of temozolomide (TMZ) has improved the prognosis for glioblastoma multiforme patients. However, TMZ resistance may be one of the main reasons why treatment fails. Although this resistance has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT) it seems that this enzyme is not the only molecular mechanism that may account for the appearance of drug resistance in glioblastoma multiforme patients as the mismatch repair (MMR) complex, P-glycoprotein, and/or the presence of cancer stem cells may also be implicated. Methods Four nervous system tumor cell lines were used to analyze the modulation of MGMT expression and MGMT promoter methylation by TMZ treatment. Furthermore, 5-aza-2’-deoxycytidine was used to demethylate the MGMT promoter and O(6)-benzylguanine to block GMT activity. In addition, MMR complex and P-glycoprotein expression were studied before and after TMZ exposure and correlated with MGMT expression. Finally, the effect of TMZ exposure on CD133 expression was analyzed. Results Our results showed two clearly differentiated groups of tumor cells characterized by low (A172 and LN229) and high (SF268 and SK-N-SH) basal MGMT expression. Interestingly, cell lines with no MGMT expression and low TMZ IC50 showed a high MMR complex expression, whereas cell lines with high MGMT expression and high TMZ IC50 did not express the MMR complex. In addition, modulation of MGMT expression in A172 and LN229 cell lines was accompanied by a significant increase in the TMZ IC50, whereas no differences were observed in SF268 and SK-N-SH cell lines. In contrast, P-glycoprotein and CD133 was found to be unrelated to TMZ resistance in these cell lines. Conclusions These results may be relevant in understanding the phenomenon of TMZ resistance, especially in glioblastoma multiforme patients laking MGMT expression, and may also aid in the design of new therapeutic strategies to improve the efficacy of TMZ in glioblastoma

  6. Clearance and binding of radiolabeled glycoproteins by cells of the murine mononuclear phagocyte system

    SciTech Connect

    Imber, M.J.

    1982-01-01

    The clearance and binding of radiolabeled lactoferrin and fast ..cap alpha../sub 2/-macroglobulin were studied. Both glycoproteins cleared rapidly following intravenous injection in mice, and both bound specifically to discrete receptors on murine peritoneal macrophages. The simultaneous presence of excess, unlabeled ligands specific for receptors recognizing terminal fucose, mannose, N-acetylglucosamine or galactose residues did not inhibit the clearance or binding of either lactoferrin or fast-..cap alpha../sub 2/M. The clearance and binding of enzymatically defucosylated lactoferrin was indistinguishable from native lactoferrin, indicating that terminal ..cap alpha..(1-3)-linked fucose on lactoferrin is not necessary for receptor recognition. The clearance and binding of two fast -..cap alpha../sub 2/M forms, ..cap alpha../sub 2/M-trypsin and ..cap alpha../sub 2/M-MeNH/sub 2/ cross compete with each other. Saturation binding studies indicated that the total binding of mannosyl -BSA, fusocyl-BSA, and N-acetylglucosaminyl-BSA to macrophages activated by BCG was approximately 15% of the levels observed with inflammatory macrophages elicited by thioglycollate broth. Cross-competition binding studies demonstrated a common surface receptor mediated binding of all three neoglycoprotein ligands and was identical to the receptor on mononuclear phagocytes that binds mannosyl- and N-acetylglucosaminyl-terminated glycoproteins. These results suggest that difference between discrete states of macrophage function may be correlated with selective changes in levels of the surface receptor for mannose-containing glycoproteins.

  7. Influence of the Cell Wall on Intracellular Delivery to Algal Cells by Electroporation and Sonication

    PubMed Central

    Azencott, Harold R.; Peter, Gary F.; Prausnitz, Mark R.

    2007-01-01

    To assess the cell wall’s role as a barrier to intracellular delivery, wild-type Chlamydomonas reinhardtii algal cells and mutant cells lacking a cell wall were exposed to electroporation or sonication. Flow cytometry determined intracellular uptake of calcein and bovine serum albumin (BSA) and loss of cell viability as functions of electroporation transmembrane potential and acoustic energy. Electroporation of wild-type cells increased calcein uptake with increasing transmembrane potential, but delivered much less BSA. Electroporation of wall-deficient cells had similar effects on calcein uptake, but increased BSA uptake as much as 7.5-fold relative to wild-type cells, which indicated that the cell wall was a significant barrier to BSA delivery during electroporation. Sonication of wild-type cells caused calcein and BSA uptake at similar levels. This suggests that the cell wall barrier to BSA delivery can be overcome by sonication. Increased electroporation transmembrane potential or acoustic energy also caused increased loss of cell viability, where wall-deficient cells were especially susceptible to lysis. Overall, we believe this is the first study to compare the effects of electroporation and sonication in a direct fashion in any cell type. Specifically, these findings suggest that electroporation primarily transports molecules across the plasma membrane, because its mechanism is specific to lipid bilayer disruption, whereas sonication transports molecules across both the plasma membrane and cell wall, because it non-specifically disrupts cell-surface barriers. PMID:17602827

  8. Cell wall structure and function in lactic acid bacteria

    PubMed Central

    2014-01-01

    The cell wall of Gram-positive bacteria is a complex assemblage of glycopolymers and proteins. It consists of a thick peptidoglycan sacculus that surrounds the cytoplasmic membrane and that is decorated with teichoic acids, polysaccharides, and proteins. It plays a major role in bacterial physiology since it maintains cell shape and integrity during growth and division; in addition, it acts as the interface between the bacterium and its environment. Lactic acid bacteria (LAB) are traditionally and widely used to ferment food, and they are also the subject of more and more research because of their potential health-related benefits. It is now recognized that understanding the composition, structure, and properties of LAB cell walls is a crucial part of developing technological and health applications using these bacteria. In this review, we examine the different components of the Gram-positive cell wall: peptidoglycan, teichoic acids, polysaccharides, and proteins. We present recent findings regarding the structure and function of these complex compounds, results that have emerged thanks to the tandem development of structural analysis and whole genome sequencing. Although general structures and biosynthesis pathways are conserved among Gram-positive bacteria, studies have revealed that LAB cell walls demonstrate unique properties; these studies have yielded some notable, fundamental, and novel findings. Given the potential of this research to contribute to future applied strategies, in our discussion of the role played by cell wall components in LAB physiology, we pay special attention to the mechanisms controlling bacterial autolysis, bacterial sensitivity to bacteriophages and the mechanisms underlying interactions between probiotic bacteria and their hosts. PMID:25186919

  9. Cell wall structure and function in lactic acid bacteria.

    PubMed

    Chapot-Chartier, Marie-Pierre; Kulakauskas, Saulius

    2014-08-29

    The cell wall of Gram-positive bacteria is a complex assemblage of glycopolymers and proteins. It consists of a thick peptidoglycan sacculus that surrounds the cytoplasmic membrane and that is decorated with teichoic acids, polysaccharides, and proteins. It plays a major role in bacterial physiology since it maintains cell shape and integrity during growth and division; in addition, it acts as the interface between the bacterium and its environment. Lactic acid bacteria (LAB) are traditionally and widely used to ferment food, and they are also the subject of more and more research because of their potential health-related benefits. It is now recognized that understanding the composition, structure, and properties of LAB cell walls is a crucial part of developing technological and health applications using these bacteria. In this review, we examine the different components of the Gram-positive cell wall: peptidoglycan, teichoic acids, polysaccharides, and proteins. We present recent findings regarding the structure and function of these complex compounds, results that have emerged thanks to the tandem development of structural analysis and whole genome sequencing. Although general structures and biosynthesis pathways are conserved among Gram-positive bacteria, studies have revealed that LAB cell walls demonstrate unique properties; these studies have yielded some notable, fundamental, and novel findings. Given the potential of this research to contribute to future applied strategies, in our discussion of the role played by cell wall components in LAB physiology, we pay special attention to the mechanisms controlling bacterial autolysis, bacterial sensitivity to bacteriophages and the mechanisms underlying interactions between probiotic bacteria and their hosts. PMID:25186919

  10. A cytoplasmic peptidoglycan amidase homologue controls mycobacterial cell wall synthesis

    PubMed Central

    Boutte, Cara C; Baer, Christina E; Papavinasasundaram, Kadamba; Liu, Weiru; Chase, Michael R; Meniche, Xavier; Fortune, Sarah M; Sassetti, Christopher M; Ioerger, Thomas R; Rubin, Eric J

    2016-01-01

    Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in Mycobacterium tuberculosis (Mtb). However, little is known about how the cell wall is regulated in stress. We found that CwlM, a protein homologous to peptidoglycan amidases, coordinates peptidoglycan synthesis with nutrient availability. Surprisingly, CwlM is sequestered from peptidoglycan (PG) by localization in the cytoplasm, and its enzymatic function is not essential. Rather, CwlM is phosphorylated and associates with MurA, the first enzyme in PG precursor synthesis. Phosphorylated CwlM activates MurA ~30 fold. CwlM is dephosphorylated in starvation, resulting in lower MurA activity, decreased cell wall metabolism, and increased tolerance to multiple antibiotics. A phylogenetic analysis of cwlM implies that localization in the cytoplasm drove the evolution of this factor. We describe a system that controls cell wall metabolism in response to starvation, and show that this regulation contributes to antibiotic tolerance. DOI: http://dx.doi.org/10.7554/eLife.14590.001 PMID:27304077

  11. Environmental stability of stem cell wall traits in alfalfa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The concentration of stem cell wall constituents in alfalfa, Medicago sativa L., herbage can affect dry matter intake and energy availability in dairy and beef production systems and impact energy conversion efficiency when alfalfa is used to produce biofuels. Stem Klason lignin, glucose, xylose, an...

  12. A cytoplasmic peptidoglycan amidase homologue controls mycobacterial cell wall synthesis.

    PubMed

    Boutte, Cara C; Baer, Christina E; Papavinasasundaram, Kadamba; Liu, Weiru; Chase, Michael R; Meniche, Xavier; Fortune, Sarah M; Sassetti, Christopher M; Ioerger, Thomas R; Rubin, Eric J

    2016-01-01

    Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in Mycobacterium tuberculosis (Mtb). However, little is known about how the cell wall is regulated in stress. We found that CwlM, a protein homologous to peptidoglycan amidases, coordinates peptidoglycan synthesis with nutrient availability. Surprisingly, CwlM is sequestered from peptidoglycan (PG) by localization in the cytoplasm, and its enzymatic function is not essential. Rather, CwlM is phosphorylated and associates with MurA, the first enzyme in PG precursor synthesis. Phosphorylated CwlM activates MurA ~30 fold. CwlM is dephosphorylated in starvation, resulting in lower MurA activity, decreased cell wall metabolism, and increased tolerance to multiple antibiotics. A phylogenetic analysis of cwlM implies that localization in the cytoplasm drove the evolution of this factor. We describe a system that controls cell wall metabolism in response to starvation, and show that this regulation contributes to antibiotic tolerance. PMID:27304077

  13. Titration of Isolated Cell Walls of Lemna minor L 1

    PubMed Central

    Morvan, Claudine; Demarty, Maurice; Thellier, Michel

    1979-01-01

    A theoretical model has been built to bypass the equation of titration of the cell wall. This equation, which is an extension of the Henderson-Hasselbach equation, underlines the importance of the exchange constant, the ionic strength as well as the rate of neutralization. The model is restricted to the case when the ionization degree is equal to the neutralization degree. The shape of the titration curve is shown to be strongly dependent on the valency of the base used. Experimental results have shown that isolated cell walls bear at least two kinds of sites. The first sites which are titrated after a short time of equilibration are attributed to polyuronic acids (capacity: 0.3 milliequivalents per gram fresh cell walls). The second sites, are obtained after a long time of equilibration (capacity: 1.2 to 1.3 milliequivalents per gram, fresh cell walls). Titrations have been performed with different bases [KOH, NaOH, and Ca(OH)2] and under different ionic strengths. The results obtained with NaOH and KOH do not exhibit any difference of selectivity. Conversely, the sites have a much bigger affinity for the Ca2+ ions than for the monovalent ones. The apparent pKa of the uronic acids was estimated to lie between 3.0 and 3.4; this is consistent with the values obtained with polyuronic acid solutions. PMID:16660868

  14. Hetero-oligomeric cell wall channels (porins) of Nocardia farcinica.

    PubMed

    Kläckta, Christian; Knörzer, Philipp; Riess, Franziska; Benz, Roland

    2011-06-01

    The cell wall of Nocardia farcinica contains a cation-selective cell wall channel, which may be responsible for the limited permeability of the cell wall of N. farcinica for negatively charged antibiotics. Based on partial sequencing of the protein responsible for channel formation derived from N. farcinica ATTC 3318 we were able to identify the corresponding genes (nfa15890 and nfa15900) within the known genome of N. farcinica IFM 10152. The corresponding genes of N. farcinica ATTC 3318 were separately expressed in the Escherichia coli BL21DE3Omp8 strain and the N-terminal His10-tagged proteins were purified to homogeneity using immobilized metal affinity chromatography. The pure proteins were designated NfpANHis and NfpBNHis, for N. farcinica porin A and N. farcinica porin B. The two proteins were checked separately for channel formation in lipid bilayers. Our results clearly indicate that the proteins NfpANHis and NfpBNHis expressed in E. coli could only together form a channel in lipid bilayer membranes. This means that the cell wall channel of N. farcinica is formed by a heterooligomer. NfpA and NfpB form together a channel that may structurally be related to MspA of Mycobacterium smegmatis based on amino acid comparison and renaturation procedure. PMID:21092733

  15. Polymer mobility in cell walls of cucumber hypocotyls

    NASA Technical Reports Server (NTRS)

    Fenwick, K. M.; Apperley, D. C.; Cosgrove, D. J.; Jarvis, M. C.

    1999-01-01

    Cell walls were prepared from the growing region of cucumber (Cucumis sativus) hypocotyls and examined by solid-state 13C NMR spectroscopy, in both enzymically active and inactivated states. The rigidity of individual polymer segments within the hydrated cell walls was assessed from the proton magnetic relaxation parameter, T2, and from the kinetics of cross-polarisation from 1H to 13C. The microfibrils, including most of the xyloglucan in the cell wall, as well as cellulose, behaved as very rigid solids. A minor xyloglucan fraction, which may correspond to cross-links between microfibrils, shared a lower level of rigidity with some of the pectic galacturonan. Other pectins, including most of the galactan side-chain residues of rhamnogalacturonan I, were much more mobile and behaved in a manner intermediate between the solid and liquid states. The only difference observed between the enzymically active and inactive cell walls, was the loss of a highly mobile, methyl-esterified galacturonan fraction, as the result of pectinesterase activity.

  16. Medicago truncatula as a Model for Dicot Cell Wall Development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Strong interest in renewable energy has promoted an upsurge of research on plant cell wall traits that influence the availability of lignocellulosic-derived sugars for fermentation in production of biofuels. We have initiated a genome-wide transcript profiling study using the model legume Medicago t...

  17. Determination of carbohydrate profile in sugarbeet (Beta vulgaris) cell walls

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sugarbeet germplasms USH20, C869, EL55, EL54 were used, and different tissues at different developmental stages were sampled, including dry seeds, germinating seedlings, developing leaves, mature leaves, petioles, hypocotyls, mature roots, flowering stems and inflorescences. Cell Wall Composition An...

  18. Glycoprotein Analysis

    NASA Astrophysics Data System (ADS)

    Fernandes, Daryl; Spencer, Daniel

    This chapter provides an overview of practical methods for glycosylation analysis of glycoprotein therapeutics . The topics include glycoprofiling methods for glycoforms, monosaccharides (neutral and N-acetylated species as well as sialic acids), oligosaccharides (chemical and enzymatic methods for glycan release, post-release purification, labeling and derivatization, different types of glycan HPLC and MS), and glycosylation site profiling.

  19. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2006-10-31

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  20. Glycoprotein synthesis

    DOEpatents

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

    2009-07-14

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  1. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-05-15

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  2. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2005-08-09

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  3. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-08-28

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  4. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2010-11-02

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  5. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-07-03

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  6. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-02-27

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  7. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2010-11-16

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  8. Glycoprotein synthesis

    DOEpatents

    Shultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-04-03

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  9. Undressing the fungal cell wall/cell membrane--the antifungal drug targets.

    PubMed

    Tada, Rui; Latgé, Jean-Paul; Aimanianda, Vishukumar

    2013-01-01

    Being external, the fungal cell wall plays a crucial role in the fungal life. By covering the underneath cell, it offers mechanical strength and acts as a barrier, thus protecting the fungus from the hostile environment. Chemically, this cell wall is composed of different polysaccharides. Because of their specific composition, the fungal cell wall and its underlying plasma membrane are unique targets for the development of drugs against pathogenic fungal species. The objective of this review is to consolidate the current knowledge on the antifungal drugs targeting the cell wall and plasma membrane, mainly of Aspergillus and Candida species - the most prevalent fungal pathogens, and also to present challenges and questions conditioning the development of new antifungal drugs targeting the cell wall. PMID:23278542

  10. Varicella-zoster virus glycoproteins B and E are major targets of CD4+ and CD8+ T cells reconstituting during zoster after allogeneic transplantation

    PubMed Central

    Kleemann, Patrick; Distler, Eva; Wagner, Eva M.; Thomas, Simone; Klobuch, Sebastian; Aue, Steffi; Schnürer, Elke; Schild, Hansjörg; Theobald, Matthias; Plachter, Bodo; Tenzer, Stefan; Meyer, Ralf G.; Herr, Wolfgang

    2012-01-01

    Background After allogeneic hematopoietic stem-cell transplantation patients are at increased risk for herpes zoster as long as varicella-zoster virus specific T-cell reconstitution is impaired. This study aimed to identify immunodominant varicella-zoster virus antigens that drive recovery of virus-specific T cells after transplantation. Design and Methods Antigens were purified from a varicella-zoster virus infected cell lysate by high-performance liquid chromatography and were identified by quantitative mass spectrometric analysis. To approximate in vivo immunogenicity for memory T cells, antigen preparations were consistently screened with ex vivo PBMC of varicella-zoster virus immune healthy individuals in sensitive interferon-γ ELISpot assays. Candidate virus antigens identified by the approach were genetically expressed in PBMC using electroporation of in vitro transcribed RNA encoding full-length proteins and were then analyzed for recognition by CD4+ and CD8+ memory T cells. Results Varicella-zoster virus encoded glycoproteins B and E, and immediate early protein 62 were identified in immunoreactive lysate material. Predominant CD4+ T-cell reactivity to these proteins was observed in healthy virus carriers. Furthermore, longitudinal screening in allogeneic stem-cell transplantation patients showed strong expansions of memory T cells recognizing glycoproteins B and E after onset of herpes zoster, while immediate early protein 62 reactivity remained moderate. Reactivity to viral glycoproteins boosted by acute zoster was mediated by both CD4+ and CD8+ T cells. Conclusions Our data demonstrate that glycoproteins B and E are major targets of varicella-zoster virus specific CD4+ and CD8+ T-cell reconstitution occurring during herpes zoster after allogeneic stem-cell transplantation. Varicella-zoster virus glycoproteins B and E might form the basis for novel non-hazardous zoster subunit vaccines suitable for immunocompromised transplant patients. PMID:22207687

  11. Comparison of P-glycoprotein expression in cell lines and xenogragraft sections using I-125 MRK-16 monoclonal antibody (MAB)

    SciTech Connect

    Mehta, B.M.; Kostakoglu, L.; Levchenko, A.

    1994-05-01

    P-glycoprotein (Pgp) is known to be associated with multidrug resistance (MDR). Quantitation of P-glycoprotein expression may permit appropriate therapy depending on Pgp expression in tumors. The present study was undertaken to evaluate the utility of quantitative autoradiography (QAR) in the quantification of MDR using MRK-16, a murine IgG mAb reactive against Pgp. Balb/c mice were xenografted with colchicine resistant BE(2)C/CHC cells. Animals with established tumors were sacrificed, and 8 {mu}m tumor sections were prepared. Mab MRK-16 was labeled with I-125 (150 {mu}Ci/0.625 nmole) by the iodogen method and subsequently purified by size exclusion chromatography. Consecutive tumor sections were incubated overnight at 4{degrees}C with serial dilutions of I-125 MRK-16. Similarly cell suspensions containing 1 X 10{sup 7} cells per ml were also incubated with serial dilutions. QAR analysis of tissue sections of BE(2)C/CHC tumors growing as xenografts in nude mice, determined the binding affinity (K{sub a}) for MRK-16 to be 1 x 10{sup 9} L/M and the number of binding sites (B{sub max}) to be 137, 700 per cell (222 picomols/g); it compared very well with the K{sub a} value of 5 x 10{sup 8} L/M and the B{sub max} value of 130,000 per cell (217 picomols/g) obtained from binding analysis with cell suspensions.

  12. Partial Chemical Characterization of Corn Root Cell Walls 1

    PubMed Central

    Dever, John E.; Bandurski, Robert S.; Kivilaan, A.

    1968-01-01

    The present study reports on chemical changes which occur in the cell wall of Zea mays during early phases of growth. Roots of seedling corn plants were divided into a meristematic zone, the zone of elongation, and the maturation zone, and the cell wall isolated from each of these zones. The wall preparations were then extracted sequentially to obtain pectin, hemicellulose, cellulose, and lignin fractions. Each of these, except for the lignin fraction, was hydrolyzed and the resultant sugars isolated, identified, and estimated quantitatively. Quantitative analysis of the products of hydrolysis of these fractions demonstrated that the classical scheme of fractionation is a valuable indicator of the changes in solubility properties which the various polysaccharide components for the wall undergo. It does not however yield definite chemical entities. For example, the “pectin” fraction contains only about 3% galacturonic acid; the bulk of it being composed of glucose, xylose, and galactose. By summation of analysis of these various fractions, it was found that substances yielding glucose and xylose upon hydrolysis increase with advancing age of the tissue. Galactose- and arabinose-yielding compounds decrease and mannose appears during maturation. Anhydrouronic acids first decrease, then increase. Most interestingly, of the total dry weight of the cell wall, only 24, 45, and 50% of the meristematic, elongation, and maturation zones respectively are accounted for as simple sugars in the acid hydrolysates. Oligosaccharides were not encountered in large amounts so that the 50 to 75% of the wall weight unaccounted for would consist of polysaccharides or oligosaccharides not precipitated by ethanol from the extracting solutions employed and by polysaccharides in the hemicellulose fraction which are resistant to acid hydrolysis. PMID:16656735

  13. Cell-Wall Polysaccharides of Developing Flax Plants.

    PubMed Central

    Gorshkova, T. A.; Wyatt, S. E.; Salnikov, V. V.; Gibeaut, D. M.; Ibragimov, M. R.; Lozovaya, V. V.; Carpita, N. C.

    1996-01-01

    Flax (Linum usitatissimum L.) fibers originate from procambial cells of the protophloem and develop in cortical bundles that encircle the vascular cylinder. We determined the polysaccharide composition of the cell walls from various organs of the developing flax plant, from fiber-rich strips peeled from the stem, and from the xylem. Ammonium oxalate-soluble polysaccharides from all tissues contained 5-linked arabinans with low degrees of branching, rhamnogalacturonans, and polygalacturonic acid. The fiber-rich peels contained, in addition, substantial amounts of a buffer-soluble, 4-linked galactan branched at the 0-2 and 0-3 positions with nonreducing terminal-galactosyl units. The cross-linking glycans from all tissues were (fucogalacto)xyloglucan, typical of type-I cell walls, xylans containing (1->)-[beta]-D-xylosyl units branched exclusively at the xylosyl O-2 with t-(4-O-methyl)-glucosyluronic acid units, and (galacto)glucomannans. Tissues containing predominantly primary cell wall contained a larger proportion of xyloglucan. The xylem cells were composed of about 60% 4-xylans, 32% cellulose, and small amounts of pectin and the other cross-linking polysaccharides. The noncellulosic polysaccharides of flax exhibit an uncommonly low degree of branching compared to similar polysaccharides from other flowering plants. Although the relative abundance of the various noncellulosic polysaccharides varies widely among the different cell types, the linkage structure and degree of branching of several of the noncellulosic polysaccharides are invariant. PMID:12226214

  14. Palmitoylation of the cysteine-rich endodomain of the SARS-coronavirus spike glycoprotein is important for spike-mediated cell fusion

    SciTech Connect

    Petit, Chad M.; Chouljenko, Vladimir N.; Iyer, Arun; Colgrove, Robin; Farzan, Michael; Knipe, David M.; Kousoulas, K.G. . E-mail: vtgusk@lsu.edu

    2007-04-10

    The SARS-coronavirus (SARS-CoV) is the etiological agent of the severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. The cytoplasmic portion of the S glycoprotein contains four cysteine-rich amino acid clusters. Individual cysteine clusters were altered via cysteine-to-alanine amino acid replacement and the modified S glycoproteins were tested for their transport to cell-surfaces and ability to cause cell fusion in transient transfection assays. Mutagenesis of the cysteine cluster I, located immediately proximal to the predicted transmembrane, domain did not appreciably reduce cell-surface expression, although S-mediated cell fusion was reduced by more than 50% in comparison to the wild-type S. Similarly, mutagenesis of the cysteine cluster II located adjacent to cluster I reduced S-mediated cell fusion by more than 60% compared to the wild-type S, while cell-surface expression was reduced by less than 20%. Mutagenesis of cysteine clusters III and IV did not appreciably affect S cell-surface expression or S-mediated cell fusion. The wild-type S was palmitoylated as evidenced by the efficient incorporation of {sup 3}H-palmitic acid in wild-type S molecules. S glycoprotein palmitoylation was significantly reduced for mutant glycoproteins having cluster I and II cysteine changes, but was largely unaffected for cysteine cluster III and IV mutants. These results show that the S cytoplasmic domain is palmitoylated and that palmitoylation of the membrane proximal cysteine clusters I and II may be important for S-mediated cell fusion.

  15. Crushing Strength of Aluminum Honeycomb with Thinning Cell Wall

    NASA Astrophysics Data System (ADS)

    Ogasawara, Nagahisa; Chiba, Norimasa; Kobayashi, Eiji; Kikuchi, Yuji

    To evaluate the crash safety of automobiles, various collision tests are performed by the auto industry. In the offset frontal collision test and the side collision test, the target is an aluminum honeycomb material which has thinning cell walls. In this study, based on the analyses of the shock absorption mechanism, a new crushing strength formula is proposed. First, load-displacement curves obtained from compression tests in quasi-static condition showed an almost linear relation between a thinning rate of cell walls and a crushing strength. Second, based on Wierzbicki's theory, a new formula was proposed, which can estimate a crushing strength of a honeycomb material with thinning wall. In addition, a correcting equation which considered an elastic deformation was also proposed. Third, parametric analyses were carried out with a FE model which can simulate a delamination between cell walls. The results obtained from the theory and FEM almost corresponded to each other for a wide range of the thinning rate. Fourth, impact tests were carried out, in which the weight was dropped freely at the speed used for the automobile tests. Those results almost agreed well with the sum of the theoretical crush strength and the inside air pressure.

  16. Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells

    PubMed Central

    Liesche, Johannes; Marek, Magdalena; Günther-Pomorski, Thomas

    2015-01-01

    Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays. PMID:25717323

  17. Resistance to antibiotics targeted to the bacterial cell wall

    PubMed Central

    Nikolaidis, I; Favini-Stabile, S; Dessen, A

    2014-01-01

    Peptidoglycan is the main component of the bacterial cell wall. It is a complex, three-dimensional mesh that surrounds the entire cell and is composed of strands of alternating glycan units crosslinked by short peptides. Its biosynthetic machinery has been, for the past five decades, a preferred target for the discovery of antibacterials. Synthesis of the peptidoglycan occurs sequentially within three cellular compartments (cytoplasm, membrane, and periplasm), and inhibitors of proteins that catalyze each stage have been identified, although not all are applicable for clinical use. A number of these antimicrobials, however, have been rendered inactive by resistance mechanisms. The employment of structural biology techniques has been instrumental in the understanding of such processes, as well as the development of strategies to overcome them. This review provides an overview of resistance mechanisms developed toward antibiotics that target bacterial cell wall precursors and its biosynthetic machinery. Strategies toward the development of novel inhibitors that could overcome resistance are also discussed. PMID:24375653

  18. Messenger Functions of the Bacterial Cell Wall-derived Muropeptides

    PubMed Central

    Boudreau, Marc A.; Fisher, Jed. F.; Mobashery, Shahriar

    2012-01-01

    Bacterial muropeptides are soluble peptidoglycan structures central to recycling of the bacterial cell wall, and messengers in diverse cell-signaling events. Bacteria sense muropeptides as signals that antibiotics targeting cell-wall biosynthesis are present, and eukaryotes detect muropeptides during the innate immune response to bacterial infection. This review summarizes the roles of bacterial muropeptides as messengers, with a special emphasis on bacterial muropeptide structures and the relationship of structure to the biochemical events that the muropeptides elicit. Muropeptide sensing and recycling in both Gram-positive and Gram-negative bacteria is discussed, followed by muropeptide sensing by eukaryotes as a crucial event to the innate immune response of insects (via peptidoglycan-recognition proteins) and mammals (through Nod-like receptors) to bacterial invasion. PMID:22409164

  19. Functional roles of membrane glycoprotein CD36.

    PubMed

    Daviet, L; McGregor, J L

    1996-01-01

    Cell-cell and cell-extracellular matrix interactions are mediated by a number of membrane glycoproteins. On the basis of structural homologies, several families of cell adhesion molecules (integrins, selectins, immunoglobulins, cadherins, leucine-rich glycoproteins) have been established. Since 1991, a new family of CD36-like proteins has been identified. CD36 is a cell surface glycoprotein that interacts with a large variety of ligands. CD36 has been implicated in thrombosis, vascular biology, lipid metabolism and atherogenesis. In this review, we aim to summarize our present knowledge on this important, multifunctional glycoprotein. PMID:21043590

  20. Stressor-dependent Alterations in Glycoprotein 130: Implications for Glial Cell Reactivity, Cytokine Signaling and Ganglion Cell Health in Glaucoma

    PubMed Central

    Echevarria, FD; Walker, CC; Abella, SK; Won, M; Sappington, RM

    2013-01-01

    Objective: The interleukin-6 (IL-6) family of cytokines is associated with retinal ganglion cell (RGC) survival and glial reactivity in glaucoma. The purpose of this study was to evaluate glaucoma-related changes in glycoprotein-130 (gp130), the common signal transducer of the IL-6 family of cytokines, as they relate to RGC health, glial reactivity and expression of IL-6 cytokine family members. Methods: For all experiments, we examined healthy retina (young C57), aged retina (aged C57), retina predisposed to glaucoma (young DBA/2) and retina with IOP-induced glaucoma (aged DBA/2). We determined retinal gene expression of gp130 and IL-6 family members, using quantitative PCR, and protein expression of gp130, using multiplex ELISA. For protein localization and cell-specific expression, we performed co-immunolabeling for gp130 and cell type-specific markers. We used quantitative microscopy to measure layer-specific expression of gp130 and its relationships to astrocyte and Müller glia reactivity and RGC axonal transport, as determined by uptake and transport of cholera toxin β-subunit (CTB). Results: Gene expression of gp130 was elevated with all glaucoma-related stressors, but only normal aging increased protein levels. In healthy retina, gp130 localized primarily to the inner retina, where it was expressed by astrocytes, Müller cells and RGCs. Layer-specific analysis of gp130 expression revealed increased expression in aging retina and decreased expression in glaucomatous retina that was eccentricity-dependent. These glaucoma-related changes in gp130 expression correlated with the level of GFAP and glutamine synthetase expression, as well as axonal transport in RGCs. The relationships between gp130, glial reactivity and RGC health could impact signaling by many IL-6 family cytokines, which exhibited overall increased expression in a stressor-dependent manner. Conclusions: Glaucoma-related stressors, including normal aging, glaucoma predisposition and IOP

  1. Regulation of Varicella-Zoster Virus-Induced Cell-to-Cell Fusion by the Endocytosis-Competent Glycoproteins gH and gE

    PubMed Central

    Pasieka, Tracy Jo; Maresova, Lucie; Shiraki, Kimiyasu; Grose, Charles

    2004-01-01

    The gH glycoprotein of varicella-zoster virus (VZV) is a major fusogen. The realigned short cytoplasmic tail of gH (18 amino acids) harbors a functional endocytosis motif (YNKI) that mediates internalization in both VZV-infected and transfected cells (T. J. Pasieka, L. Maresova, and C. Grose, J. Virol. 77: 4194-4202, 2003). During subsequent confocal microscopy studies of endocytosis-deficient gH mutants, we observed that cells transfected with the gH tail mutants exhibited marked fusion. Therefore, we postulated that VZV gH endocytosis served to regulate cell-to-cell fusion. Subsequent analyses of gH+gL transfection fusion assays by the Kolmogorov-Smirnov statistical test demonstrated that expression of the endocytosis-deficient gH mutants resulted in a statistically significant enhancement of cell-to-cell fusion (P < 0.0001) compared to wild-type gH. On the other hand, coexpression of VZV gE, another endocytosis-competent VZV glycoprotein, was able to temper the fusogenicity of the gH endocytosis mutants by facilitating internalization of the mutant gH protein from the cell surface. When the latter results were similarly analyzed, there was no longer any enhanced fusion by the endocytosis-deficient gH mutant protein. In summary, these studies support a role for gH endocytosis in regulating the cell surface expression of gH and thereby regulating gH-mediated fusion. The data also confirm and extend prior observations of a gE-gH interaction during viral glycoprotein trafficking in a VZV transfection system. PMID:14990707

  2. Artificial Formation and Tuning of Glycoprotein Networks on Live Cell Membranes: A Single-Molecule Tracking Study.

    PubMed

    Möckl, Leonhard; Lindhorst, Thisbe K; Bräuchle, Christoph

    2016-03-16

    We present a method to artificially induce network formation of membrane glycoproteins and show the precise tuning of their interconnection on living cells. For this, membrane glycans are first metabolically labeled with azido sugars and then tagged with biotin by copper-free click chemistry. Finally, these biotin-tagged membrane proteins are interconnected with streptavidin (SA) to form an artificial protein network in analogy to a lectin-induced lattice. The degree of network formation can be controlled by the concentration of SA, its valency, and the concentration of biotin on membrane proteins. This was verified by investigation of the spatiotemporal dynamics of the SA-protein networks employing single-molecule tracking. It was also proven that this network formation strongly influences the biologically relevant process of endocytosis as it is known from natural lattices on the cell surface. PMID:26698366

  3. P-Glycoprotein/MDR1 regulates pokemon gene transcription through p53 expression in human breast cancer cells.

    PubMed

    He, Shengnan; Liu, Feng; Xie, Zhenhua; Zu, Xuyu; Xu, Wei; Jiang, Yuyang

    2010-01-01

    P-glycoprotein (Pgp), encoded by the multidrug resistance 1 (MDR1) gene, is an efflux transporter and plays an important role in pharmacokinetics. In this study, we demonstrated that the pokemon promoter activity, the pokemon mRNA and protein expression can be significantly inhibited by Pgp. Chromatin immunoprecipitation assay showed that Pgp can bind the pokemon prompter to repress pokemon transcription activity. Furthermore, Pgp regulated pokemon transcription activity through expression of p53 as seen by use of p53 siRNA transfected MCF-7 cells or p53 mutated MDA-MB-231 cells. Moreover, p53 was detected to bind with Pgp in vivo using immunoprecipitation assay. Taken together, we conclude that Pgp can regulate the expression of pokemon through the presence of p53, suggesting that Pgp is a potent regulator and may offer an effective novel target for cancer therapy. PMID:20957096

  4. P-Glycoprotein/MDR1 Regulates Pokemon Gene Transcription Through p53 Expression in Human Breast Cancer Cells

    PubMed Central

    He, Shengnan; Liu, Feng; Xie, Zhenhua; Zu, Xuyu; Xu, Wei; Jiang, Yuyang

    2010-01-01

    P-glycoprotein (Pgp), encoded by the multidrug resistance 1 (MDR1) gene, is an efflux transporter and plays an important role in pharmacokinetics. In this study, we demonstrated that the pokemon promoter activity, the pokemon mRNA and protein expression can be significantly inhibited by Pgp. Chromatin immunoprecipitation assay showed that Pgp can bind the pokemon prompter to repress pokemon transcription activity. Furthermore, Pgp regulated pokemon transcription activity through expression of p53 as seen by use of p53 siRNA transfected MCF-7 cells or p53 mutated MDA-MB-231 cells. Moreover, p53 was detected to bind with Pgp in vivo using immunoprecipitation assay. Taken together, we conclude that Pgp can regulate the expression of pokemon through the presence of p53, suggesting that Pgp is a potent regulator and may offer an effective novel target for cancer therapy. PMID:20957096

  5. Identification of Cell Wall Synthesis Regulatory Genes Controlling Biomass Characteristics and Yield in Rice (Oryza Sativa)

    SciTech Connect

    Peng, Zhaohua PEng; Ronald, Palmela; Wang, Guo-Liang

    2013-04-26

    This project aims to identify the regulatory genes of rice cell wall synthesis pathways using a cell wall removal and regeneration system. We completed the gene expression profiling studies following the time course from cell wall removal to cell wall regeneration in rice suspension cells. We also completed, total proteome, nuclear subproteome and histone modification studies following the course from cell wall removal and cell wall regeneration process. A large number of differentially expressed regulatory genes and proteins were identified. Meanwhile, we generated RNAi and over-expression transgenic rice for 45 genes with at least 10 independent transgenic lines for each gene. In addition, we ordered T-DNA and transposon insertion mutants for 60 genes from Korea, Japan, and France and characterized the mutants. Overall, we have mutants and transgenic lines for over 90 genes, exceeded our proposed goal of generating mutants for 50 genes. Interesting Discoveries a) Cell wall re-synthesis in protoplasts may involve a novel cell wall synthesis mechanism. The synthesis of the primary cell wall is initiated in late cytokinesis with further modification during cell expansion. Phragmoplast plays an essential role in cell wall synthesis. It services as a scaffold for building the cell plate and formation of a new cell wall. Only one phragmoplast and one new cell wall is produced for each dividing cell. When the cell wall was removed enzymatically, we found that cell wall re-synthesis started from multiple locations simultaneously, suggesting that a novel mechanism is involved in cell wall re-synthesis. This observation raised many interesting questions, such as how the starting sites of cell wall synthesis are determined, whether phragmoplast and cell plate like structures are involved in cell wall re-synthesis, and more importantly whether the same set of enzymes and apparatus are used in cell wall re-synthesis as during cytokinesis. Given that many known cell wall

  6. Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures.

    PubMed

    Mélida, Hugo; Largo-Gosens, Asier; Novo-Uzal, Esther; Santiago, Rogelio; Pomar, Federico; García, Pedro; García-Angulo, Penélope; Acebes, José Luis; Álvarez, Jesús; Encina, Antonio

    2015-04-01

    Maize (Zea mays L.) suspension-cultured cells with up to 70% less cellulose were obtained by stepwise habituation to dichlobenil (DCB), a cellulose biosynthesis inhibitor. Cellulose deficiency was accompanied by marked changes in cell wall matrix polysaccharides and phenolics as revealed by Fourier transform infrared (FTIR) spectroscopy. Cell wall compositional analysis indicated that the cellulose-deficient cell walls showed an enhancement of highly branched and cross-linked arabinoxylans, as well as an increased content in ferulic acid, diferulates and p-coumaric acid, and the presence of a polymer that stained positive for phloroglucinol. In accordance with this, cellulose-deficient cell walls showed a fivefold increase in Klason-type lignin. Thioacidolysis/GC-MS analysis of cellulose-deficient cell walls indicated the presence of a lignin-like polymer with a Syringyl/Guaiacyl ratio of 1.45, which differed from the sensu stricto stress-related lignin that arose in response to short-term DCB-treatments. Gene expression analysis of these cells indicated an overexpression of genes specific for the biosynthesis of monolignol units of lignin. A study of stress signaling pathways revealed an overexpression of some of the jasmonate signaling pathway genes, which might trigger ectopic lignification in response to cell wall integrity disruptions. In summary, the structural plasticity of primary cell walls is proven, since a lignification process is possible in response to cellulose impoverishment. PMID:25735403

  7. Viscoelastic properties of cell walls of single living plant cells determined by dynamic nanoindentation

    PubMed Central

    Hayot, Céline M.; Forouzesh, Elham; Goel, Ashwani; Avramova, Zoya; Turner, Joseph A.

    2012-01-01

    Plant development results from controlled cell divisions, structural modifications, and reorganizations of the cell wall. Thereby, regulation of cell wall behaviour takes place at multiple length scales involving compositional and architectural aspects in addition to various developmental and/or environmental factors. The physical properties of the primary wall are largely determined by the nature of the complex polymer network, which exhibits time-dependent behaviour representative of viscoelastic materials. Here, a dynamic nanoindentation technique is used to measure the time-dependent response and the viscoelastic behaviour of the cell wall in single living cells at a micron or sub-micron scale. With this approach, significant changes in storage (stiffness) and loss (loss of energy) moduli are captured among the tested cells. The results reveal hitherto unknown differences in the viscoelastic parameters of the walls of same-age similarly positioned cells of the Arabidopsis ecotypes (Col 0 and Ws 2). The technique is also shown to be sensitive enough to detect changes in cell wall properties in cells deficient in the activity of the chromatin modifier ATX1. Extensive computational modelling of the experimental measurements (i.e. modelling the cell as a viscoelastic pressure vessel) is used to analyse the influence of the wall thickness, as well as the turgor pressure, at the positions of our measurements. By combining the nanoDMA technique with finite element simulations quantifiable measurements of the viscoelastic properties of plant cell walls are achieved. Such techniques are expected to find broader applications in quantifying the influence of genetic, biological, and environmental factors on the nanoscale mechanical properties of the cell wall. PMID:22291130

  8. Viscoelastic properties of cell walls of single living plant cells determined by dynamic nanoindentation.

    PubMed

    Hayot, Céline M; Forouzesh, Elham; Goel, Ashwani; Avramova, Zoya; Turner, Joseph A

    2012-04-01

    Plant development results from controlled cell divisions, structural modifications, and reorganizations of the cell wall. Thereby, regulation of cell wall behaviour takes place at multiple length scales involving compositional and architectural aspects in addition to various developmental and/or environmental factors. The physical properties of the primary wall are largely determined by the nature of the complex polymer network, which exhibits time-dependent behaviour representative of viscoelastic materials. Here, a dynamic nanoindentation technique is used to measure the time-dependent response and the viscoelastic behaviour of the cell wall in single living cells at a micron or sub-micron scale. With this approach, significant changes in storage (stiffness) and loss (loss of energy) moduli are captured among the tested cells. The results reveal hitherto unknown differences in the viscoelastic parameters of the walls of same-age similarly positioned cells of the Arabidopsis ecotypes (Col 0 and Ws 2). The technique is also shown to be sensitive enough to detect changes in cell wall properties in cells deficient in the activity of the chromatin modifier ATX1. Extensive computational modelling of the experimental measurements (i.e. modelling the cell as a viscoelastic pressure vessel) is used to analyse the influence of the wall thickness, as well as the turgor pressure, at the positions of our measurements. By combining the nanoDMA technique with finite element simulations quantifiable measurements of the viscoelastic properties of plant cell walls are achieved. Such techniques are expected to find broader applications in quantifying the influence of genetic, biological, and environmental factors on the nanoscale mechanical properties of the cell wall. PMID:22291130

  9. Cell Wall-Associated Protein Antigens of Streptococcus salivarius: Purification, Properties, and Function in Adherence

    PubMed Central

    Weerkamp, Anton H.; Jacobs, Ton

    1982-01-01

    Three cell wall-associated protein antigens (antigens b, c, and d) were isolated from mutanolysin-solubilized cell walls of Streptococcus salivarius HB and purified to apparent homogeneity by a combination of ion-exchange chromatography, gel filtration, and immunoadsorption chromatography. Antigens b and c were also isolated from culture supernatants. Antigen b consisted of more than 80% protein and had an apparent molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 320,000. Antigen c consisted of 57% protein, about 30% neutral sugar, and about 13% amino sugar, and its glycoprotein nature was confirmed by specific staining techniques. During sodium dodecyl sulfate-polyacrylamide gel electrophoresis antigen c resolved into two or more bands, depending on the source or the isolation procedure, in the molecular weight range from 220,000 to 280,000. Antigen d consisted of 95% protein and was observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two bands with molecular weights of 129,000 and 121,000. Under nondenaturing conditions all three antigens had molecular weights in the range from 1 × 106 to 3 × 106 as determined by gel filtration. The amino acid compositions of antigens b, c, and d were characterized by low amounts of basic amino acids and relatively high levels of nonpolar amino acids. Among oral streptococcal species antigens b and c were virtually restricted to strains of S. salivarius and most often to serotype I strains. Antigen b was recognized as the factor that mediates coaggregation of S. salivarius with Veillonella strains. The purified protein retained its biological activity. Antigen c could be linked to functions relating to adhesion of the streptococci to host tissues on the basis of its absence in mutant strains and blocking by specific antisera. The purified molecule had no detectable biological activity. Antigen d could not be linked to an established adhesion function. Images

  10. Establishment and characterization of an MDCK cell line stably-transfected with chicken Abcb1 encoding P-glycoprotein.

    PubMed

    Sun, Yong; Guo, Tingting; Guo, Dawei; Guo, Li; Chen, Li; Zhang, Yu; Wang, Liping

    2016-06-01

    Chicken P-glycoprotein (chP-gp), encoded by Abcb1, determines the bioavailability because of its effect on pharmacokinetics of various drugs. However, comprehensive studies on chP-gp are still limited. In this study, the chicken full-length cDNA was first successfully cloned and then stably expressed in MDCK cell line. The open reading frame of chicken Abcb1 consists of 3864 nucleotides, encoding for a 1287-amino acid protein. Sequence alignments analysis showed that chicken P-gp had high identities with the homologues of turkey (95%), human (72%), pig (72%), rat (71%) and cattle (68%). The efflux ratio of rhodamine123 (Rho123, a human P-gp substrate) in chAbcb1 transfected MDCK cells was significantly higher than that in the wild type MDCK cell (6.24 vs 1.64, P<0.05), suggesting a good transporting function of chicken P-gp overexpressed in the transfected cell. Importantly, MDCK-chAbcb1 cells, unlike Caco-2 cells, exhibited biphasic saturation kinetics in transporting Rho123. In conclusion, an MDCK cell line stably expressing chAbcb1 was successfully established, which could provide a new cell model to screen its substrates and inhibitors and study the drug-drug interaction medicated via chicken P-gp. PMID:27234533

  11. (Rapid regulatory control of plant cell expansion and wall relaxation)

    SciTech Connect

    Cosgrove, D.J.

    1990-01-01

    This section presents a brief overview of accomplishments related to this project in the past 3-year period. Our work has focused on the basic mechanisms of plant cell expansion, particularly on the interrelations of water and solute transport with cell wall relaxation and expansion. To study these processes, we have developed new methods and used these methods to analyze the dynamic behavior of growth processes and to examine how various agents (GA, drought, light, genetic lesions) alter the growth machinery of the cell.

  12. Orbital wall infarction in child with sickle cell disease.

    PubMed

    Janssens, C; Claeys, L; Maes, P; Boiy, T; Wojciechowski, M

    2015-12-01

    We present the case of a 17-year-old boy, known with homozygous sickle cell disease, who was admitted because of generalised pain. He developed bilateral periorbital oedema and proptosis, without pain or visual disturbances. In addition to hyperhydration, oxygen and analgesia IV antibiotics were started, to cover a possible osteomyelitis. Patients with sickle cell disease are at risk for vaso-occlusive crises, when the abnormally shaped red blood cells aggregate and block the capillaries. Such a crisis typically presents at a location with high bone marrow activity, as the vertebrae and long bones. At an early age, the bone marrow is still active at other sites, for example the orbital wall, and thus infarction can also occur there. Thus, in young persons with sickle cell disease, it is important to consider orbital wall infarction in the differential diagnosis, since the approach is different from osteomyelitis. If the disease is complicated by an orbital compression syndrome, corticosteroids or surgical intervention may be necessary to preserve the vision. In our patient, an MRI of the orbitae demonstrated periorbital oedema with bone anomalies in the orbital and frontal bones, confirming orbital wall infarction. Ophthalmological examination revealed no signs of pressure on the nervus opticus. The patient recovered gradually with conservative treatment. PMID:26790559

  13. Herpes simplex virus (HSV)-specific proliferative and cytotoxic T-cell responses in humans immunized with an HSV type 2 glycoprotein subunit vaccine.

    PubMed Central

    Zarling, J M; Moran, P A; Brewer, L; Ashley, R; Corey, L

    1988-01-01

    Studies were undertaken to determine whether immunization of humans with a herpes simplex virus type 2 (HSV-2) glycoprotein-subunit vaccine would result in the priming of both HSV-specific proliferating cells and cytotoxic T cells. Peripheral blood lymphocytes (PBL) from all eight vaccines studied responded by proliferating after stimulation with HSV-2, HSV-1, and glycoprotein gB-1. The PBL of five of these eight vaccines proliferated following stimulation with gD-2, whereas stimulation with gD-1 resulted in relatively low or no proliferative responses. T-cell clones were generated from HSV-2-stimulated PBL of three vaccinees who demonstrated strong proliferative responses to HSV-1 and HSV-2. Of 12 clones studied in lymphoproliferative assays, 9 were found to be cross-reactive for HSV-1 and HSV-2. Of the approximately 90 T-cell clones isolated, 14 demonstrated HSV-specific cytotoxic activity. Radioimmunoprecipitation-polyacrylamide gel electrophoresis analyses confirmed that the vaccinees had antibodies only to HSV glycoproteins, not to proteins which are absent in the subunit vaccine, indicating that these vaccinees had not become infected with HSV. Immunization of humans with an HSV-2 glycoprotein-subunit vaccine thus results in the priming of T cells that proliferate in response to stimulation with HSV and its glycoproteins and T cells that have cytotoxic activity against HSV-infected cells. Such HSV-specific memory T cells were detected as late as 2 years following the last boost with the subunit vaccine. Images PMID:2846864

  14. Generation of a drug resistance profile by quantitation of mdr-1/P-glycoprotein in the cell lines of the National Cancer Institute Anticancer Drug Screen.

    PubMed Central

    Alvarez, M; Paull, K; Monks, A; Hose, C; Lee, J S; Weinstein, J; Grever, M; Bates, S; Fojo, T

    1995-01-01

    Identifying new chemotherapeutic agents and characterizing mechanisms of resistance may improve cancer treatment. The Anticancer Drug Screen of the National Cancer Institute uses 60 cell lines to identify new agents. Expression of mdr-1/P-glycoprotein was measured by quantitative PCR. Expression was detected in 39 cell lines; the highest levels were in renal and colon carcinomas. Expression was also detected in all melanomas and central nervous system tumors, but in only one ovarian carcinoma and one leukemia cell line. Using a modified version of the COMPARE program, a high correlation was found between expression of mdr-1 and cellular resistance to a large number of compounds. Evidence that these compounds are P-glycoprotein substrates includes: (a) enhancement of cytotoxicity by verapamil; (b) demonstration of cross-resistance in a multidrug-resistant cell line, (c) ability to antagonize P-glycoprotein, increasing vinblastine accumulation by decreasing efflux; and (d) inhibition of photoaffinity labeling by azidopine. Identification of many heretofore unrecognized compounds as substrates indicates that P-glycoprotein has a broader substrate specificity than previously recognized. This study confirms the validity of this novel approach and provides the basis for similar studies examining a diverse group of gene products, including other resistance mechanisms, putative drug targets, and genes involved in the cell cycle and apoptosis. Images PMID:7738186

  15. Consequences of cell-to-cell P-glycoprotein transfer on acquired multidrug resistance in breast cancer: a cell population dynamics model

    PubMed Central

    2011-01-01

    Background Cancer is a proliferation disease affecting a genetically unstable cell population, in which molecular alterations can be somatically inherited by genetic, epigenetic or extragenetic transmission processes, leading to a cooperation of neoplastic cells within tumoural tissue. The efflux protein P-glycoprotein (P-gp) is overexpressed in many cancer cells and has known capacity to confer multidrug resistance to cytotoxic therapies. Recently, cell-to-cell P-gp transfers have been shown. Herein, we combine experimental evidence and a mathematical model to examine the consequences of an intercellular P-gp trafficking in the extragenetic transfer of multidrug resistance from resistant to sensitive cell subpopulations. Methodology and Principal Findings We report cell-to-cell transfers of functional P-gp in co-cultures of a P-gp overexpressing human breast cancer MCF-7 cell variant, selected for its resistance towards doxorubicin, with the parental sensitive cell line. We found that P-gp as well as efflux activity distribution are progressively reorganized over time in co-cultures analyzed by flow cytometry. A mathematical model based on a Boltzmann type integro-partial differential equation structured by a continuum variable corresponding to P-gp activity describes the cell populations in co-culture. The mathematical model elucidates the population elements in the experimental data, specifically, the initial proportions, the proliferative growth rates, and the transfer rates of P-gp in the sensitive and resistant subpopulations. Conclusions We confirmed cell-to-cell transfer of functional P-gp. The transfer process depends on the gradient of P-gp expression in the donor-recipient cell interactions, as they evolve over time. Extragenetically acquired drug resistance is an additional aptitude of neoplastic cells which has implications in the diagnostic value of P-gp expression and in the design of chemotherapy regimens. Reviewers This article was reviewed by

  16. Accumulation of Autophagosomes in Semliki Forest Virus-Infected Cells Is Dependent on Expression of the Viral Glycoproteins

    PubMed Central

    Eng, Kai Er; Panas, Marc D.; Murphy, Deirdre; Karlsson Hedestam, Gunilla B.

    2012-01-01

    Autophagy is a cellular process that sequesters cargo in double-membraned vesicles termed autophagosomes and delivers this cargo to lysosomes to be degraded. It is enhanced during nutrient starvation to increase the rate of amino acid turnover. Diverse roles for autophagy have been reported for viral infections, including the assembly of viral replication complexes on autophagic membranes and protection of host cells from cell death. Here, we show that autophagosomes accumulate in Semliki Forest virus (SFV)-infected cells. Despite this, disruption of autophagy had no effect on the viral replication rate or formation of viral replication complexes. Also, viral proteins rarely colocalized with autophagosome markers, suggesting that SFV did not utilize autophagic membranes for its replication. Further, we found that SFV infection, unlike nutrient starvation, did not inactivate the constitutive negative regulator of autophagosome formation, mammalian target of rapamycin, suggesting that SFV-dependent accumulation of autophagosomes was not a result of enhanced autophagosome formation. In starved cells, addition of NH4Cl, an inhibitor of lysosomal acidification, caused a dramatic accumulation of starvation-induced autophagosomes, while in SFV-infected cells, NH4Cl did not further increase levels of autophagosomes. These results suggest that accumulation of autophagosomes in SFV-infected cells is due to an inhibition of autophagosome degradation rather than enhanced rates of autophagosome formation. Finally, we show that the accumulation of autophagosomes in SFV-infected cells is dependent on the expression of the viral glycoprotein spike complex. PMID:22438538

  17. Dynamics of cell wall elasticity pattern shapes the cell during yeast mating morphogenesis.

    PubMed

    Goldenbogen, Björn; Giese, Wolfgang; Hemmen, Marie; Uhlendorf, Jannis; Herrmann, Andreas; Klipp, Edda

    2016-09-01

    The cell wall defines cell shape and maintains integrity of fungi and plants. When exposed to mating pheromone, Saccharomyces cerevisiae grows a mating projection and alters in morphology from spherical to shmoo form. Although structural and compositional alterations of the cell wall accompany shape transitions, their impact on cell wall elasticity is unknown. In a combined theoretical and experimental approach using finite-element modelling and atomic force microscopy (AFM), we investigated the influence of spatially and temporally varying material properties on mating morphogenesis. Time-resolved elasticity maps of shmooing yeast acquired with AFM in vivo revealed distinct patterns, with soft material at the emerging mating projection and stiff material at the tip. The observed cell wall softening in the protrusion region is necessary for the formation of the characteristic shmoo shape, and results in wider and longer mating projections. The approach is generally applicable to tip-growing fungi and plants cells. PMID:27605377

  18. Hydrolyzable Tannins (Chebulagic Acid and Punicalagin) Target Viral Glycoprotein-Glycosaminoglycan Interactions To Inhibit Herpes Simplex Virus 1 Entry and Cell-to-Cell Spread▿

    PubMed Central

    Lin, Liang-Tzung; Chen, Ting-Ying; Chung, Chueh-Yao; Noyce, Ryan S.; Grindley, T. Bruce; McCormick, Craig; Lin, Ta-Chen; Wang, Guey-Horng; Lin, Chun-Ching; Richardson, Christopher D.

    2011-01-01

    Herpes simplex virus 1 (HSV-1) is a common human pathogen that causes lifelong latent infection of sensory neurons. Non-nucleoside inhibitors that can limit HSV-1 recurrence are particularly useful in treating immunocompromised individuals or cases of emerging acyclovir-resistant strains of herpesvirus. We report that chebulagic acid (CHLA) and punicalagin (PUG), two hydrolyzable tannins isolated from the dried fruits of Terminalia chebula Retz. (Combretaceae), inhibit HSV-1 entry at noncytotoxic doses in A549 human lung cells. Experiments revealed that both tannins targeted and inactivated HSV-1 viral particles and could prevent binding, penetration, and cell-to-cell spread, as well as secondary infection. The antiviral effect from either of the tannins was not associated with induction of type I interferon-mediated responses, nor was pretreatment of the host cell protective against HSV-1. Their inhibitory activities targeted HSV-1 glycoproteins since both natural compounds were able to block polykaryocyte formation mediated by expression of recombinant viral glycoproteins involved in attachment and membrane fusion. Our results indicated that CHLA and PUG blocked interactions between cell surface glycosaminoglycans and HSV-1 glycoproteins. Furthermore, the antiviral activities from the two tannins were significantly diminished in mutant cell lines unable to produce heparan sulfate and chondroitin sulfate and could be rescued upon reconstitution of heparan sulfate biosynthesis. We suggest that the hydrolyzable tannins CHLA and PUG may be useful as competitors for glycosaminoglycans in the management of HSV-1 infections and that they may help reduce the risk for development of viral drug resistance during therapy with nucleoside analogues. PMID:21307190

  19. Effect of Yeast Cell Morphology, Cell Wall Physical Structure and Chemical Composition on Patulin Adsorption.

    PubMed

    Luo, Ying; Wang, Jianguo; Liu, Bin; Wang, Zhouli; Yuan, Yahong; Yue, Tianli

    2015-01-01

    The capability of yeast to adsorb patulin in fruit juice can aid in substantially reducing the patulin toxic effect on human health. This study aimed to investigate the capability of yeast cell morphology and cell wall internal structure and composition to adsorb patulin. To compare different yeast cell morphologies, cell wall internal structure and composition, scanning electron microscope, transmission electron microscope and ion chromatography were used. The results indicated that patulin adsorption capability of yeast was influenced by cell surface areas, volume, and cell wall thickness, as well as 1,3-β-glucan content. Among these factors, cell wall thickness and 1,3-β-glucan content serve significant functions. The investigation revealed that patulin adsorption capability was mainly affected by the three-dimensional network structure of the cell wall composed of 1,3-β-glucan. Finally, patulin adsorption in commercial kiwi fruit juice was investigated, and the results indicated that yeast cells could adsorb patulin from commercial kiwi fruit juice efficiently. This study can potentially simulate in vitro cell walls to enhance patulin adsorption capability and successfully apply to fruit juice industry. PMID:26295574

  20. Cell wall bound anionic peroxidases from asparagus byproducts.

    PubMed

    Jaramillo-Carmona, Sara; López, Sergio; Vazquez-Castilla, Sara; Jimenez-Araujo, Ana; Rodriguez-Arcos, Rocio; Guillen-Bejarano, Rafael

    2014-10-01

    Asparagus byproducts are a good source of cationic soluble peroxidases (CAP) useful for the bioremediation of phenol-contaminated wastewaters. In this study, cell wall bound peroxidases (POD) from the same byproducts have been purified and characterized. The covalent forms of POD represent >90% of the total cell wall bound POD. Isoelectric focusing showed that whereas the covalent fraction is constituted primarily by anionic isoenzymes, the ionic fraction is a mixture of anionic, neutral, and cationic isoenzymes. Covalently bound peroxidases were purified by means of ion exchange chromatography and affinity chromatography. In vitro detoxification studies showed that although CAP are more effective for the removal of 4-CP and 2,4-DCP, anionic asparagus peroxidase (AAP) is a better option for the removal of hydroxytyrosol (HT), the main phenol present in olive mill wastewaters. PMID:25195693

  1. Compounds active against cell walls of medically important fungi.

    PubMed Central

    Hector, R F

    1993-01-01

    A number of substances that directly or indirectly affect the cell walls of fungi have been identified. Those that actively interfere with the synthesis or degradation of polysaccharide components share the property of being produced by soil microbes as secondary metabolites. Compounds specifically interfering with chitin or beta-glucan synthesis have proven effective in studies of preclinical models of mycoses, though they appear to have a restricted spectrum of coverage. Semisynthetic derivatives of some of the natural products have offered improvements in activity, toxicology, or pharmacokinetic behavior. Compounds which act on the cell wall indirectly or by a secondary mechanism of action, such as the azoles, act against diverse fungi but are usually fungistatic in nature. Overall, these compounds are attractive candidates for further development. PMID:8457977

  2. Human cytomegalovirus glycoprotein B contains autonomous determinants for vectorial targeting to apical membranes of polarized epithelial cells.

    PubMed

    Tugizov, S; Maidji, E; Xiao, J; Zheng, Z; Pereira, L

    1998-09-01

    We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is vectorially transported to apical membranes of CMV-infected polarized human retinal pigment epithelial cells propagated on permeable filter supports and that virions egress predominantly from the apical membrane domain. In the present study, we investigated whether gB itself contains autonomous information for apical transport by expressing the molecule in stably transfected Madine-Darby canine kidney (MDCK) cells grown on permeable filter supports. Laser scanning confocal immunofluorescence microscopy and domain-selective biotinylation of surface membrane domains showed that CMV gB was transported to apical membranes independently of other envelope glycoproteins and that it colocalized with proteins in transport vesicles of the biosynthetic and endocytic pathways. Determinants for trafficking to apical membranes were located by evaluating the targeting of gB derivatives with deletions in the lumen, transmembrane (TM) anchor, and carboxyl terminus. Derivative gB(Delta717-747), with an internal deletion in the luminal juxtamembrane sequence that preserved the N- and O-glycosylation sites, retained vectorial transport to apical membranes. In contrast, derivatives that lacked the TM anchor and cytosolic domain (gBDelta646-906) or the TM anchor alone (gBDelta751-771) underwent considerable basolateral targeting. Likewise, derivatives lacking the entire cytosolic domain (gBDelta772-906) or the last 73 amino acids (gBDelta834-906) showed disrupted apical transport. Site-specific mutations that deleted or altered the cluster of acidic residues with a casein kinase II phosphorylation site at the extreme carboxyl terminus, which can serve as an internalization signal, caused partial missorting of gB to basolateral membranes. Our studies indicate that CMV gB contains autonomous information for apical targeting in luminal, TM anchor, and cytosolic domain sequences, forming distinct structural

  3. Human Cytomegalovirus Glycoprotein B Contains Autonomous Determinants for Vectorial Targeting to Apical Membranes of Polarized Epithelial Cells

    PubMed Central

    Tugizov, Sharof; Maidji, Ekaterina; Xiao, Jianqiao; Zheng, Zhenwei; Pereira, Lenore

    1998-01-01

    We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is vectorially transported to apical membranes of CMV-infected polarized human retinal pigment epithelial cells propagated on permeable filter supports and that virions egress predominantly from the apical membrane domain. In the present study, we investigated whether gB itself contains autonomous information for apical transport by expressing the molecule in stably transfected Madine-Darby canine kidney (MDCK) cells grown on permeable filter supports. Laser scanning confocal immunofluorescence microscopy and domain-selective biotinylation of surface membrane domains showed that CMV gB was transported to apical membranes independently of other envelope glycoproteins and that it colocalized with proteins in transport vesicles of the biosynthetic and endocytic pathways. Determinants for trafficking to apical membranes were located by evaluating the targeting of gB derivatives with deletions in the lumen, transmembrane (TM) anchor, and carboxyl terminus. Derivative gB(Δ717-747), with an internal deletion in the luminal juxtamembrane sequence that preserved the N- and O-glycosylation sites, retained vectorial transport to apical membranes. In contrast, derivatives that lacked the TM anchor and cytosolic domain (gBΔ646-906) or the TM anchor alone (gBΔ751-771) underwent considerable basolateral targeting. Likewise, derivatives lacking the entire cytosolic domain (gBΔ772-906) or the last 73 amino acids (gBΔ834-906) showed disrupted apical transport. Site-specific mutations that deleted or altered the cluster of acidic residues with a casein kinase II phosphorylation site at the extreme carboxyl terminus, which can serve as an internalization signal, caused partial missorting of gB to basolateral membranes. Our studies indicate that CMV gB contains autonomous information for apical targeting in luminal, TM anchor, and cytosolic domain sequences, forming distinct structural elements that

  4. Phosphorylation of the multidrug resistance associated glycoprotein

    SciTech Connect

    Mellado, W.; Horwitz, S.B.

    1987-11-03

    Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistant phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl/sub 2/ was enhanced a minimum of 2-fold by 10 ..mu..M cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by (..gamma..-/sup 32/P)ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance.

  5. Phosphorylation of the multidrug resistance associated glycoprotein.

    PubMed

    Mellado, W; Horwitz, S B

    1987-11-01

    Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistance phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl2 was enhanced a minimum of 2-fold by 10 microM cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by [gamma-32P]ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance. PMID:3427052

  6. Chemical and in situ characterization of macromolecular components of the cell walls from the green seaweed Codium fragile.

    PubMed

    Estevez, José Manuel; Fernández, Paula Virginia; Kasulin, Luciana; Dupree, Paul; Ciancia, Marina

    2009-03-01

    A comprehensive analysis of the carbohydrate-containing macromolecules from the coencocytic green seaweed Codium fragile and their arrangement in the cell wall was carried out. Cell walls in this seaweed are highly complex structures composed of 31% (w/w) of linear (1-->4)-beta-D-mannans, 9% (w/w) of pyruvylated arabinogalactan sulfates (pAGS), and low amounts of hydroxyproline rich-glycoprotein epitopes (HRGP). In situ chemical imaging by synchrotron radiation Fourier transform infrared (SR-FTIR) microspectroscopy and by immunolabeling using antibodies against specific cell wall carbohydrate epitopes revealed that beta-d-mannans and pAGS are placed in the middle part of the cell wall, whereas HRGP epitopes (arabinogalactan proteins (AGPs) and extensins) are located on the wall boundaries, especially in the utricle apical zone. pAGS are sulfated at C-2 and/or C-4 of the 3-linked beta-L-arabinopyranose units and at C-4 and/or C-6 of the 3-linked beta-D-galactopyranose residues. In addition, high levels of ketals of pyruvic acid were found mainly at 3,4- of some terminal beta-D-Galp units forming a five-membered ring. Ramification was found at some C-6 of the 3-linked beta-D-Galp units. In agreement with the immunolabeled AGP epitopes, a nonsulfated branched furanosidic arabinan with 5-linked alpha-L-Araf, 3,5-linked alpha-L-Araf, and terminal alpha-L-Araf units and a nonsulfated galactan structure composed of 3-(3,6)-linked beta-D-Galp residues, both typical of type-II AG glycans were found, suggesting that AGP structures are present at low levels in the cell walls of this seaweed. Based on this study, it is starting to emerge that Codium has developed unique cell wall architecture, when compared, not only with that of vascular plants, but also with other related green seaweeds and algae. PMID:18832454

  7. Inhibition of fucosylation of cell wall components by 2-fluoro 2-deoxy-L-fucose induces defects in root cell elongation.

    PubMed

    Dumont, Marie; Lehner, Arnaud; Bardor, Muriel; Burel, Carole; Vauzeilles, Boris; Lerouxel, Olivier; Anderson, Charles T; Mollet, Jean-Claude; Lerouge, Patrice

    2015-12-01

    Screening of commercially available fluoro monosaccharides as putative growth inhibitors in Arabidopsis thaliana revealed that 2-fluoro 2-l-fucose (2F-Fuc) reduces root growth at micromolar concentrations. The inability of 2F-Fuc to affect an Atfkgp mutant that is defective in the fucose salvage pathway indicates that 2F-Fuc must be converted to its cognate GDP nucleotide sugar in order to inhibit root growth. Chemical analysis of cell wall polysaccharides and glycoproteins demonstrated that fucosylation of xyloglucans and of N-linked glycans is fully inhibited by 10 μm 2F-Fuc in Arabidopsis seedling roots, but genetic evidence indicates that these alterations are not responsible for the inhibition of root development by 2F-Fuc. Inhibition of fucosylation of cell wall polysaccharides also affected pectic rhamnogalacturonan-II (RG-II). At low concentrations, 2F-Fuc induced a decrease in RG-II dimerization. Both RG-II dimerization and root growth were partially restored in 2F-Fuc-treated seedlings by addition of boric acid, suggesting that the growth phenotype caused by 2F-Fuc was due to a deficiency of RG-II dimerization. Closer investigation of the 2F-Fuc-induced growth phenotype demonstrated that cell division is not affected by 2F-Fuc treatments. In contrast, the inhibitor suppressed elongation of root cells and promoted the emergence of adventitious roots. This study further emphasizes the importance of RG-II in cell elongation and the utility of glycosyltransferase inhibitors as new tools for studying the functions of cell wall polysaccharides in plant development. Moreover, supplementation experiments with borate suggest that the function of boron in plants might not be restricted to RG-II cross-linking, but that it might also be a signal molecule in the cell wall integrity-sensing mechanism. PMID:26565655

  8. Enzyme Amplified Detection of Microbial Cell Wall Components

    NASA Technical Reports Server (NTRS)

    Wainwright, Norman R.

    2004-01-01

    This proposal is MBL's portion of NASA's Johnson Space Center's Astrobiology Center led by Principal Investigator, Dr. David McKay, entitled: 'Institute for the Study of Biomarkers in Astromaterials.' Dr. Norman Wainwright is the principal investigator at MBL and is responsible for developing methods to detect trace quantities of microbial cell wall chemicals using the enzyme amplification system of Limulus polyphemus and other related methods.

  9. Bacterial Cell Wall Polymer-Induced Granulomatous Inflammation

    PubMed

    Sartor; Herfarth; Van Tol EAF

    1996-04-01

    Local or systemic injection of peptidoglycan-polysaccharide polymers, which are the primary structural components of cell walls of nearly all bacteria, leads to acute inflammation, which can develop into chronic, spontaneously relapsing, granulomatous inflammation in a number of organs. Evolution into chronic granulomatous inflammation is dependent upon persistence of poorly biodegradable cell wall polymers within tissues, genetically determined host susceptibility, and generation of a T-lymphocyte-mediated immune response. Intraperitoneal injection of peptidoglycan-polysaccharide fragments from group A streptococci or selected intestinal bacteria into susceptible Lewis rats leads to chronic, spontaneously reactivating erosive arthritis and hepatic granulomas. Subserosal (intramural) injection of poorly biodegradable cell wall fragments into the distal intestine of Lewis rats induces chronic, spontaneously relapsing granulomatous enterocolitis with associated arthritis, hepatic granulomas, anemia, and leukocytosis. Chronic inflammation does not occur in T-lymphocyte-deficient rats and is prevented by cyclosporin-A therapy and degradation of peptidoglycan by the muralytic enzyme, mutanolysin. Moreover, resistant Buffalo and Fischer F344 rats, the latter sharing identical MHC antigens with Lewis rats, develop only acute inflammation with no chronic granulomatous response. Peptidoglycan-polysaccharide polymers activate almost every limb of the inflammatory response. Blockade of specific pathways suggests that interleukin-1, transforming growth factor-beta, plasma kallikrein, and T lymphocytes are dominant mediators of peptidoglycan-polysaccharide-induced arthritis, hepatic granulomas, and enterocolitis. Because of the similarity of immune mechanisms of these rat models to human disease, bacterial cell wall-induced inflammation provides unique opportunities to study pathogenic mechanisms of granuloma formation in response to ubiquitous microbial agents and to test

  10. Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli.

    PubMed

    Cho, Won Kyong; Hyun, Tae Kyung; Kumar, Dhinesh; Rim, Yeonggil; Chen, Xiong Yan; Jo, Yeonhwa; Kim, Suwha; Lee, Keun Woo; Park, Zee-Yong; Lucas, William J; Kim, Jae-Yean

    2015-08-01

    Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Based on bioinformatics analyses, 389 classical rice cell wall proteins, possessing a signal peptide, and 334 putative non-classical cell wall proteins, lacking a signal peptide, were identified. By combining previously established rice cell wall protein databases with current data for the classical rice cell wall proteins, a comprehensive rice cell wall proteome, comprised of 496 proteins, was constructed. A comparative analysis of the rice and Arabidopsis cell wall proteomes revealed a high level of homology, suggesting a predominant conservation between monocot and eudicot cell wall proteins. This study importantly increased information on cell wall proteins, which serves for future functional analyses of these identified rice cell wall proteins. PMID:26194822

  11. Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli

    PubMed Central

    Cho, Won Kyong; Hyun, Tae Kyung; Kumar, Dhinesh; Rim, Yeonggil; Chen, Xiong Yan; Jo, Yeonhwa; Kim, Suwha; Lee, Keun Woo; Park, Zee-Yong; Lucas, William J.; Kim, Jae-Yean

    2015-01-01

    Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Based on bioinformatics analyses, 389 classical rice cell wall proteins, possessing a signal peptide, and 334 putative non-classical cell wall proteins, lacking a signal peptide, were identified. By combining previously established rice cell wall protein databases with current data for the classical rice cell wall proteins, a comprehensive rice cell wall proteome, comprised of 496 proteins, was constructed. A comparative analysis of the rice and Arabidopsis cell wall proteomes revealed a high level of homology, suggesting a predominant conservation between monocot and eudicot cell wall proteins. This study importantly increased information on cell wall proteins, which serves for future functional analyses of these identified rice cell wall proteins. PMID:26194822

  12. Aspergillus Enzymes Involved in Degradation of Plant Cell Wall Polysaccharides

    PubMed Central

    de Vries, Ronald P.; Visser, Jaap

    2001-01-01

    Degradation of plant cell wall polysaccharides is of major importance in the food and feed, beverage, textile, and paper and pulp industries, as well as in several other industrial production processes. Enzymatic degradation of these polymers has received attention for many years and is becoming a more and more attractive alternative to chemical and mechanical processes. Over the past 15 years, much progress has been made in elucidating the structural characteristics of these polysaccharides and in characterizing the enzymes involved in their degradation and the genes of biotechnologically relevant microorganisms encoding these enzymes. The members of the fungal genus Aspergillus are commonly used for the production of polysaccharide-degrading enzymes. This genus produces a wide spectrum of cell wall-degrading enzymes, allowing not only complete degradation of the polysaccharides but also tailored modifications by using specific enzymes purified from these fungi. This review summarizes our current knowledge of the cell wall polysaccharide-degrading enzymes from aspergilli and the genes by which they are encoded. PMID:11729262

  13. Phosphate-containing cell wall polymers of bacilli.

    PubMed

    Potekhina, N V; Streshinskaya, G M; Tul'skaya, E M; Kozlova, Yu I; Senchenkova, S N; Shashkov, A S

    2011-07-01

    Anionic phosphate-containing cell wall polymers of bacilli are represented by teichoic acids and poly(glycosyl 1-phosphates). Different locations of phosphodiester bonds in the main chain of teichoic acids as well as the nature and combination of the constituent structural elements underlie their structural diversity. Currently, the structures of teichoic acids of bacilli can be classified into three types, viz. poly(polyol phosphates) with glycerol or ribitol as the polyol; poly(glycosylpolyol phosphates), mainly glycerol-containing polymers; and poly(acylglycosylglycerol phosphate), in which the components are covalently linked through glycosidic, phosphodiester, and amide bonds. In addition to teichoic acids, poly(glycosyl 1-phosphates) with mono- and disaccharide residues in the repeating units have been detected in cell walls of several Bacillus subtilis and Bacillus pumilus strains. The known structures of teichoic acids and poly(glycosyl 1-phosphates) of B. subtilis, B. atrophaeus, B. licheniformis, B. pumilus, B. stearothermophilus, B. coagulans, B. cereus as well as oligomers that link the polymers to peptidoglycan are surveyed. The reported data on the structures of phosphate-containing polymers of different strains of B. subtilis suggest heterogeneity of the species and may be of interest for the taxonomy of bacilli to allow differentiation of closely related organisms according to the "structures and composition of cell wall polymers" criterion. PMID:21999535

  14. A mannose receptor mediates mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth muscle cells.

    PubMed Central

    Lew, D B; Songu-Mize, E; Pontow, S E; Stahl, P D; Rattazzi, M C

    1994-01-01

    The putative mannose receptor (MR), previously implicated in mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth muscle (ASM) cells, was studied to determine its properties. Specific binding of the mitogenic neoglycoprotein, mannosylated bovine serum albumin (Man-BSA) to ASM cells was saturable, with an apparent Kd = 5.0 x 10(-8) M. Cell-bound ManBSA-colloidal gold conjugate was localized by electron microscopy to clathrin-coated pits on the cell surface, and was found to undergo internalization to endosomes; this was inhibitable by weak bases and swainsonine, that also inhibited ligand-induced mitogenesis. The ASM-MR, isolated by mannose-affinity chromatography, had the same apparent molecular mass as the macrophage (Mø) MR (M(r) = 175 kD), and was immunoprecipitated by an anti-MøMR immune serum. This antiserum blocked 125I-labeled-ManBSA binding to intact ASM cells, stimulated mitogenesis, and immunolocalized the ASM-MR in cytoplasmic vesicles compatible with endosomes. A monoclonal antibody directed against the MøMR also reacted with the ASM-MR; like the polyclonal antibodies, it stimulated mitogenesis as effectively as beta-hexosaminidases. These data indicate that the ASM-MR shares a number of functional and structural properties with the MøMR and suggest that similar receptors may have different main functions in different cells. Images PMID:7962531

  15. The Nectin-1α Transmembrane Domain, But Not The Cytoplasmic Tail, Influences Cell Fusion Induced by HSV-1 Glycoproteins

    PubMed Central

    Subramanian, Ravi P.; Dunn, Jennifer E.; Geraghty, Robert J.

    2006-01-01

    Nectin-1 is a receptor for herpes simplex virus (HSV), a member of the immunoglobulin superfamily, and a cellular adhesion molecule. To study domains of nectin-1α involved in cell fusion, we measured the ability of nectin-1α/nectin-2α chimeras, nectin-1α/CD4 chimeras, and transmembrane domain and cytoplasmic tail mutants of nectin-1α to promote cell fusion induced by HSV-1 glycoproteins. Our results demonstrate that only chimeras and mutants containing the entire V-like domain and a link to the plasma membrane conferred cell-fusion activity. The transmembrane domain and cytoplasmic tail of nectin-1 were not required for any viral receptor or cell adhesion function tested. Cellular cytoplasmic factors that bind to the nectin-1α cytoplasmic tail, therefore, did not influence virus entry or cell fusion. Interestingly, the efficiency of cell fusion was reduced when membrane spanning domains of nectin-1α and gD were replaced by glycosylphosphatidylinositol tethers, indicating that transmembrane domains may play a modulatory role in the gD/nectin-1α interaction in fusion. PMID:16005040

  16. The nectin-1{alpha} transmembrane domain, but not the cytoplasmic tail, influences cell fusion induced by HSV-1 glycoproteins

    SciTech Connect

    Subramanian, Ravi P.; Dunn, Jennifer E.; Geraghty, Robert J. . E-mail: rgeragh@uky.edu

    2005-09-01

    Nectin-1 is a receptor for herpes simplex virus (HSV), a member of the immunoglobulin superfamily, and a cellular adhesion molecule. To study domains of nectin-1{alpha} involved in cell fusion, we measured the ability of nectin-1{alpha}/nectin-2{alpha} chimeras, nectin-1{alpha}/CD4 chimeras, and transmembrane domain and cytoplasmic tail mutants of nectin-1{alpha} to promote cell fusion induced by HSV-1 glycoproteins. Our results demonstrate that only chimeras and mutants containing the entire V-like domain and a link to the plasma membrane conferred cell-fusion activity. The transmembrane domain and cytoplasmic tail of nectin-1 were not required for any viral receptor or cell adhesion function tested. Cellular cytoplasmic factors that bind to the nectin-1{alpha} cytoplasmic tail, therefore, did not influence virus entry or cell fusion. Interestingly, the efficiency of cell fusion was reduced when membrane-spanning domains of nectin-1{alpha} and gD were replaced by glycosylphosphatidylinositol tethers, indicating that transmembrane domains may play a modulatory role in the gD/nectin-1{alpha} interaction in fusion.

  17. Modeling of thin, back-wall silicon solar cells

    NASA Technical Reports Server (NTRS)

    Baraona, C. R.

    1979-01-01

    The performance of silicon solar cells with p-n junctions on the nonilluminated surface (i.e., upside-down or back-wall cells) was calculated. These structures consisted of a uniformly shaped p-type substrate layer, a p(+)-type field layer on the front (illuminated) surface, and a shallow, n-type junction on the back (nonilluminated) surface. A four-layer solar cell model was used to calculate efficiency, open-circuit voltage, and short-circuit current. The effect on performance of p-layer thickness and resistivity was determined. The diffusion length was varied to simulate the effect of radiation damage. The results show that peak initial efficiencies greater than 15 percent are possible for cell thicknesses or 100 micrometers or less. After 10 years of radiation damage in geosynchronous orbit, thin (25 to 50 micrometers thick) cells made from 10 to 100 ohm cm material show the smallest decrease (approximately 10 percent) in performance.

  18. The Cellular Localization of Human Cytomegalovirus Glycoprotein Expression Greatly Influences the Frequency and Functional Phenotype of Specific CD4+ T Cell Responses

    PubMed Central

    Zuo, Jianmin; Ryan, Gordon B.; Begum, Jusnara; Moss, Paul A. H.

    2015-01-01

    CMV infection is a significant cause of morbidity and mortality in immunocompromised individuals, and the development of a vaccine is of high priority. Glycoprotein B (gB) is a leading vaccine candidate but the glycoprotein H (gH) pentameric complex is now recognized as the major target for neutralizing Abs. However, little is known about the T cell immune response against gH and glycoprotein L (gL) and this is likely to be an important attribute for vaccine immunogenicity. In this study, we examine and contrast the magnitude and phenotype of the T cell immune response against gB, gH, and gL within healthy donors. gB-specific CD4+ T cells were found in 95% of donors, and 29 epitopes were defined with gB-specific response sizes ranging from 0.02 to 2.88% of the CD4+ T cell pool. In contrast, only 20% of donors exhibited a T cell response against gH or gL. Additionally, gB-specific CD4+ T cells exhibited a more cytotoxic phenotype, with high levels of granzyme B expression. Glycoproteins were effectively presented following delivery to APCs but only gB-derived epitopes were presented following endogenous synthesis. gB expression was observed exclusively within vesicular structures colocalizing with HLA-DM whereas gH was distributed evenly throughout the cytoplasm. Grafting of the C-terminal domain from gB onto gH could not transfer this pattern of presentation. These results reveal that gB is a uniquely immunogenic CMV glycoprotein and this is likely to reflect its unique pattern of endogenous Ag presentation. Consideration may be required toward mechanisms that boost cellular immunity to gH and gL within future subunit vaccines. PMID:26363059

  19. Evaluating fundamental position-dependent differences in wood cell wall adhesion using nanoindentation

    PubMed Central

    Obersriebnig, Michael; Konnerth, Johannes; Gindl-Altmutter, Wolfgang

    2013-01-01

    Spruce wood specimens were bonded with one-component polyurethane (PUR) and urea-formaldehyde (UF) adhesive, respectively. The adhesion of the adhesives to the wood cell wall was evaluated at two different locations by means of a new micromechanical assay based on nanoindentation. One location tested corresponded to the interface between the adhesive and the natural inner cell wall surface of the secondary cell wall layer 3 (S3), whereas the second location corresponded to the interface between the adhesive and the freshly cut secondary cell wall layer 2 (S2). Overall, a trend towards reduced cell wall adhesion was found for PUR compared to UF. Position-resolved examination revealed excellent adhesion of UF to freshly cut cell walls (S2) but significantly diminished adhesion to the inner cell wall surface (S3). In contrast, PUR showed better adhesion to the inner cell wall surface and less adhesion to freshly cut cell walls. Atomic force microscopy revealed a less polar character for the inner cell wall surface (S3) compared to freshly cut cell walls (S2). It is proposed that differences in the polarity of the used adhesives and the surface chemistry of the two cell wall surfaces examined account for the observed trends.

  20. Proteomic analysis of cell walls of two developmental stages of alfalfa stems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cell walls are important for the growth and development of all plants. They are also valuable resources for feed and fiber, and more recently as a potential feedstock for bioenergy production. Cell wall proteins comprise only a fraction of the cell wall, but play important roles in establishing the ...

  1. Stress analysis for wall structure in mobile hot cell design

    NASA Astrophysics Data System (ADS)

    Bahrin, Muhammad Hannan; Rahman, Anwar Abdul; Hamzah, Mohd Arif; Mamat, Mohd Rizal; Azman, Azraf; Hasan, Hasni

    2016-01-01

    Malaysian Nuclear Agency is developing a Mobile Hot Cell (MHC) in order to handle and manage Spent High Activity Radioactive Sources (SHARS) such as teletherapy heads and irradiators. At present, there are only two units of MHC in the world, in South Africa and China. Malaysian Mobile Hot cell is developed by Malaysian Nuclear Agency with the assistance of IAEA expert, based on the design of South Africa and China, but with improved features. Stress analysis has been performed on the design in order to fulfil the safety requirement in operation of MHC. This paper discusses the loading analysis effect from the sand to the MHC wall structure.

  2. Endothelial cell injury in vitro is associated with increased secretion of an Mr 43,000 glycoprotein ligand.

    PubMed

    Sage, H; Tupper, J; Bramson, R

    1986-06-01

    A novel, serum albumin-binding glycoprotein of molecular weight (mw) 43,000 (43K protein) was initially purified from the culture medium of bovine aortic endothelial (BAE) cells (Sage, H., Johnson, C., and Bornstein, P., J. Biol. Chem. 259:3993-4007, 1984). Its secretion by normal mesenchymal cells and by transformed cells of both ectodermal and endodermal origin suggested a general role in cellular function. To examine the effect of sublethal injury in vitro on the biosynthesis of 43K protein, BAE cells were exposed to endotoxin. At concentrations which produced minimal cell detachment and lysis, the cells secreted 70-100% more protein compared to control cultures, and the relative increase in 43K protein over total protein was approximately three-fold. A second type of cellular injury, manifested by rapid cellular proliferation and migration in response to sparse plating density (a condition that we have termed 'culture shock'), was also accompanied by a significant increase in the secretion of 43K protein. Pulse-chase studies revealed that the initial product secreted within 1.5 h was of Mr 38,000, and that between 6 and 21 h this molecule was converted to the final form of Mr 43,000. The 43K protein was not associated with RNA or glycosaminoglycan, but appeared to be linked to complex oligosaccharides containing peripheral sialosyl residues. Treatment with tunicamycin produced lower mw forms that displayed reduced affinity for albumin. By immunologic criteria, peptide mapping, and amino acid analysis, the 43K protein was shown to be structurally distinct from several proteins of Mr 40,000-50,000 associated with endothelium or with serum, including tissue factor, a plasminogen anti-activator, and several apolipoproteins. In addition, the 43K protein was not present in the extracellular matrices of endothelial, fibroblastic, or smooth muscle cells, nor was it found in plasma, serum, platelet releasate, or alveolar lavage fluids. These studies identify a unique Mr

  3. Astragaloside IV reduces the expression level of P-glycoprotein in multidrug-resistant human hepatic cancer cell lines

    PubMed Central

    WANG, PEI-PEI; XU, DU-JUAN; HUANG, CAN; WANG, WEI-PING; XU, WEN-KE

    2014-01-01

    Astragaloside is a saponin widely used in traditional Chinese medicine and has been reported to be a potent multidrug resistance (MDR) reversal agent. The present study investigated the role of astragaloside IV (ASIV) in the regulation of P-glycoprotein (P-gp, encoded by the mdr1 gene) and its effect on the reversal of MDR. The activity of ASIV was evaluated using human hepatic cancer cells Bel-7402 and the corresponding 5-fluorouracil (5-FU) resistant cells Bel-7402/FU. ASIV (0.08 mg/ml) potentiated the cytotoxicity of 5-FU which was demonstrated using the MTT assay on Bel-7402/FU cells. ASIV reduced the expression of P-gp as was revealed by immunocytochemistry. Accumulation and efflux studies with the P-gp substrate, rhodamine 123 (Rh123), demonstrated that ASIV inhibited P-gp-mediated drug efflux. Furthermore, it was demonstrated that ASIV enhanced the drug accumulation of 5-FU using a high performance liquid chromatography (HPLC) assay for drug resistant cells. Furthermore, ASIV may downregulate the expression of P-gp, which was examined using western blot analysis and polymerase chain reaction. In conclusion, the results of the present study indicated that ASIV reverses the drug resistance of Bel-7402/FU cells by downregulating the expression of mdr1. ASIV may represent a potent modulator of P-gp-mediated MDR in hepatic cancer therapy. PMID:24676670

  4. Astragaloside Ⅳ reduces the expression level of P-glycoprotein in multidrug-resistant human hepatic cancer cell lines.

    PubMed

    Wang, Pei-Pei; Xu, Du-Juan; Huang, Can; Wang, Wei-Ping; Xu, Wen-Ke

    2014-06-01

    Astragaloside is a saponin widely used in traditional Chinese medicine and has been reported to be a potent multidrug resistance (MDR) reversal agent. The present study investigated the role of astragaloside Ⅳ (ASIV) in the regulation of P-glycoprotein (P-gp, encoded by the mdr1 gene) and its effect on the reversal of MDR. The activity of ASIV was evaluated using human hepatic cancer cells Bel-7402 and the corresponding 5-fluorouracil (5-FU) resistant cells Bel-7402/FU. ASIV (0.08 mg/ml) potentiated the cytotoxicity of 5-FU which was demonstrated using the MTT assay on Bel-7402/FU cells. ASIV reduced the expression of P-gp as was revealed by immunocytochemistry. Accumulation and efflux studies with the P-gp substrate, rhodamine 123 (Rh123), demonstrated that ASIV inhibited P-gp-mediated drug efflux. Furthermore, it was demonstrated that ASⅣ enhanced the drug accumulation of 5-FU using a high performance liquid chromatography (HPLC) assay for drug resistant cells. Furthermore, ASIV may downregulate the expression of P-gp, which was examined using western blot analysis and polymerase chain reaction. In conclusion, the results of the present study indicated that ASIV reverses the drug resistance of Bel-7402/FU cells by downregulating the expression of mdr1. ASIV may represent a potent modulator of P-gp-mediated MDR in hepatic cancer therapy. PMID:24676670

  5. Gangliosides are functional nerve cell ligands for myelin-associated glycoprotein (MAG), an inhibitor of nerve regeneration

    PubMed Central

    Vyas, Alka A.; Patel, Himatkumar V.; Fromholt, Susan E.; Heffer-Lauc, Marija; Vyas, Kavita A.; Dang, Jiyoung; Schachner, Melitta; Schnaar, Ronald L.

    2002-01-01

    Myelin-associated glycoprotein (MAG) binds to the nerve cell surface and inhibits nerve regeneration. The nerve cell surface ligand(s) for MAG are not established, although sialic acid-bearing glycans have been implicated. We identify the nerve cell surface gangliosides GD1a and GT1b as specific functional ligands for MAG-mediated inhibition of neurite outgrowth from primary rat cerebellar granule neurons. MAG-mediated neurite outgrowth inhibition is attenuated by (i) neuraminidase treatment of the neurons; (ii) blocking neuronal ganglioside biosynthesis; (iii) genetically modifying the terminal structures of nerve cell surface gangliosides; and (iv) adding highly specific IgG-class antiganglioside mAbs. Furthermore, neurite outgrowth inhibition is mimicked by highly multivalent clustering of GD1a or GT1b by using precomplexed antiganglioside Abs. These data implicate the nerve cell surface gangliosides GD1a and GT1b as functional MAG ligands and suggest that the first step in MAG inhibition is multivalent ganglioside clustering. PMID:12060784

  6. Temozolomide down-regulates P-glycoprotein in human blood-brain barrier cells by disrupting Wnt3 signaling.

    PubMed

    Riganti, Chiara; Salaroglio, Iris C; Pinzòn-Daza, Martha L; Caldera, Valentina; Campia, Ivana; Kopecka, Joanna; Mellai, Marta; Annovazzi, Laura; Couraud, Pierre-Olivier; Bosia, Amalia; Ghigo, Dario; Schiffer, Davide

    2014-02-01

    Low delivery of many anticancer drugs across the blood-brain barrier (BBB) is a limitation to the success of chemotherapy in glioblastoma. This is because of the high levels of ATP-binding cassette transporters like P-glycoprotein (Pgp/ABCB1), which effluxes drugs back to the bloodstream. Temozolomide is one of the few agents able to cross the BBB; its effects on BBB cells permeability and Pgp activity are not known. We found that temozolomide, at therapeutic concentration, increased the transport of Pgp substrates across human brain microvascular endothelial cells and decreased the expression of Pgp. By methylating the promoter of Wnt3 gene, temozolomide lowers the endogenous synthesis of Wnt3 in BBB cells, disrupts the Wnt3/glycogen synthase kinase 3/β-catenin signaling, and reduces the binding of β-catenin on the promoter of mdr1 gene, which encodes for Pgp. In co-culture models of BBB cells and human glioblastoma cells, pre-treatment with temozolomide increases the delivery, cytotoxicity, and antiproliferative effects of doxorubicin, vinblastine, and topotecan, three substrates of Pgp that are usually poorly delivered across BBB. Our work suggests that temozolomide increases the BBB permeability of drugs that are normally effluxed by Pgp back to the bloodstream. These findings may pave the way to new combinatorial chemotherapy schemes in glioblastoma. PMID:23771630

  7. Overexpression and purification of HSV-2 glycoprotein D in suspension CHO cells with serum-free medium and immunogenicity analysis.

    PubMed

    Qi, Yong; Xu, Yueyue; Pan, Ying; Li, Suqin; Li, Bingjun; Pan, Mingjie; Zhang, Shumin; Li, Yuexi

    2016-05-01

    Glycoprotein D (gD2) is the most important candidate antigen for herpes simplex virus type 2 (HSV-2) vaccine development. Establishment of a stable eukaryotic cell line to overexpress gD2 and an efficient purification process to purify is essential for the development of subunit vaccine against HSV-2. The DNA sequence of the extracellular epitope-rich fragment of gD2 was optimized, chemically synthesized, and cloned into plasmid pMD902. The recombinant plasmid pMD902-gD was stably transfected into CHO-DG44 cells, and cell lines with high levels of expression of gD2 were established. The recombinant gD2 was purified efficiently using an anion exchange column and a Sephadex G-25 desalting column. The yield of the purified gD2 was 57 mg/L of serum-free culture medium, and its purity was determined to be about 95% by HPLC analysis. Finally, the immunogenicity of the purified gD2 was measured and it induced strong and specific humoral immunity and higher level of cellular immune response than gD2 expressed in prokaryotic cells. We established a stable, secretory, and high-yield gD2-expression cell line and an easy and efficient gD2-purification process, which lays the foundation for preparation of large amount of gD2 that is essential for HSV-2 subunit vaccine development. PMID:25906680

  8. Chromosome breakage at a major fragile site associated with P-glycoprotein gene amplification in multidrug-resistant CHO cells.

    PubMed Central

    Kuo, M T; Vyas, R C; Jiang, L X; Hittelman, W N

    1994-01-01

    Recent studies of several drug-resistant Chinese hamster cell lines suggested that a breakage-fusion-bridge mechanism is frequently involved in the amplification of drug resistance genes. These observations underscore the importance of chromosome breakage in the initiation of DNA amplification in mammalian cells. However, the mechanism of this breakage is unknown. Here, we propose that the site of chromosome breakage consistent with the initial event of P-glycoprotein (P-gp) gene amplification via the breakage-fusion-bridge cycle in three independently established multidrug-resistant CHO cells was located at 1q31. This site is a major chromosome fragile site that can be induced by methotrexate and aphidicolin treatments. Pretreatments of CHO cells with methotrexate or aphidicolin enhanced the frequencies of resistance to vinca alkaloid and amplification of the P-gp gene. These observations suggest that chromosome fragile sites play a pivotal role in DNA amplification in mammalian cells. Our data are also consistent with the hypothesis that gene amplification can be initiated by stress-induced chromosome breakage that is independent of modes of action of cytotoxic agents. Drug-resistant variants may arise by their growth advantage due to overproduction of cellular target molecules via gene amplification. Images PMID:7913517

  9. Chromosome breakage at a major fragile site associated with P-glycoprotein gene amplification in multidrug-resistant CHO cells.

    PubMed

    Kuo, M T; Vyas, R C; Jiang, L X; Hittelman, W N

    1994-08-01

    Recent studies of several drug-resistant Chinese hamster cell lines suggested that a breakage-fusion-bridge mechanism is frequently involved in the amplification of drug resistance genes. These observations underscore the importance of chromosome breakage in the initiation of DNA amplification in mammalian cells. However, the mechanism of this breakage is unknown. Here, we propose that the site of chromosome breakage consistent with the initial event of P-glycoprotein (P-gp) gene amplification via the breakage-fusion-bridge cycle in three independently established multidrug-resistant CHO cells was located at 1q31. This site is a major chromosome fragile site that can be induced by methotrexate and aphidicolin treatments. Pretreatments of CHO cells with methotrexate or aphidicolin enhanced the frequencies of resistance to vinca alkaloid and amplification of the P-gp gene. These observations suggest that chromosome fragile sites play a pivotal role in DNA amplification in mammalian cells. Our data are also consistent with the hypothesis that gene amplification can be initiated by stress-induced chromosome breakage that is independent of modes of action of cytotoxic agents. Drug-resistant variants may arise by their growth advantage due to overproduction of cellular target molecules via gene amplification. PMID:7913517

  10. Human hepatoma cells rich in P-glycoprotein are sensitive to aclarubicin and resistant to three other anthracyclines.

    PubMed Central

    Lehne, G.; De Angelis, P.; Clausen, O. P.; Rugstad, H. E.

    1996-01-01

    Drug resistance is a major obstacle to successful chemotherapy of primary liver cancer, which is associated with high expression of the multidrug resistance (MDR) gene product P-glycoprotein (Pgp), a multidrug efflux transporter. The most effective single agents in treatment of primary liver carcinoma belong to the anthracycline family, yet several anthracyclines are known to be substrates for Pgp. In the present study, we compared four anthracyclines with respect to cell growth inhibition, intracellular accumulation and cellular efflux using the HB8065/R human hepatoma cell line which is rich in Pgp, and the Pgp-poor parental line HB8065/S. The anthracyclines were also administered in conjunction with the Pgp-modifying agents verapamil and SDZ PSC 833 to assess modulation of resistance. The HB8065/R cells were sensitive to aclarubicin (ACL) and highly resistant to epirubicin (EPI), doxorubicin (DOX) and daunorubicin (DNR). SDZ PSC 833 enhanced accumulation, decreased efflux and increased cytotoxicity of EPI, DOX and DNR in the HB8065/R cells, but none of these effects was seen with ACL. In conclusion, ACL is apparently not transported by Pgp and retains its activity in a multidrug-resistant human hepatoma cell line; such properties can be exploited for clinical purposes. Images Figure 5 PMID:8956784

  11. P-glycoprotein in adriamycin-resistant cells functions as an efflux pump for benzopyrene, a chemical carcinogen

    SciTech Connect

    Chao Yeh, G.; Poore, C.M.; Lopaczynska, J.; Phang, J.M. )

    1991-03-15

    The physiological function of multidrug resistant gene (MDR 1) coded P-glycoprotein 170 (P-gp) in normal tissues remains unknown. The authors propose that P-gp functions as an efflux pump in normal tissues for benzopyrene and other xenobiotic substances. To examine their hypothesis the authors used a series of MDR human breast cancer MCF-7 cells with increasing degrees of drug resistance, expression of MDR and levels of P-gp. First, they found the IC{sub 50} for benzopyrene is linearly correlated with the levels of P-gp at different stages of adriamycin resistant MCF-7 cells. Using P-gp ({sup 3}H)azidopine labeling as a measurement of P-gp they found benzopyrene competes for labeling of P-gp. Finally, they directly measured cellular efflux of benzopyrene with adherent cell laser cytometry and found that resistant cells expressing high levels of P-gp showed rapid efflux of benzopyrene. By contrast, drug sensitive wild type cells with undetectable P-gp showed negligible efflux. They conclude that P-gp can function as an efflux pump for benzopyrene and suggest that P-gp may be a cellular mechanism for resistance to carcinogens.

  12. Local Nanomechanical Motion of the Cell Wall of Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Pelling, Andrew E.; Sehati, Sadaf; Gralla, Edith B.; Valentine, Joan S.; Gimzewski, James K.

    2004-08-01

    We demonstrate that the cell wall of living Saccharomyces cerevisiae (baker's yeast) exhibits local temperature-dependent nanomechanical motion at characteristic frequencies. The periodic motions in the range of 0.8 to 1.6 kHz with amplitudes of ~3 nm were measured using the cantilever of an atomic force microscope (AFM). Exposure of the cells to a metabolic inhibitor causes the periodic motion to cease. From the strong frequency dependence on temperature, we derive an activation energy of 58 kJ/mol, which is consistent with the cell's metabolism involving molecular motors such as kinesin, dynein, and myosin. The magnitude of the forces observed (~10 nN) suggests concerted nanomechanical activity is operative in the cell.

  13. Impact of processing on the noncovalent interactions between procyanidin and apple cell wall.

    PubMed

    Le Bourvellec, Carine; Watrelot, Aude A; Ginies, Christian; Imberty, Anne; Renard, Catherine M G C

    2012-09-19

    Procyanidins can bind cell wall material in raw product, and it could be supposed that the same mechanism of retention of procyanidins by apple cell walls takes place in cooked products. To evaluate the influence of cell wall composition and disassembly during cooking on the cell walls' capacity to interact with procyanidins, four cell wall materials differing in their protein contents and physical characteristics were prepared: cell wall with proteins, cell wall devoid of protein, and two processed cell walls differing by their drying method. Protein contents varied from 23 to 99 mg/g and surface areas from 1.26 to 3.16 m(2)/g. Apple procyanidins with an average polymerization degree of 8.7 were used. The adsorption of apple procyanidins on solid cell wall material was quantified using the Langmuir isotherm formulation. The protein contents in cell wall material had no effect on procyanidin/cell wall interactions, whereas modification of the cell wall material by boiling, which reduces pectin content, and drying decreased the apparent affinity and increased the apparent saturation levels when constants were expressed relative to cell wall weight. However, boiling and drying increased apparent saturation levels and had no effect on apparent affinity when the same data were expressed per surface units. Isothermal titration calorimetry indicated strong affinity (K(a) = 1.4 × 10(4) M(-1)) between pectins solubilized by boiling and procyanidins. This study higllights the impact of highly methylated pectins and drying, that is, composition and structure of cell wall in the cell wall/procyanidin interactions. PMID:22861056

  14. High affinity binding of beta 2-glycoprotein I to human endothelial cells is mediated by annexin II.

    PubMed

    Ma, K; Simantov, R; Zhang, J C; Silverstein, R; Hajjar, K A; McCrae, K R

    2000-05-19

    Beta(2)-glycoprotein I (beta(2)GPI) is an abundant plasma phospholipid-binding protein and an autoantigen in the antiphospholipid antibody syndrome. Binding of beta(2)GPI to endothelial cells targets them for activation by anti-beta(2)GPI antibodies, which circulate and are associated with thrombosis in patients with the antiphospholipid antibody syndrome. However, the binding of beta(2)GPI to endothelial cells has not been characterized and is assumed to result from association of beta(2)GPI with membrane phospholipid. Here, we characterize the binding of beta(2)GPI to endothelial cells and identify the beta(2)GPI binding site. (125)I-beta(2)GPI bound with high affinity (K(d) approximately 18 nm) to human umbilical vein endothelial cells (HUVECs). Using affinity purification, we isolated beta(2)GPI-binding proteins of approximately 78 and approximately 36 kDa from HUVECs and EAHY.926 cells. Amino acid sequences of tryptic peptides from each of these were identical to sequences within annexin II. A role for annexin II in binding of beta(2)GPI to cells was confirmed by the observations that annexin II-transfected HEK 293 cells bound approximately 10-fold more (125)I-beta(2)GPI than control cells and that anti-annexin II antibodies inhibited the binding of (125)I-beta(2)GPI to HUVECs by approximately 90%. Finally, surface plasmon resonance studies revealed high affinity binding between annexin II and beta(2)GPI. These results demonstrate that annexin II mediates the binding of beta(2)GPI to endothelial cells. PMID:10809787

  15. Seed coat mucilage cells of Arabidopsis thaliana as a model for plant cell wall research.

    PubMed

    Arsovski, Andrej A; Haughn, George W; Western, Tamara L

    2010-07-01

    Plant cells are encased within a complex polysaccharide wall that strengthens the cell and has key roles in all aspects of plant cell growth, differentiation, and interaction with the environment. This dynamic structure is under continual modification during plant development, and its synthesis and modification require the activity of a myriad of enzymes. The mucilage secretory cells (MSCs) of the Arabidopsis thaliana seed coat provide a model for the discovery of novel genes involved in the synthesis, secretion and modification of cell wall components, particularly pectin. These cells synthesize copious amounts of pectinaceous mucilage during development and, upon hydration of the desiccated seed, the mucilage rapidly swells, bursts from the MSCs and surrounds the seed in a gelatinous capsule. Several genes affecting MSC differentiation, pectin synthesis, and mucilage release have been identified and additional genes involved in these and related processes including pectin secretion and the mechanical alteration of cell walls await to be discovered. PMID:20505351

  16. Cell wall pH and auxin transport velocity

    NASA Technical Reports Server (NTRS)

    Hasenstein, K. H.; Rayle, D.

    1984-01-01

    According to the chemiosmotic polar diffusion hypothesis, auxin pulse velocity and basal secretion should increase with decreasing cell wall pH. Experiments were designed to test this prediction. Avena coleoptile sections were preincubated in either fusicoccin (FC), cycloheximide, pH 4.0, or pH 8.0 buffer and subsequently their polar transport capacities were determined. Relative to controls, FC enhanced auxin (IAA) uptake while CHI and pH 8.0 buffer reduced IAA uptake. Nevertheless, FC reduced IAA pulse velocity while cycloheximide increased velocity. Additional experiments showed that delivery of auxin to receivers is enhanced by increased receiver pH. This phenomenon was overcome by a pretreatment of the tissue with IAA. Our data suggest that while acidic wall pH values facilitate cellular IAA uptake, they do not enhance pulse velocity or basal secretion. These findings are inconsistent with the chemiosmotic hypothesis for auxin transport.

  17. Cell wall pH and auxin transport velocity.

    PubMed Central

    Hasenstein, K H; Rayle, D

    1984-01-01

    According to the chemiosmotic polar diffusion hypothesis, auxin pulse velocity and basal secretion should increase with decreasing cell wall pH. Experiments were designed to test this prediction. Avena coleoptile sections were preincubated in either fusicoccin (FC), cycloheximide, pH 4.0, or pH 8.0 buffer and subsequently their polar transport capacities were determined. Relative to controls, FC enhanced auxin (IAA) uptake while CHI and pH 8.0 buffer reduced IAA uptake. Nevertheless, FC reduced IAA pulse velocity while cycloheximide increased velocity. Additional experiments showed that delivery of auxin to receivers is enhanced by increased receiver pH. This phenomenon was overcome by a pretreatment of the tissue with IAA. Our data suggest that while acidic wall pH values facilitate cellular IAA uptake, they do not enhance pulse velocity or basal secretion. These findings are inconsistent with the chemiosmotic hypothesis for auxin transport. PMID:11540807

  18. Enzymology and molecular biology of cell wall biosynthesis. Progress report

    SciTech Connect

    Ray, P.M.

    1993-03-20

    In order to be able to explore the control of cell wall polysaccharide synthesis at the molecular level, which inter alia might eventually lead to means for useful modification of plant biomass polysaccharide production, the immediate goals of this project are to identify polypeptides responsible for wall polysaccharide synthase activities and to obtain clones of the genes that encode them. We are concentrating on plasma membraneassociated (1,3)-{beta}-glucan synthase (glucan synthase-II or GS-II) and Golgi-associated (1,4)-{beta}-glucan synthase (glucan synthase-I or GS-I), of growing pea stem tissue. Our progress has been much more rapid with respect to GS-II than regarding GS-I.

  19. Scattering properties of microalgae: the effect of cell size and cell wall

    NASA Astrophysics Data System (ADS)

    Svensen, Øyvind; Frette, Øyvind; Rune Erga, Svein

    2007-08-01

    The main objective of this work was to investigate how the cell size and the presence of a cell wall influence the scattering properties of the green microalgae Chlamydomonas reinhardtii. The growth cycle of two strains, one with a cell wall and one without, was synchronized to be in the same growth phase. Measurements were conducted at two different phases of the growth cycle on both strains of the algae. It was found that the shape of the scattering phase function was very similar for both strains at both growth phases, but the regular strain with a cell wall scatters more strongly than the wall-less mutant. It was also found that the mutant strain has a stronger increase in scattering than the regular strain, as the algae grow, and that the scattering from the regular strain is more wavelength dependent than from the mutant strain.

  20. A Molecular Switch Abrogates Glycoprotein 100 (gp100) T-cell Receptor (TCR) Targeting of a Human Melanoma Antigen*

    PubMed Central

    Bianchi, Valentina; Bulek, Anna; Fuller, Anna; Lloyd, Angharad; Attaf, Meriem; Rizkallah, Pierre J.; Dolton, Garry; Sewell, Andrew K.; Cole, David K.

    2016-01-01

    Human CD8+ cytotoxic T lymphocytes can mediate tumor regression in melanoma through the specific recognition of HLA-restricted peptides. Because of the relatively weak affinity of most anti-cancer T-cell receptors (TCRs), there is growing emphasis on immunizing melanoma patients with altered peptide ligands in order to induce strong anti-tumor immunity capable of breaking tolerance toward these self-antigens. However, previous studies have shown that these immunogenic designer peptides are not always effective. The melanocyte differentiation protein, glycoprotein 100 (gp100), encodes a naturally processed epitope that is an attractive target for melanoma immunotherapies, in particular peptide-based vaccines. Previous studies have shown that substitutions at peptide residue Glu3 have a broad negative impact on polyclonal T-cell responses. Here, we describe the first atomic structure of a natural cognate TCR in complex with this gp100 epitope and highlight the relatively high affinity of the interaction. Alanine scan mutagenesis performed across the gp100280–288 peptide showed that Glu3 was critically important for TCR binding. Unexpectedly, structural analysis demonstrated that the Glu3 → Ala substitution resulted in a molecular switch that was transmitted to adjacent residues, abrogating TCR binding and T-cell recognition. These findings help to clarify the mechanism of T-cell recognition of gp100 during melanoma responses and could direct the development of altered peptides for vaccination. PMID:26917722

  1. Molecular characterization of E2 glycoprotein of classical swine fever virus: adaptation and propagation in porcine kidney cells.

    PubMed

    Kumar, Rakesh; Barman, Nagendra N; Khatoon, Elina; Rajbongshi, Gitika; Deka, Nipu; Morla, Sudhir; Kumar, Sachin

    2015-05-01

    Classical swine fever virus (CSFV) is the causative agent of a highly contagious disease, hog cholera in pigs. The disease is endemic in many parts of the world, and vaccination is the only way to protect the animals from CSFV infection. The lapinized vaccine strains are occasionally not protective because of animal to animal passage, inadequate vaccination strategy, suboptimal vaccine dose, and emergence of new variants. The surface glycoprotein E2 of CSFV is a major antigenic determinant and can modulate the disease outcome in pigs. In the present study, we characterized the CSFV in porcine kidney cells. The CSFV vaccine strains showed enhanced replication following 15 passages in porcine kidney cells. Nucleotide sequence analysis of the E2 protein gene of the cell culture-adapted vaccine strain of CSFV showed a mutation in putative amino acid sequences that are identical to its virulent counterpart. The study suggests the possibility of exaltation in vaccine strains following its adaptation in host cells and paves the way for a further exploration of the biology of its outbreak. PMID:25552311

  2. Identification of a surface glycoprotein on African green monkey kidney cells as a receptor for hepatitis A virus.

    PubMed Central

    Kaplan, G; Totsuka, A; Thompson, P; Akatsuka, T; Moritsugu, Y; Feinstone, S M

    1996-01-01

    Very little is known about the mechanism of cell entry of hepatitis A virus (HAV), and the identification of cellular receptors for this picornavirus has been elusive. Here we describe the molecular cloning of a cellular receptor for HAV using protective monoclonal antibodies raised against susceptible African green monkey kidney (AGMK) cells as probes. Monoclonal antibodies 190/4, 235/4 and 263/6, which reacted against similar epitopes, specifically protected AGMK cells against HAV infection by blocking the binding of HAV. Expression cloning and nucleotide sequence analysis of the cDNA coding for epitope 190/4 revealed a novel mucin-like class I integral membrane glycoprotein of 451 amino acids, the HAV cellular receptor 1 (HAVcr-1). Immunofluorescence analysis indicated that mouse Ltk- cells transfected with HAVcr-1 cDNA gained limited susceptibility to HAV infection, which was blocked by treatment with monoclonal antibody 190/4. Our results demonstrate that the HAVcr-1 polypeptide is an attachment receptor for HAV and strongly suggest that it is also a functional receptor which mediates HAV infection. This report constitutes the first identification of a cellular receptor for HAV. Images PMID:8861957

  3. Clofazimine and B4121 sensitize an intrinsically resistant human colon cancer cell line to P-glycoprotein substrates.

    PubMed

    van Rensburg, C E; Joone, G K; O'Sullivan, J F

    2000-01-01

    The potential of B4121 to sensitize three intrinsically resistant human colon cancer cell lines (CaCo2, ATCC HTB 37; COLO 32 DM, ATCC CCL 220; HT-29, ATCC HTB 38) to vinblastine, doxorubicin, daunorubicin, paclitaxel, taxotere and cisplatin at a non-toxic, therapeutically relevant concentration of 0.25 microg/ml was compared with that of clofazimine at a similar concentration. The cell line expressing high levels of P-glycoprotein (P-gp), COLO 320 DM, was susceptible to chemosensitization by the experimental agents for the P-gp substrates (paclitaxel, taxotere, daunorubicin, vinblastine and doxorubicin) but not for cisplatin. CaCo2 cells expressed lower levels of P-gp and were only marginally susceptible to sensitization by any one of these drugs, except in the case of sensitization by B4121 for doxorubicin and taxotere, whereas the HT-29, a P-gp negative cell line, was unaffected. The riminophenazines, especially B4121, might prove useful as combination treatment in circumventing P-gp mediated resistance of colon cancers. PMID:10601617