Science.gov

Sample records for encoding putative cell-surface

  1. SurfaceomeDB: a cancer-orientated database for genes encoding cell surface proteins.

    PubMed

    de Souza, Jorge Estefano Santana; Galante, Pedro Alexandre Favoretto; de Almeida, Renan Valieris Bueno; da Cunha, Julia Pinheiro Chagas; Ohara, Daniel Takatori; Ohno-Machado, Lucila; Old, Lloyd J; de Souza, Sandro José

    2012-01-01

    Cell surface proteins (CSPs) are excellent targets for the development of diagnostic and therapeutic reagents, and it is estimated that 10-20% of all genes in the human genome encode CSPs. In an effort to integrate all data publicly available for genes encoding cell surface proteins, a database (SurfaceomeDB) was developed. SurfaceomeDB is a gene-centered portal containing different types of information, including annotation for gene expression, protein domains, somatic mutations in cancer, and protein-protein interactions for all human genes encoding CSPs. SurfaceomeDB was implemented as an integrative and relational database in a user-friendly web interface, where users can search for gene name, gene annotation, or keywords. There is also a streamlined graphical representation of all data provided and links to the most important data repositories and databases, such as NCBI, UCSC Genome Browser, and EBI. PMID:23390370

  2. Characterization and purification of proteins which bind high-density lipoprotein. A putative cell-surface receptor.

    PubMed Central

    Bond, H M; Morrone, G; Venuta, S; Howell, K E

    1991-01-01

    High-density lipoprotein (HDL) is shown by ligand blotting to bind membrane-associated polypeptides with sizes of 60, 100 and 210 kDa. Binding was concentration-dependent and competed by excess unlabelled HDL. All the major apolipoproteins of HDL, apoA-I, apoA-II and apoA-IV, bound independently. The 100 kDa and 210 kDa HDL-binding activities were purified from membranes of Hep3B tumour cells by ion-exchange chromatography and gel filtration. The binding activities at 100 kDa and 210 kDa co-purified. After treatment with disulphide-reducing reagent, the 210 kDa band was no longer present and an increase was observed in the amount and binding ability of the 100 kDa polypeptide. The 100 kDa binding protein labelled at the cell surface with 125I could be immunoprecipitated after cross-linking to cell-surface-bound HDL. It is proposed that this HDL-binding activity, a putative cell-surface receptor for HDL, exists totally or in part as a high-molecular-mass complex composed of 100 kDa subunits. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:1659384

  3. A genetically encoded alkyne directs palladium-mediated protein labeling on live mammalian cell surface.

    PubMed

    Li, Nan; Ramil, Carlo P; Lim, Reyna K V; Lin, Qing

    2015-02-20

    The merging of site-specific incorporation of small bioorthogonal functional groups into proteins via amber codon suppression with bioorthogonal chemistry has created exciting opportunities to extend the power of organic reactions to living systems. Here we show that a new alkyne amino acid can be site-selectively incorporated into mammalian proteins via a known orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair and directs an unprecedented, palladium-mediated cross-coupling reaction-driven protein labeling on live mammalian cell surface. A comparison study with the alkyne-encoded proteins in vitro indicated that this terminal alkyne is better suited for the palladium-mediated cross-coupling reaction than the copper-catalyzed click chemistry. PMID:25347611

  4. Expression and function of a putative cell surface receptor for fibronectin in hamster and human cell lines

    SciTech Connect

    Brown, P.J.; Juliano, R.L.

    1986-10-01

    The authors have previously reported the use of monoclonal antibodies to identify a 140-kD cell surface glycoprotein in mammalian cells that is specifically involved in fibronectin-mediated cell adhesion. They now report the purification of this molecule using immunoaffinity chromatography and the subsequent generation of polyclonal antibodies that selectively immunoprecipitate 140-kD putative fibronectin receptor glycoprotein (gp140) extracted from rodent or human cells; these antibodies also specifically block fibronectin-mediated cell adhesion but not adhesion mediated by other factors in serum. Expression of gp140-like molecules was detected on the surfaces of several adherent human cell lines (HDF, WISH, and EFC) but not on erythrocytes; however, gp140 was also detected on a nonadherent human lymphoid line (DAUDI). Analysis of gp140 on nonreducing SDS gels revealed two closely migrating bands. Protease digestion and peptide mapping suggests that the two bnads are closely related polypeptides.

  5. The Role of Putative Phosphatidylserine-Interactive Residues of Tissue Factor on Its Coagulant Activity at the Cell Surface

    PubMed Central

    Ansari, Shabbir A.; Pendurthi, Usha R.; Sen, Prosenjit; Rao, L. Vijaya Mohan

    2016-01-01

    Exposure of phosphatidylserine (PS) on the outer leaflet of the cell membrane is thought to play a critical role in tissue factor (TF) decryption. Recent molecular dynamics simulation studies suggested that the TF ectodomain may directly interact with PS. To investigate the potential role of TF direct interaction with the cell surface phospholipids on basal TF activity and the enhanced TF activity following the decryption, one or all of the putative PS-interactive residues in the TF ectodomain were mutated and tested for their coagulant activity in cell systems. Out of the 9 selected TF mutants, five of them -TFS160A, TFS161A, TFS162A, TFK165A, and TFD180A- exhibited a similar TF coagulant activity to that of the wild-type TF. The specific activity of three mutants, TFK159A, TFS163A, and TFK166A, was reduced substantially. Mutation of the glycine residue at the position 164 markedly abrogated the TF coagulant activity, resulting in ~90% inhibition. Mutation of all nine lipid binding residues together did not further decrease the activity of TF compared to TFG164A. A similar fold increase in TF activity was observed in wild-type TF and all TF mutants following the treatment of THP-1 cells with either calcium ionomycin or HgCl2, two agents that are commonly used to decrypt TF. Overall, our data show that a few select TF residues that are implicated in interacting with PS contribute to the TF coagulant activity at the cell surface. However, our data also indicate that TF regions outside of the putative lipid binding region may also contribute to PS-dependent decryption of TF. PMID:27348126

  6. The Role of Putative Phosphatidylserine-Interactive Residues of Tissue Factor on Its Coagulant Activity at the Cell Surface.

    PubMed

    Ansari, Shabbir A; Pendurthi, Usha R; Sen, Prosenjit; Rao, L Vijaya Mohan

    2016-01-01

    Exposure of phosphatidylserine (PS) on the outer leaflet of the cell membrane is thought to play a critical role in tissue factor (TF) decryption. Recent molecular dynamics simulation studies suggested that the TF ectodomain may directly interact with PS. To investigate the potential role of TF direct interaction with the cell surface phospholipids on basal TF activity and the enhanced TF activity following the decryption, one or all of the putative PS-interactive residues in the TF ectodomain were mutated and tested for their coagulant activity in cell systems. Out of the 9 selected TF mutants, five of them -TFS160A, TFS161A, TFS162A, TFK165A, and TFD180A- exhibited a similar TF coagulant activity to that of the wild-type TF. The specific activity of three mutants, TFK159A, TFS163A, and TFK166A, was reduced substantially. Mutation of the glycine residue at the position 164 markedly abrogated the TF coagulant activity, resulting in ~90% inhibition. Mutation of all nine lipid binding residues together did not further decrease the activity of TF compared to TFG164A. A similar fold increase in TF activity was observed in wild-type TF and all TF mutants following the treatment of THP-1 cells with either calcium ionomycin or HgCl2, two agents that are commonly used to decrypt TF. Overall, our data show that a few select TF residues that are implicated in interacting with PS contribute to the TF coagulant activity at the cell surface. However, our data also indicate that TF regions outside of the putative lipid binding region may also contribute to PS-dependent decryption of TF. PMID:27348126

  7. Tomato Ve disease resistance genes encode cell surface-like receptors

    PubMed Central

    Kawchuk, Lawrence M.; Hachey, John; Lynch, Dermot R.; Kulcsar, Frank; van Rooijen, Gijs; Waterer, Doug R.; Robertson, Albert; Kokko, Eric; Byers, Robert; Howard, Ronald J.; Fischer, Rainer; Prüfer, Dirk

    2001-01-01

    In tomato, Ve is implicated in race-specific resistance to infection by Verticillium species causing crop disease. Characterization of the Ve locus involved positional cloning and isolation of two closely linked inverted genes. Expression of individual Ve genes in susceptible potato plants conferred resistance to an aggressive race 1 isolate of Verticillium albo-atrum. The deduced primary structure of Ve1 and Ve2 included a hydrophobic N-terminal signal peptide, leucine-rich repeats containing 28 or 35 potential glycosylation sites, a hydrophobic membrane-spanning domain, and a C-terminal domain with the mammalian E/DXXXLφ or YXXφ endocytosis signals (φ is an amino acid with a hydrophobic side chain). A leucine zipper-like sequence occurs in the hydrophobic N-terminal signal peptide of Ve1 and a Pro-Glu-Ser-Thr (PEST)-like sequence resides in the C-terminal domain of Ve2. These structures suggest that the Ve genes encode a class of cell-surface glycoproteins with receptor-mediated endocytosis-like signals and leucine zipper or PEST sequences. PMID:11331751

  8. Crystal Structure of a Virus-Encoded Putative Glycosyltransferase

    SciTech Connect

    Xiang, Ye; Baxa, Ulrich; Zhang, Ying; Steven, Alasdair C.; Lewis, Gentry L.; Van Etten, James L.; Rossmann, Michael G.

    2010-11-22

    The chloroviruses (family Phycodnaviridae), unlike most viruses, encode some, if not most, of the enzymes involved in the glycosylation of their structural proteins. Annotation of the gene product B736L from chlorovirus NY-2A suggests that it is a glycosyltransferase. The structure of the recombinantly expressed B736L protein was determined by X-ray crystallography to 2.3-{angstrom} resolution, and the protein was shown to have two nucleotide-binding folds like other glycosyltransferase type B enzymes. This is the second structure of a chlorovirus-encoded glycosyltransferase and the first structure of a chlorovirus type B enzyme to be determined. B736L is a retaining enzyme and belongs to glycosyltransferase family 4. The donor substrate was identified as GDP-mannose by isothermal titration calorimetry and was shown to bind into the cleft between the two domains in the protein. The active form of the enzyme is probably a dimer in which the active centers are separated by about 40 {angstrom}.

  9. Phylogeny of Algal Sequences Encoding Carbohydrate Sulfotransferases, Formylglycine-Dependent Sulfatases, and Putative Sulfatase Modifying Factors

    PubMed Central

    Ho, Chai-Ling

    2015-01-01

    Many algae are rich sources of sulfated polysaccharides with biological activities. The physicochemical/rheological properties and biological activities of sulfated polysaccharides are affected by the pattern and number of sulfate moieties. Sulfation of carbohydrates is catalyzed by carbohydrate sulfotransferases (CHSTs) while modification of sulfate moieties on sulfated polysaccharides was presumably catalyzed by sulfatases including formylglycine-dependent sulfatases (FGly-SULFs). Post-translationally modification of Cys to FGly in FGly-SULFs by sulfatase modifiying factors (SUMFs) is necessary for the activity of this enzyme. The aims of this study are to mine for sequences encoding algal CHSTs, FGly-SULFs and putative SUMFs from the fully sequenced algal genomes and to infer their phylogenetic relationships to their well characterized counterparts from other organisms. Algal sequences encoding CHSTs, FGly-SULFs, SUMFs, and SUMF-like proteins were successfully identified from green and brown algae. However, red algal FGly-SULFs and SUMFs were not identified. In addition, a group of SUMF-like sequences with different gene structure and possibly different functions were identified for green, brown and red algae. The phylogeny of these putative genes contributes to the corpus of knowledge of an unexplored area. The analyses of these putative genes contribute toward future production of existing and new sulfated carbohydrate polymers through enzymatic synthesis and metabolic engineering. PMID:26635861

  10. Bacillus anthracis pXO1 plasmid encodes a putative membrane-bound bacteriocin

    PubMed Central

    Perlińska, Agata

    2014-01-01

    Evolutionary advantages over cousin cells in bacterial pathogens may decide about the success of a specific cell in its environment. Bacteria use a plethora of methods to defend against other cells and many devices to attack their opponents when competing for resources. Bacteriocins are antibacterial proteins that are used to eliminate competition. We report the discovery of a putative membrane-bound bacteriocin encoded by the Bacillus anthracis pathogenic pXO1 plasmid. We analyze the genomic structure of the bacteriocin operon. The proposed mechanisms of action predestine this operon as a potent competitive advantage over cohabitants of the same niche. PMID:25426338

  11. A Micropeptide Encoded by a Putative Long Non-coding RNA Regulates Muscle Performance

    PubMed Central

    Anderson, Douglas M.; Anderson, Kelly M.; Chang, Chi-Lun; Makarewich, Catherine A.; Nelson, Benjamin R.; McAnally, John R.; Kasaragod, Prasad; Shelton, John M.; Liou, Jen; Bassel-Duby, Rhonda; Olson, Eric N.

    2015-01-01

    Summary Functional micropeptides can be concealed within RNAs that appear to be non-coding. We discovered a conserved micropeptide, that we named myoregulin (MLN), encoded by a skeletal muscle-specific RNA annotated as a putative long non-coding RNA. MLN shares structural and functional similarity with phospholamban (PLN) and sarcolipin (SLN), which inhibit SERCA, the membrane pump that controls muscle relaxation by regulating Ca2+ uptake into the sarcoplasmic reticulum (SR). MLN interacts directly with SERCA and impedes Ca2+ uptake into the SR. In contrast to PLN and SLN, which are expressed in cardiac and slow skeletal muscle in mice, MLN is robustly expressed in all skeletal muscle. Genetic deletion of MLN in mice enhances Ca2+ handling in skeletal muscle and improves exercise performance. These findings identify MLN as an important regulator of skeletal muscle physiology and highlight the possibility that additional micropeptides are encoded in the many RNAs currently annotated as non-coding. PMID:25640239

  12. An endogenous hybrid mRNA encodes TWE-PRIL, a functional cell surface TWEAK–APRIL fusion protein

    PubMed Central

    Pradet-Balade, B.; Medema, J.P.; López-Fraga, M.; Lozano, J.C.; Kolfschoten, G.M.; Picard, A.; Martínez-A., C.; Garcia-Sanz, J.A.; Hahne, M.

    2002-01-01

    TWEAK and APRIL are two recently identified tumour necrosis factor (TNF) ligand family members, implicated in angiogenesis and immune regulation, respectively. TWEAK is a transmembrane protein expressed on the cell surface, whereas APRIL acts solely as a secreted factor. In this report, using RACE, RT–PCR, cDNA library screening and an RNase protection assay, we characterize a hybrid transcript between TWEAK and APRIL mRNAs. The encoded TWE-PRIL protein is composed of TWEAK cytoplasmic and transmembrane domains fused to the APRIL C-terminal domain. TWE-PRIL mRNA is expressed and translated in human primary T cells and monocytes, and endogenous TWE-PRIL protein was detected in primary human T lymphocytes and monocytic cell lines. TWE-PRIL is membrane anchored and presents the APRIL receptor-binding domain at the cell surface. It is a biologically active ligand, as it stimulates cycling in T- and B-lymphoma cell lines. Much like membrane-bound and secreted TNF-α, the different cellular localizations of TWE-PRIL and APRIL suggest that they exert distinct biological roles. PMID:12411489

  13. Molecular Characterization of the Mycoplasma gallisepticum pvpA Gene Which Encodes a Putative Variable Cytadhesin Protein

    PubMed Central

    Boguslavsky, S.; Menaker, D.; Lysnyansky, I.; Liu, T.; Levisohn, S.; Rosengarten, R.; García, M.; Yogev, D.

    2000-01-01

    A putative cytadhesin-related protein (PvpA) undergoing variation in its expression was identified in the avian pathogen Mycoplasma gallisepticum. The pvpA gene was cloned, expressed in Escherichia coli, and sequenced. It exhibits 54 and 52% homology with the P30 and P32 cytadhesin proteins of the human pathogens Mycoplasma pneumoniae and Mycoplasma genitalium, respectively. In addition, 50% homology was found with the MGC2 cytadhesin of M. gallisepticum and 49% homology was found with a stretch of 205 amino acids of the cytadherence accessory protein HMW3 of M. pneumoniae. The PvpA molecule possesses a proline-rich carboxy-terminal region (28%) containing two identical directly repeated sequences of 52 amino acids and a tetrapeptide motif (Pro-Arg-Pro-X) which is repeated 14 times. Genetic analysis of several clonal isolates representing different expression states of the PvpA product ruled out chromosomal rearrangement as the mechanism for PvpA phase variation. The molecular basis of PvpA variation was revealed in a short tract of repeated GAA codons, encoding five successive glutamate resides, located in the N-terminal region and subject to frequent mutation generating an in-frame UAA stop codon. Size variation of the PvpA protein was observed among M. gallisepticum strains, ranging from 48 to 55 kDa and caused by several types of deletions occurring at the PvpA C-terminal end and within the two directly repeated sequences. By immunoelectron microscopy, the PvpA protein was localized on the mycoplasma cell surface, in particular on the terminal tip structure. Collectively, these findings suggest that PvpA is a newly identified variable surface cytadhesin protein of M. gallisepticum. PMID:10858209

  14. Molecular characterization of the Mycoplasma gallisepticum pvpA gene which encodes a putative variable cytadhesin protein.

    PubMed

    Boguslavsky, S; Menaker, D; Lysnyansky, I; Liu, T; Levisohn, S; Rosengarten, R; García, M; Yogev, D

    2000-07-01

    A putative cytadhesin-related protein (PvpA) undergoing variation in its expression was identified in the avian pathogen Mycoplasma gallisepticum. The pvpA gene was cloned, expressed in Escherichia coli, and sequenced. It exhibits 54 and 52% homology with the P30 and P32 cytadhesin proteins of the human pathogens Mycoplasma pneumoniae and Mycoplasma genitalium, respectively. In addition, 50% homology was found with the MGC2 cytadhesin of M. gallisepticum and 49% homology was found with a stretch of 205 amino acids of the cytadherence accessory protein HMW3 of M. pneumoniae. The PvpA molecule possesses a proline-rich carboxy-terminal region (28%) containing two identical directly repeated sequences of 52 amino acids and a tetrapeptide motif (Pro-Arg-Pro-X) which is repeated 14 times. Genetic analysis of several clonal isolates representing different expression states of the PvpA product ruled out chromosomal rearrangement as the mechanism for PvpA phase variation. The molecular basis of PvpA variation was revealed in a short tract of repeated GAA codons, encoding five successive glutamate resides, located in the N-terminal region and subject to frequent mutation generating an in-frame UAA stop codon. Size variation of the PvpA protein was observed among M. gallisepticum strains, ranging from 48 to 55 kDa and caused by several types of deletions occurring at the PvpA C-terminal end and within the two directly repeated sequences. By immunoelectron microscopy, the PvpA protein was localized on the mycoplasma cell surface, in particular on the terminal tip structure. Collectively, these findings suggest that PvpA is a newly identified variable surface cytadhesin protein of M. gallisepticum. PMID:10858209

  15. A 38-kilobase pathogenicity island specific for Mycobacterium avium subsp. paratuberculosis encodes cell surface proteins expressed in the host.

    PubMed

    Stratmann, Janin; Strommenger, Birgit; Goethe, Ralph; Dohmann, Karen; Gerlach, Gerald-F; Stevenson, Karen; Li, Ling-Ling; Zhang, Qing; Kapur, Vivek; Bull, Tim J

    2004-03-01

    We have used representational difference analysis to identify a novel Mycobacterium avium subsp. paratuberculosis-specific ABC transporter operon (mpt), which comprises six open reading frames designated mptA to -F and is immediately preceded by two putative Fur boxes. Functional genomics revealed that the mpt operon is flanked on one end by a fep cluster encoding proteins involved in the uptake of Fe(3+) and on the other end by a sid cluster encoding non-ribosome-dependent heterocyclic siderophore synthases. Together these genes form a 38-kb M. avium subsp. paratuberculosis-specific locus flanked by an insertion sequence similar to IS1110. Expression studies using Western blot analyses showed that MptC is present in the envelope fraction of M. avium subsp. paratuberculosis. The MptD protein was shown to be surface exposed, using a specific phage (fMptD) isolated from a phage-peptide library, by differential screening of Mycobacterium smegmatis transformants. The phage fMptD-derived peptide could be used in a peptide-mediated capture PCR with milk from infected dairy herds, thereby showing surface-exposed expression of the MptD protein in the host. Together, these data suggest that the 38-kb locus constitutes an M. avium subsp. paratuberculosis pathogenicity island. PMID:14977927

  16. Leafy head2, which encodes a putative RNA-binding protein, regulates shoot development of rice.

    PubMed

    Xiong, Guo Sheng; Hu, Xing Ming; Jiao, Yong Qing; Yu, Yan Chun; Chu, Cheng Cai; Li, Jia Yang; Qian, Qian; Wang, Yong Hong

    2006-03-01

    During vegetative development, higher plants continuously form new leaves in regular spatial and temporal patterns. Mutants with abnormal leaf developmental patterns not only provide a great insight into understanding the regulatory mechanism of plant architecture, but also enrich the ways to its modification by which crop yield could be improved. Here, we reported the characterization of the rice leafy-head2 (lhd2) mutant that exhibits shortened plastochron, dwarfism, reduced tiller number, and failure of phase transition from vegetative to reproductive growth. Anatomical and histological study revealed that the rapid emergence of leaves in lhd2 was resulted from the rapid initiation of leaf primordia whereas the reduced tiller number was a consequence of the suppression of the tiller bud outgrowth. The molecular and genetic analysis showed that LHD2 encodes a putative RNA binding protein with 67% similarity to maize TE1. Comparison of genome-scale expression profiles between wild-type and lhd2 plants suggested that LHD2 may regulate rice shoot development through KNOX and hormone-related genes. The similar phenotypes caused by LHD2 mutation and the conserved expression pattern of LHD2 indicated a conserved mechanism in controlling the temporal leaf initiation in grass. PMID:16541125

  17. Identification of genes encoding putative nucleoporins and transport factors in the fission yeast Schizosaccharomyces pombe: a deletion analysis.

    PubMed

    Chen, Xue Qin; Du, Xianming; Liu, Jianhua; Balasubramanian, Mohan K; Balasundaram, David

    2004-04-30

    In a systematic approach to study genes that are related to nucleocytoplasmic trafficking in the fission yeast Schizosaccharomyces pombe, the open reading frames (ORFs) of 26 putative nucleoporins and transport factors were deleted. Here we report the initial characterization of these deletion mutants. Of the 26 putative genes deleted, 14 were found to be essential for viability. Null mutations of essential genes resulted in failure to either complete one round or to sustain cell division. Four of the 14 essential genes, SPBC582.11c, SPBC17G9.04c, SPBC3B9.16c and SPCC162.08c, encode putative nucleoporins and a myosin-like protein with homologues NUP84, NUP85, NUP120 and MLP1, respectively, that are not required for viability in Saccharomyces cerevisiae, suggesting that their gene products perform critical functions in Sz. pombe. On the basis of combined drug sensitivity assays and genetic analysis we have identified five non-essential null mutants that were hypersensitive to the microtubule depolymerizing drug thiabendazole (TBZ) and exhibited a cut phenotype upon TBZ treatment, suggesting possible involvement in microtubule function. Three of the corresponding ORFs, SPCC18B5.07c, nup40 and SPAC1805.04, encode putative nucleoporins with low similarity to the S. cerevisiae nucleoporins NUP2p, NUP53p and NUP133p, respectively. Further genetic analysis revealed that one of the nucleoporin genes, nup40, and another gene, SPCC1322.06, encoding a putative importin-beta/Cse1p superfamily protein may have a spindle checkpoint function. PMID:15116432

  18. In silico cloning of genes encoding neuropeptides, neurohormones and their putative G-protein coupled receptors in a spider mite.

    PubMed

    Veenstra, Jan A; Rombauts, Stephane; Grbić, Miodrag

    2012-04-01

    The genome of the spider mite was prospected for the presence of genes coding neuropeptides, neurohormones and their putative G-protein coupled receptors. Fifty one candidate genes were found to encode neuropeptides or neurohormones. These include all known insect neuropeptides and neurohormones, with the exception of sulfakinin, corazonin, neuroparsin and PTTH. True orthologs of adipokinetic hormone (AKH) were neither found, but there are three genes encoding peptides similar in structure to both AKH and the AKH-corazonin-related peptide. We were also unable to identify the precursors for pigment dispersing factor (PDF) or the recently discovered trissin. However, the spider mite probably does have such genes, as we found their putative receptors. A novel arthropod neuropeptide gene was identified that shows similarity to previously described molluscan neuropeptide genes and was called EFLamide. A total of 65 putative neuropeptide GPCR genes were also identified, of these 58 belong to the A-family and 7 to the B-family. Phylogenetic analysis showed that 50 of them are closely related to insect GPCRs, which allowed the identification of their putative ligand in 39 cases with varying degrees of certainty. Other spider mite GPCRs however have no identifiable orthologs in the genomes of the four holometabolous insect species best analyzed. Whereas some of the latter have orthologs in hemimetabolous insect species, crustaceans or ticks, for others such arthropod homologs are currently unknown. PMID:22214827

  19. Isolation and characterization of 17 different genes encoding putative endopolygalacturonase genes from Rhizopus oryzae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polygalacturonase enzymes are a valuable aid in the retting of flax for production of linens and, more recently, production of biofuels from citrus wastes. In a search of the recently sequenced Rhizopus oryzae strain 99-880 genome database, 18 putative endopolygalacturonase genes were identified, w...

  20. Characterization of the BLR1 gene encoding a putative blue-light regulator in the phytopathogenic fungus Bipolaris oryzae.

    PubMed

    Kihara, Junichi; Moriwaki, Akihiro; Tanaka, Nozomi; Ueno, Makoto; Arase, Sakae

    2007-01-01

    Bipolaris oryzae is a filamentous ascomycetous fungus that causes brown leaf spot disease in rice. We isolated and characterized BLR1, a gene that encodes a putative blue-light regulator similar to Neurospora crassa white-collar 1 (WC-1). The deduced amino acid sequence of BLR1 showed high degrees of similarity to other fungal blue-light regulator protein. Disruption of the BLR1 gene demonstrated that this gene is essential for conidial development after conidiophore formation and for near-UV radiation-enhanced photolyase gene expression. PMID:17233721

  1. Virus-encoded chemokine receptors--putative novel antiviral drug targets.

    PubMed

    Rosenkilde, Mette M

    2005-01-01

    Large DNA viruses, in particular herpes- and poxviruses, have evolved proteins that serve as mimics or decoys for endogenous proteins in the host. The chemokines and their receptors serve key functions in both innate and adaptive immunity through control of leukocyte trafficking, and have as such a paramount role in the antiviral immune responses. It is therefore not surprising that viruses have found ways to exploit and subvert the chemokine system by means of molecular mimicry. By ancient acts of molecular piracy and by induction and suppression of endogenous genes, viruses have utilized chemokines and their receptors to serve a variety of roles in viral life-cycle. This review focuses on the pharmacology of virus-encoded chemokine receptors, yet also the family of virus-encoded chemokines and chemokine-binding proteins will be touched upon. Key properties of the virus-encoded receptors, compared to their closest endogenous homologs, are interactions with a wider range of chemokines, which can act as agonists, antagonists and inverse agonists, and the exploitation of many signal transduction pathways. High constitutive activity is another key property of some--but not all--of these receptors. The chemokine receptors belong to the superfamily of G-protein coupled 7TM receptors that per se are excellent drug targets. At present, non-peptide antagonists have been developed against many chemokine receptors. The potentials of the virus-encoded chemokine receptors as drug targets--ie. as novel antiviral strategies--will be highlighted here together with the potentials of the virus-encoded chemokines and chemokine-binding proteins as novel anti-inflammatory biopharmaceutical strategies. PMID:15617722

  2. Functional analysis of putative genes encoding the PIP2 water channel subfamily in Populus trichocarpa.

    PubMed

    Secchi, Francesca; MacIver, Bryce; Zeidel, Mark L; Zwieniecki, Maciej A

    2009-11-01

    We located fully sequenced putative genes of the plasma membrane intrinsic proteins (PIPs) family in the Populus trichocarpa (Torr. Gray), genome. Of 23 gene candidates, we assigned eight genes to the PIP2 subfamily. All eight putative genes were expressed in vegetative tissues (roots, leaves, bark and wood), and all of them showed water channel activity after being expressed in Xenopus oocytes. Six of eight proteins were affected by mercury ions. No proteins were affected by the presence of nickel or tungsten ions, or by lowering the pH of bathing external solution from 7.4 to 6.5. The presence of copper ions caused seven of eight PIP2 proteins to increase their water transport capacity by as much as 50%. This systematic study of the PIP2 subfamily of proteins in P. trichocarpa provides a basic overview of their activity as water channels and will be a useful reference for future physiological studies of plant water relations that use P. trichocarpa as a model system. PMID:19808706

  3. Cloning and characterization of CSP37, a novel gene encoding a putative membrane protein of Candida albicans.

    PubMed Central

    Sentandreu, M; Nieto, A; Iborra, A; Elorza, M V; Ponton, J; Fonzi, W A; Sentandreu, R

    1997-01-01

    In the course of an analysis of the functions and assembly of the cell wall of Candida albicans, we have cloned and characterized a gene, which we designated CSP37 (cell surface protein), encoding a 37-kDa polypeptide which is a membrane-associated protein. The gene was isolated by immunological screening of a DNA library constructed from mycelial cells with a polyclonal serum raised against cell walls of this morphology. Analysis of the nucleotide sequence of a corresponding genomic DNA fragment revealed a single open reading frame which encodes a predicted protein of 321 amino acids with no significant homology to others in the databases. Disruption of the CSP37 gene by the method described by Fonzi and Irwin (Genetics 134:717-728, 1993) eliminated expression of the Csp37 protein. The mutant strains showed no apparent defect in cell viability, growth, or cell wall assembly but displayed attenuated virulence in systemic infections induced in mice and reduced the ability to adhere to polystyrene. PMID:9244249

  4. Genes encoding putative natural killer cell C-type lectin receptors in teleostean fishes

    PubMed Central

    Sato, Akie; Mayer, Werner E.; Overath, Peter; Klein, Jan

    2003-01-01

    Mammalian natural killer (NK) cells are cytotoxic lymphocytes that express receptors specific for MHC class I molecules. The NK cell receptors belong to two structurally unrelated families, the killer cell Ig-like receptors and the killer cell C-type lectin receptors. We describe a cDNA clone derived from the bony (cichlid) fish Paralabidochromis chilotes and show that it encodes a protein related to the CD94/NK cell group 2 (NKG2) subfamily of the killer cell C-type lectin receptors. The gene encoding this receptor in a related species, Oreochromis niloticus, has a similar structure to the human CD94/NKG2 genes and is a member of a multigene cluster that resembles the mammalian NK cell gene complex. Thus, the CD94/NKG2 subfamily of NK cell receptors must have arisen before the divergence of fish and tetrapods and may have retained its function (possibly monitoring the expression of MHC class I molecules) for >400 million years. PMID:12802013

  5. Characterization of a putative S locus encoded receptor protein and its role in self-incompatibility

    SciTech Connect

    Not Available

    1991-01-01

    Monoclonal antibodies (MAb) were raised against purified SLSG and polyclonal antisera were raised against purified trpE/SLR1 fusion proteins. MAbH8 reacts with a protein epitope on SLSG. MAbH8 and the anit-SLR1 antisera were used with immunogold labeling to show SLSG and SLR1 proteins are localized in papillar cell walls in the stigma. MAbH8 reacts with SLSG from CRM+ cells but not CRM-cells; amino acid sequence identity between the two classes was only 65%, vs. 80% within the CRM+ class. SLSG is necessary but not sufficient for self-incompatibility. Variable molecular weight (MW) SLSG proteins are derived from the same SLG gene. MW variations in both SLSG and SLR1 are due to changes in glycosylation and phosphorylation state. SLSG is not detectable in mature pollen, but is expressed during microspore development. Using a SLG probe, a gene for a putative receptor with protein kinase activity was identified.

  6. A new gene encoding a putative transcription factor regulated by the Drosophila circadian clock.

    PubMed

    Rouyer, F; Rachidi, M; Pikielny, C; Rosbash, M

    1997-07-01

    Circadian rhythms of locomotor activity and eclosion in Drosophila depend upon the reciprocal autoregulation of the period (per) and timeless (tim) genes. As part of this regulatory loop, per and tim mRNA levels oscillate in a circadian fashion. Other cycling transcripts may participate in this central pacemaker mechanism or represent outputs of the clock. In this paper, we report the isolation of Crg-1, a new circadianly regulated gene. Like per and tim transcript levels, Crg-1 transcript levels oscillate with a 24 h period in light:dark (LD) conditions, with a maximal abundance at the beginning of the night. These oscillations persist in complete darkness and depend upon per and tim proteins. The putative CRG-1 proteins show some sequence similarity with the DNA-binding domain of the HNF3/fork head family of transcription factors. In the adult head, in situ hybridization analysis reveals that per and Crg-1 have similar expression patterns in the eyes and optic lobes. PMID:9233804

  7. Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea

    SciTech Connect

    Stein, J.C.; Howlett, B.; Boyes, D.C.; Nasrallah, M.E.; Nasrallah, J.B. )

    1991-10-01

    Self-recognition between pollen and stigma during pollination in Brassica oleracea is genetically controlled by the multiallelic self-incompatibility locus (S). The authors describe the S receptor kinase (SRK) gene, a previously uncharacterized gene that residues at the S locus. The nucleotide sequences of genomic DNA and of cDNAs corresponding to SRK predict a putative transmembrane receptor having serine/threonine-specific protein kinase activity. Its extracellular domain exhibits striking homology to the secreted product of the S-locus genotypes are highly polymorphic and have apparently evolved in unison with genetically linked alleles of SLG. SRK directs the synthesis of several alternative transcripts, which potentially encode different protein products, and these transcripts were detected exclusively in reproductive organs. The identification of SRK may provide new perspectives into the signal transduction mechanism underlying pollen recognition.

  8. Characterization and expression of two genes encoding isoforms of a putative Na, K-ATPase in the chytridiomycete Blastocladiella emersonii.

    PubMed

    Fietto, Luciano Gomes; Pugliese, Luciana; Gomes, Suely Lopes

    2002-06-01

    A P-type ATPase gene (BePAT1) from the aquatic fungus Blastocladiella emersonii, which surprisingly showed high similarity with the alpha-subunit of Na, K-ATPases from animal cells, has been reported recently [Biochim. Biophys. Acta 1383 (1998) 183]. In the present study, we describe the characterization of a second gene, denominated BePAT2, and show that these two genes have a different intron-exon structure but encode putative proteins with greater than 90% amino acid identity. Northern blot and multiplex reverse transcription and polymerase chain reaction (RT-PCR) assays have revealed that BePAT1 and BePAT2 genes have a non-coordinate, developmentally regulated expression during B. emersonii life cycle. Phosphoenzyme formation experiments using the immunopurified enzymes have indicated the presence of a Na, K-ATPase-like activity. Furthermore, immunofluorescence studies using B. emersonii zoospores localized the ATPases on the plasma membrane of these cells. PMID:12031485

  9. A Putative Gene Cluster from a Lyngbya wollei Bloom that Encodes Paralytic Shellfish Toxin Biosynthesis

    PubMed Central

    Mihali, Troco K.; Carmichael, Wayne W.; Neilan, Brett A.

    2011-01-01

    Saxitoxin and its analogs cause the paralytic shellfish-poisoning syndrome, adversely affecting human health and coastal shellfish industries worldwide. Here we report the isolation, sequencing, annotation, and predicted pathway of the saxitoxin biosynthetic gene cluster in the cyanobacterium Lyngbya wollei. The gene cluster spans 36 kb and encodes enzymes for the biosynthesis and export of the toxins. The Lyngbya wollei saxitoxin gene cluster differs from previously identified saxitoxin clusters as it contains genes that are unique to this cluster, whereby the carbamoyltransferase is truncated and replaced by an acyltransferase, explaining the unique toxin profile presented by Lyngbya wollei. These findings will enable the creation of toxin probes, for water monitoring purposes, as well as proof-of-concept for the combinatorial biosynthesis of these natural occurring alkaloids for the production of novel, biologically active compounds. PMID:21347365

  10. A putative novel nuclear-encoded subunit of the cytochrome c oxidase complex in trypanosomatids.

    PubMed

    Maslov, Dmitri A; Zíková, Alena; Kyselová, Iveta; Lukes, Julius

    2002-01-01

    A relatively large nuclear-encoded polypeptide, designated trCOIV, is found in the cytochrome c oxidase (CO) complex of trypanosomatids. In order to determine if this polypeptide represents a bona fide subunit of the complex, we have characterized the cDNA and the gene for this polypeptide in Leishmania tarentolae. Its nuclear gene has no sequence similarity to mammalian COIV. The trCOIV preprotein has a long mitochondrial targeting sequence of 31 residues. The mature polypeptide cofractionates with kinetoplast-mitochondria and its preferential mitochondrial localization was confirmed by immunofluorescence and immunoelectron microscopy. Based on the hydropathy plot analysis, the protein lacks pronounced transmembrane domains and likely occupies a peripheral position within the CO complex. The corresponding genes are also present in the sequenced portions of the Trypanosoma cruzi, Trypanosoma brucei and Leishmania major genomes, and the same polypeptide is found in cytochrome oxidase isolated from procyclic T. brucei and promastigote Leishmania mexicana amazonensis. However, the trCOIV gene, the mRNA and the polypeptide could not be detected in a respiration-deficient trypanosomatid Phytomonas serpens. PMID:12467979

  11. Arabidopsis Fragile Fiber8, Which Encodes a Putative Glucuronyltransferase, Is Essential for Normal Secondary Wall Synthesis

    PubMed Central

    Zhong, Ruiqin; Peña, Maria J.; Zhou, Gong-Ke; Nairn, C. Joseph; Wood-Jones, Alicia; Richardson, Elizabeth A.; Morrison, W. Herbert; Darvill, Alan G.; York, William S.; Ye, Zheng-Hua

    2005-01-01

    Secondary walls in vessels and fibers of dicotyledonous plants are mainly composed of cellulose, xylan, and lignin. Although genes involved in biosynthesis of cellulose and lignin have been intensively studied, little is known about genes participating in xylan synthesis. We found that Arabidopsis thaliana fragile fiber8 (fra8) is defective in xylan synthesis. The fra8 mutation caused a dramatic reduction in fiber wall thickness and a decrease in stem strength. FRA8 was found to encode a member of glycosyltransferase family 47 and exhibits high sequence similarity to tobacco (Nicotiana plumbaginifolia) pectin glucuronyltransferase. FRA8 is expressed specifically in developing vessels and fiber cells, and FRA8 is targeted to Golgi. Comparative analyses of cell wall polysaccharide fractions from fra8 and wild-type stems showed that the xylan and cellulose contents are drastically reduced in fra8, whereas xyloglucan and pectin are elevated. Further structural analysis of cell walls revealed that although wild-type xylans contain both glucuronic acid and 4-O-methylglucuronic acid residues, xylans from fra8 retain only 4-O-methylglucuronic acid, indicating that the fra8 mutation results in a specific defect in the addition of glucuronic acid residues onto xylans. These findings suggest that FRA8 is a glucuronyltransferase involved in the biosynthesis of glucuronoxylan during secondary wall formation. PMID:16272433

  12. Monoclonal antibody against a putative myristoylated membrane protein encoded by grouper iridovirus 59L gene.

    PubMed

    Chen, Zhi-Yu; Chiou, Pinwen Peter; Liou, Chian-Jiun; Lai, Yu-Shen

    2015-04-01

    Groupers (Epinephelus spp.) are economically important fish species worldwide, and ranaviruses are major viral pathogens causing heavy economic losses in grouper aquaculture. In this study, the 59L gene of grouper iridovirus (GIV-59L) was cloned and characterized. This gene is 1521 bp and encodes a protein of 506 amino acids with a predicted molecular mass of 53.9 kDa. Interestingly, GIV-59L and its homologs are found in all genera of the family Iridoviridae. A mouse monoclonal antibody specific for the C-terminal domain (amino acid positions 254-506) of the GIV-59L protein, GIV-59L(760-1518)-MAb-21, was produced and proved to be well suited for use in a number of GIV immunoassays. RT-PCR, Western blotting, and cycloheximide and cytosine arabinoside drug inhibition analyses indicated that GIV-59L is a viral late gene in GIV-infected grouper kidney cells. Immunofluorescence analysis revealed that GIV-59L protein mainly accumulates in the cytoplasm of infected cells and is finally packed into a whole virus particle. The GIV-59L(760-1518)-MAb-21 characterized in this study could have widespread application in GIV immunodiagnostics and other research on GIV. In addition, the results presented here offer important insights into the pathogenesis of GIV. PMID:25850399

  13. Characterization and Expression of DNA Sequences Encoding Putative Type-II Metallothioneins in the Seagrass Posidonia oceanica1

    PubMed Central

    Giordani, Tommaso; Natali, Lucia; Maserti, Bianca Elena; Taddei, Sonia; Cavallini, Andrea

    2000-01-01

    Posidonia oceanica is a marine phanerogam, largely widespread in the Mediterranean sea, representing an important food substrate for many marine organisms. A progressive reduction of P. oceanica meadows has been reported, due to anthropogenic coastal activity. Studying mechanisms by which this species responds to environmental stresses, three DNA sequences putatively encoding metallothioneins (MTs) have been isolated, by PCR. Two sequences, Pomt2a (accession no. AJ249603) and Pomt2b (accession no. AJ249602), show high similarities with genes encoding type-II MTs and are interrupted by two and one intron, respectively. The third sequence, Pomt2c (accession no. AJ249604), is supposed to be a pseudogene, originated by retrotranscription of the Pomt2b mRNA. These sequences belong to a multigene family with at least five members. Northern hybridizations indicated that MT transcripts accumulation is constitutive and seasonally regulated. MT encoding RNAs increase after rhyzome harvesting and (at a lesser extent) after 15 d of cultivation in an aquarium. As for animal MTs, transcripts accumulation is observed also after exposure to trace metals such as copper and cadmium. In the case of copper, the effect depends on concentration. Finally, taking into consideration the great interest in studying the biogeochemical cycle of mercury in the Mediterranean basin and since P. oceanica is commonly considered a bioindicator of this metal, the effect of mercury treatments on the accumulation of MT transcripts has been analyzed: in only a few experiments a small increase in the level of transcripts was recorded, suggesting that MTs are not key elements in the mercury accumulation by this species. PMID:10938373

  14. The BAT1 gene in the MHC encodes an evolutionarily conserved putative nuclear RNA helicase of the DEAD family

    SciTech Connect

    Peelman, L.J.; Van Zeveren, A.; Coppeiters, W.

    1995-03-20

    The BAT1 gene has previously been identified about 30 kb upstream from the tumor necrosis factor (TNF) locus and close to a NF{sub kb}-related gene of the nuclear factor family in the major histocompatibility complex (MHC) of human, mouse, and pig. We now show that the BAT1 translation product is the homolog of the rat p47 nuclear protein, the WM6 Drosophila gene product, and probably also Ce08102 of Caenorhabditis elegans, all members of the DEAD protein family of ATP-dependent RNA helicases. This family has more than 40 members, including the eukaryotic translation initiation factor-4A (eIF-4A), the human nuclear protein p68, and the Drosophila oocyte polar granule component vasa. BAT1 spans about 10 kb, is split into 10 exons of varying length, and encodes a protein of 428 amino acids ({approximately}48 kDa). Human and pig BAT1 cDNAs display 95.6% identity in the coding region and 80% identity in the 5{prime} and 3{prime} noncoding regions. Several repeat sequences of different types were identified in introns of the porcine BAT1 gene. Three different mRNAs, 4.1,1.7, and 0.9 kb, respectively, were detected in all tissues analyzed upon hybridization with porcine BAT1 cDNA. Transfection and expression of human BAT1 cDNA after tagging with a heterologous antibody recognition epitope revealed a nuclear localization of the hybrid protein. An MspI RFLP was detected in an SLA class I typed family, confirming the localization of the BAT1 gene in the porcine MHC. BAT1 thus encodes a putative nuclear ATP-dependent RNA helicase and is likely to have an indispensable function. 35 refs., 6 figs., 1 tab.

  15. Molecular and Functional Characterization of cDNAs Putatively Encoding Carboxylesterases from the Migratory Locust, Locusta migratoria

    PubMed Central

    Zhang, Jianqin; Li, Daqi; Ge, Pingting; Guo, Yaping; Zhu, Kun Yan; Ma, Enbo; Zhang, Jianzhen

    2014-01-01

    Carboxylesterases (CarEs) belong to a superfamily of metabolic enzymes encoded by a number of genes and are widely distributed in microbes, plants and animals including insects. These enzymes play important roles in detoxification of insecticides and other xenobiotics, degradation of pheromones, regulation of neurodevelopment, and control of animal development. In this study, we characterized a total of 39 full-length cDNAs putatively encoding different CarEs from the migratory locust, Locusta migratoria, one of the most severe insect pests in many regions of the world, and evaluated the role of four CarE genes in insecticide detoxification. Our phylogenetic analysis grouped the 39 CarEs into five different clades including 20 CarEs in clade A, 3 in D, 13 in E, 1 in F and 2 in I. Four CarE genes (LmCesA3, LmCesA20, LmCesD1, LmCesE1), representing three different clades (A, D and E), were selected for further analyses. The transcripts of the four genes were detectable in all the developmental stages and tissues examined. LmCesA3 and LmCesE1 were mainly expressed in the fat bodies and Malpighian tubules, whereas LmCesA20 and LmCesD1 were predominately expressed in the muscles and hemolymph, respectively. The injection of double-stranded RNA (dsRNA) synthesized from each of the four CarE genes followed by the bioassay with each of four insecticides (chlorpyrifos, malathion, carbaryl and deltamethrin) increased the nymphal mortalities by 37.2 and 28.4% in response to malathion after LmCesA20 and LmCesE1 were silenced, respectively. Thus, we proposed that both LmCesA20 and LmCesE1 played an important role in detoxification of malathion in the locust. These results are expected to help researchers reveal the characteristics of diverse CarEs and assess the risk of insecticide resistance conferred by CarEs in the locust and other insect species. PMID:24722667

  16. The putative cell cycle gene, enhancer of rudimentary, encodes a highly conserved protein found in plants and animals.

    PubMed

    Gelsthorpe, M; Pulumati, M; McCallum, C; Dang-Vu, K; Tsubota, S I

    1997-02-28

    The enhancer of rudimentary gene, e(r), in Drosophila melanogaster encodes a protein, ER, whose function has been implicated in pyrimidine biosynthesis and the cell cycle (Wojcik et al. (1994) Genetics 138, 1163-1170). In order to identify conserved regions of the protein and potentially important functional domains, the e(r) gene was cloned and sequenced from two other insects (Drosophila virilis and Aedes aegypti) and three vertebrates (Homo sapiens, Mus musculus, and Brachydanio rerio) and sequenced from a flowering plant (Arabidopsis thaliana). These sequences along with those of a nematode (Caenorhabditis elegans) exhibit a high degree of identity. ER of Drosophila melanogaster is 76% identical to the three vertebrate proteins, 49% identical to the nematode protein, and 40% identical to the plant protein. There is high evolutionary conservation among the vertebrates. The mouse and human proteins are identical and differ from that of the zebrafish by a single conservative amino-acid change (valine for isoleucine). A dramatic sequence conservation is seen in the position of the hydrophobic amino acids. Of the 27 positions occupied by hydrophobic amino acids in ER of Drosophila melanogaster, 25 of the corresponding positions in the human protein, 23 of the positions in Caenorhabditis elegans, and 20 of the positions in Arabidopsis thaliana have hydrophobic amino acids. Most of these residues are present in three conserved amphipathic alpha-helices, which are proposed to function in protein-protein interactions. Two phosphorylation sites for casein kinase II (CKII) have also been conserved within the animal groups. Purified ER from Drosophila melanogaster is phosphorylated in vitro by CKII, arguing that these two sites are functional in vivo. A putative shift in the secondary structure of ER caused by the phosphorylation of these sites suggests that CKII may be regulating the activity of the ER in vivo. PMID:9074495

  17. A Putatively Functional Haplotype in the Gene Encoding Transforming Growth Factor Beta-1 as a Potential Biomarker for Radiosensitivity

    SciTech Connect

    Schirmer, Markus A.; Brockmoeller, Juergen; Rave-Fraenk, Margret; Virsik, Patricia; Wilken, Barbara; Kuehnle, Elna; Campean, Radu; Hoffmann, Arne O.; Mueller, Katarina; Goetze, Robert G.; Neumann, Michael; Janke, Joerg H.; Nasser, Fatima; Wolff, Hendrik A.; Ghadimi, B. Michael; Schmidberger, Heinz; Hess, Clemens F.; Christiansen, Hans; Hille, Andrea

    2011-03-01

    Purpose: To determine whether genetic variability in TGFB1 is related to circulating transforming growth factor-{beta}1 (TGF-{beta}1) plasma concentrations after radiotherapy and to radiosensitivity of lymphoid cells. Patients and Methods: Transforming growth factor-{beta}1 plasma concentrations (n = 79) were measured in patients 1 year after radiotherapy and chromosomal aberrations (n = 71) ex vivo before therapy start. Furthermore, TGF-{beta}1 secretion and apoptosis were measured in isolated peripheral blood mononuclear cells of 55 healthy volunteers. These phenotypes were analyzed in relation to five germline polymorphisms in the 5' region of the TGFB1 gene. Because of high linkage disequilibrium, these five polymorphisms reflect frequent genetic variation in this region. A presumed impact of TGF-{beta}1 on DNA damage or repair was measured as micronucleus formation in 30 lymphoblastoid cell lines. Results: We identified a hypofunctional genetic haplotype termed H3 tagging the 5' region of the TGFB1 gene encoding TGF-{beta}1. H3 was associated with lower TGF-{beta}1 plasma concentrations in patients (p = 0.01) and reduced TGF-{beta}1 secretion in irradiated peripheral blood mononuclear cells (p = 0.003). Furthermore, cells with H3 were less prone to induction of chromosomal aberrations (p = 0.001) and apoptosis (p = 0.003) by irradiation. The hypothesis that high TGF-{beta}1 could sensitize cells to DNA damage was further supported by increased micronuclei formation in 30 lymphoblastoid cell lines when preincubated with TGF-{beta}1 before irradiation (p = 0.04). Conclusions: On the basis of TGF-{beta}1 plasma levels and radiation sensitivity of lymphoid cells, this study revealed a putatively hypofunctional TGFB1 haplotype. The significance of this haplotype and the suggested link between TGF-{beta}1 function and DNA integrity should be further explored in other cell types, as well as other experimental and clinical conditions.

  18. Identification of genes encoding zinc finger proteins, non-histone chromosomal HMG protein homologue, and a putative GTP phosphohydrolase in the genome of Chilo iridescent virus.

    PubMed Central

    Schnitzler, P; Hug, M; Handermann, M; Janssen, W; Koonin, E V; Delius, H; Darai, C

    1994-01-01

    Five RNA transcripts of about 1.2 to 1.7 kilobases were mapped to a part of the genome of insect iridescent virus type 6 (Chilo iridescent virus; CIV) between genome coordinates 0.832 and 0.856 within the EcoRI DNA fragment F. The nucleotide sequence of this particular region (5702 base pairs) of the CIV genome was determined. The DNA sequence contains a number of perfect direct, inverted, and palindromic repeats including three clusters of tandemly organized repetitive DNA elements located between the nucleotide positions 1534 to 1566, 3720 to 3780, and 4350 to 4450. Eight long open reading frames (ORFs; EF1 to 8) were detected in the sequenced region of the CIV genome. ORF EF1 encodes a putative protein of 221 amino acid residues (aa) that is closely related to eukaryotic nonhistone chromosomal proteins of the high mobility group (HMG) superfamily. Virus encoded homologues of HMG proteins have not been reported so far. The EF2 gene product (145 aa) contains a specific zinc finger motif and belongs to a distinct group of identified and putative zinc finger proteins including a second putative protein (239 aa) of CIV encoded in the EcoRI DNA fragment Y (1984 bp; 0.381 to 0.391 viral map units). The product of EF6 (127 aa) is related to D250 ORF product of African swine fever virus (ASFV) and belongs to the recently described protein family sharing a highly conserved sequence motif with bacterial antimutator GTP phosphohydrolase MutT. Thus the sequenced region of the CIV genome encodes three putative proteins which may be directly involved in the replication and/or transcription of the viral DNA. Images PMID:8121799

  19. TOM1, an Arabidopsis gene required for efficient multiplication of a tobamovirus, encodes a putative transmembrane protein.

    PubMed

    Yamanaka, T; Ohta, T; Takahashi, M; Meshi, T; Schmidt, R; Dean, C; Naito, S; Ishikawa, M

    2000-08-29

    Host-encoded factors play an important role in virus multiplication, acting in concert with virus-encoded factors. However, information regarding the host factors involved in this process is limited. Here we report the map-based cloning of an Arabidopsis thaliana gene, TOM1, which is necessary for the efficient multiplication of tobamoviruses, positive-strand RNA viruses infecting a wide variety of plants. The TOM1 mRNA is suggested to encode a 291-aa polypeptide that is predicted to be a multipass transmembrane protein. The Sos recruitment assay supported the hypothesis that TOM1 is associated with membranes, and in addition, that TOM1 interacts with the helicase domain of tobamovirus-encoded replication proteins. Taken into account that the tobamovirus replication complex is associated with membranes, we propose that TOM1 participates in the in vivo formation of the replication complex by serving as a membrane anchor. PMID:10944200

  20. Trypanosoma rangeli and Trypanosoma cruzi: molecular characterization of genes encoding putative calcium-binding proteins, highly conserved in trypanosomatids.

    PubMed

    Porcel, B M; Bontempi, E J; Henriksson, J; Rydåker, M; Aslund, L; Segura, E L; Pettersson, U; Ruiz, A M

    1996-12-01

    Genes encoding a 29-kDa flagellar calcium-binding protein (F29) in Trypanosoma cruzi, strongly homologous to EF-hand calcium-binding protein-encoding genes previously reported in this parasite, were isolated by immunoscreening. F29 is encoded by a number of very similar genes, highly conserved among different T. cruzi isolates. The genes are located on a pair of homologous chromosomes, arranged in one or two clusters of tandem repeats. PCR amplification of Trypanosoma rangeli genomic DNA, using primers derived from the T. cruzi F29 sequence made it possible to isolate the homologous gene in T. rangeli, encoding a 23-kDa protein called TrCaBP. Gene sequence comparisons showed homology to EF-hand calcium-binding proteins from T. cruzi (82.8%), Trypanosoma brucei brucei (60.2%), and Entamoeba histolytica (28.4%). Northern blot analysis revealed that the TrCaBP gene is expressed in T. rangeli as a polyadenylated transcript. The TrCaBP-encoding genes are present in at least 20 copies per cell, organized in tandem arrays, on large T. rangeli chromosomes in some isolates and on two smaller ones in others. This gene, however, seems to be absent from Leishmania. PMID:8948328

  1. A Putative Type III Secretion System Effector Encoded by the MA20_12780 Gene in Bradyrhizobium japonicum Is-34 Causes Incompatibility with Rj4 Genotype Soybeans.

    PubMed

    Tsurumaru, Hirohito; Hashimoto, Syougo; Okizaki, Kouhei; Kanesaki, Yu; Yoshikawa, Hirofumi; Yamakawa, Takeo

    2015-09-01

    The nodulation of Bradyrhizobium japonicum Is-34 is restricted by Rj4 genotype soybeans (Glycine max). To identify the genes responsible for this incompatibility, Tn5 mutants of B. japonicum Is-34 that were able to overcome this nodulation restriction were obtained. Analysis of the Tn5 mutants revealed that Tn5 was inserted into a region containing the MA20_12780 gene. In addition, direct disruption of this gene using marker exchange overcame the nodulation restriction by Rj4 genotype soybeans. The MA20_12780 gene has a tts box motif in its upstream region, indicating a possibility that this gene encodes a type III secretion system (T3SS) effector protein. Bioinformatic characterization revealed that the MA20_12780 protein contains the small ubiquitin-like modifier (SUMO) protease domain of the C48 peptidase (ubiquitin-like protease 1 [Ulp1]) family. The results of the present study indicate that a putative T3SS effector encoded by the MA20_12780 gene causes the incompatibility with Rj4 genotype soybeans, and they suggest the possibility that the nodulation restriction of B. japonicum Is-34 may be due to Rj4 genotype soybeans recognizing the putative T3SS effector (MA20_12780 protein) as a virulence factor. PMID:26092458

  2. Overexpression, crystallization and preliminary X-ray crystallographic analysis of a putative transposase from Thermoplasma acidophilum encoded by the Ta0474 gene

    SciTech Connect

    Kang, Ji Yong; Lee, Hyung Ho; Kim, Do Jin; Han, Sang Hee; Kim, Olesya; Kim, Hyoun Sook; Lee, Sang Jae; Suh, Se Won

    2006-11-01

    A putative transposase from T. acidophilum encoded by the Ta0474 gene was crystallized and X-ray diffraction data were collected to 1.78 Å. IS200 transposases, originally identified in Salmonella typhimurium LT2, are present in many bacteria and archaea and are distinct from other groups of transposases. To facilitate further structural comparisons among IS200-like transposases, structural analysis has been initiated of a putative transposase from Thermoplasma acidophilum encoded by the Ta0474 gene. Its 137-residue polypeptide shows high levels of sequence similarity to other members of the IS200 transposase family. The protein was overexpressed in intact form in Escherichia coli and crystallized at 297 K using a reservoir solution consisting of 100 mM Na HEPES pH 7.5 and 20%(v/v) ethanol. X-ray diffraction data were collected to 1.78 Å. The crystals belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 65.00, b = 34.07, c = 121.58 Å, α = 90, β = 100.20, γ = 90°. Four monomers, representing two copies of a dimeric molecule, are present in the asymmetric unit, giving a crystal volume per protein weight (V{sub M}) of 2.02 Å{sup 3} Da{sup −1} and a solvent content of 39.2%.

  3. A Putative Type III Secretion System Effector Encoded by the MA20_12780 Gene in Bradyrhizobium japonicum Is-34 Causes Incompatibility with Rj4 Genotype Soybeans

    PubMed Central

    Hashimoto, Syougo; Okizaki, Kouhei; Kanesaki, Yu; Yoshikawa, Hirofumi; Yamakawa, Takeo

    2015-01-01

    The nodulation of Bradyrhizobium japonicum Is-34 is restricted by Rj4 genotype soybeans (Glycine max). To identify the genes responsible for this incompatibility, Tn5 mutants of B. japonicum Is-34 that were able to overcome this nodulation restriction were obtained. Analysis of the Tn5 mutants revealed that Tn5 was inserted into a region containing the MA20_12780 gene. In addition, direct disruption of this gene using marker exchange overcame the nodulation restriction by Rj4 genotype soybeans. The MA20_12780 gene has a tts box motif in its upstream region, indicating a possibility that this gene encodes a type III secretion system (T3SS) effector protein. Bioinformatic characterization revealed that the MA20_12780 protein contains the small ubiquitin-like modifier (SUMO) protease domain of the C48 peptidase (ubiquitin-like protease 1 [Ulp1]) family. The results of the present study indicate that a putative T3SS effector encoded by the MA20_12780 gene causes the incompatibility with Rj4 genotype soybeans, and they suggest the possibility that the nodulation restriction of B. japonicum Is-34 may be due to Rj4 genotype soybeans recognizing the putative T3SS effector (MA20_12780 protein) as a virulence factor. PMID:26092458

  4. Sequencing and characterization of six cDNAs putatively encoding three pairs of pheromone receptors in two sibling species, Helicoverpa armigera and Helicoverpa assulta.

    PubMed

    Zhang, Dan-Dan; Zhu, Kun Yan; Wang, Chen-Zhu

    2010-06-01

    Pheromone receptors (PRs) on male antennae mediate specific and sensitive detection of sex pheromone components in lepidopterans. In this study, we identified and sequenced six putative cDNAs encoding PRs from sibling species, namely HarmOR1, HarmOR2 and HarmOR3 in Helicoverpa armigera and HassOR1, HassOR2 and HassOR3 in Helicoverpa assulta, which appeared to be orthologs of Heliothis virescens putative PR genes HvOR13, HvOR11 and HvOR16, respectively. Expression patterns of the six PR genes were evaluated by quantitative real-time PCR (qRT-PCR). All the putative PR genes exhibited male-biased expression patterns in adult antennae except for HarmOR2 and HassOR2 that showed similar expression levels in male and female antennae. Expression level of HarmOR1 was significantly higher than those of HarmOR2 and HarmOR3 in male antennae of H. armigera, but the three corresponding PR genes in male antennae of H. assulta showed similar expression levels. This implies the role of the PR encoded by HarmOR1 for interacting with Z11-16:Ald. The level of HarmOR1 transcript was significantly higher than that of HassOR1. These results were consistent with the ratio of Z11-16:Ald in their sex pheromone blends and the abundance of sensilla tuned to Z11-16:Ald on antennae of male adults of the two species. PMID:19962987

  5. Identification and transcriptional analysis of a Treponema pallidum operon encoding a putative ABC transport system, an iron-activated repressor protein homolog, and a glycolytic pathway enzyme homolog.

    PubMed

    Hardham, J M; Stamm, L V; Porcella, S F; Frye, J G; Barnes, N Y; Howell, J K; Mueller, S L; Radolf, J D; Weinstock, G M; Norris, S J

    1997-09-15

    We have characterized a 5.2-kilobase (kb) putative transport related operon (tro) locus of Treponema pallidum subsp. pallidum (Nichols strain) (Tp) encoding six proteins: TroA, TroB, TroC, TroD, TroR and Phosphoglycerate mutase (Pgm). Four of these gene products (TroA-TroD) are homologous to members of the ATP-Binding Cassette (ABC) superfamily of bacterial transport proteins. TroA (previously identified as Tromp1) has significant sequence similarity to a family of Gram-negative periplasmic substrate-binding proteins and to a family of streptococcal proteins that may have dual roles as substrate binding proteins and adhesins. TroB is homologous to the ATP-binding protein component, whereas TroC and TroD are related to the hydrophobic membrane protein components of ABC transport systems. TroR is similar to Gram-positive iron-activated repressor proteins (DesR, DtxR, IdeR, and SirR). The last open reading frame (ORF) of the tro operon encodes a protein that is highly homologous to the glycolytic pathway enzyme, Pgm. Primer extension results demonstrated that the tro operon is transcribed from a sigma 70-type promoter element. Northern analysis and reverse transcriptase-polymerase chain reactions provided evidence for the presence of a primary 1-kb troA transcript and a secondary, less abundant, troA-pgm transcript. The tro operon is flanked by a Holliday structure DNA helicase homolog (upstream) and two ORFs representing a purine nucleoside phosphorylase homolog and tpp15, a previously characterized gene encoding a membrane lipoprotein (downstream). The presence of a complex operon containing a putative ABC transport system and a DtxR homolog indicates a possible linkage between transport and gene regulation in Tp. PMID:9332349

  6. Molecular cloning and sequencing of a cDNA encoding partial putative molt-inhibiting hormone from Penaeus chinensis

    NASA Astrophysics Data System (ADS)

    Wang, Zai-Zhao; Xiang, Jian-Hai

    2002-09-01

    Total RNA was extracted from eyestalks of shrimp Penaeus chinensis. Eyestalk cDNA was obtained from total RNA by reverse transcription. Reverse transcriptase-polymerase chain reaction (RT-PCR) was initiated using eyestalk cDNA and degenerate primers designed from the amino acid sequence of molt-inhibiting hormone from shrimp Penaeus japonicus. A specific cDNA was obtained and cloned into a T vector for sequencing. The cDNA consisted of 201 base pairs and encoding for a peptide of 67 amino acid residues. The peptide of P. chinensis had the highest identity with molt-inhibiting hormones of P. japonicus. The cDNA could be a partial gene of molt-inhibiting hormones from P. chinensis. This paper reports for the first time cDNA encoding for neuropeptide of P. chinensis.

  7. The putative ABC transporter encoded by the orf19.4531 plays a role in the sensitivity of Candida albicans cells to azole antifungal drugs.

    PubMed

    Jiang, Linghuo; Xu, Dayong; Chen, Zhen; Cao, Yongbing; Gao, Pinghui; Jiang, Yuanying

    2016-05-01

    ATP-binding cassette (ABC) transporters constitute a large superfamily of integral membrane proteins in prokaryotic and eukaryotic cells. In the human fungal pathogen Candida albicans, there are 28 genes encoding ABC transporters and many of them have not been characterized so far. The orf19.4531 (also known as IPF7530) encodes a putative ABC transporter. In this study, we have demonstrated that disruption of orf19.4531 causes C. albicans cells to become tolerant to azoles, but not to polyene antifungals and terbinafine. Therefore, the protein encoded by orf19.4531 is involved in azole sensitivity and we name it as ROA1, the regulator of azole sensitivity 1 gene. Consistently, we show that the expression of ROA1 is responsive to treatment of either fluconazole or ketoconazole inC. albicans In addition, through a GFP tagging approach, Roa1 is localized in a small punctuate compartment adjacent to the vacuolar membrane. However, ROA1 is not essential for the in vitro filamentation of C. albicans cells. PMID:26975389

  8. The indigenous Pseudomonas plasmid pQBR103 encodes plant-inducible genes, including three putative helicases.

    PubMed

    Zhang, Xue-Xian; Lilley, Andrew K; Bailey, Mark J; Rainey, Paul B

    2004-12-27

    Plasmid pQBR103 ( approximately 400 kb) is representative of many self-transmissible, mercury resistant plasmids observed in the Pseudomonas community colonising the phytosphere of sugar beet. A promoter trapping strategy (IVET) was employed to identify pQBR103 genes showing elevated levels of expression on plant surfaces. Thirty-seven different plant-inducible gene fusions were isolated that were silent in laboratory media, but active in the plant environment. Three of the fusions were to DNA sequences whose protein products show significant homology to DNA-unwinding helicases. The three helicase-like genes, designated helA, helB and helC, are restricted to a defined group of related Pseudomonas plasmids. They are induced in both the root and shoot environments of sugar beet seedlings. Sequence analysis of the three plasmid-encoded helicase-like genes shows that they are phylogenetically distinct and likely to have independent evolutionary histories. The helA gene is predicted to encode a protein of 1121 amino acids, containing conserved domains found in the ultraviolet (UV) resistance helicase, UvrD. A helA knockout mutant was constructed and no phenotypic changes were found with plasmid-conferred UV resistance or plasmid conjugation. The other 34 fusions are unique with no homologues in the public gene databases, including the Pseudomonas genomes. These data demonstrate the presence of plant responsive genes in plasmid DNA comprising a component of the genomes of plant-associated bacteria. PMID:16329852

  9. Deletion of potD, encoding a putative spermidine-binding protein, results in a complex phenotype in Legionella pneumophila.

    PubMed

    Nasrallah, Gheyath K; Abdelhady, Hany; Tompkins, Nicholas P; Carson, Kaitlyn R; Garduño, Rafael A

    2014-07-01

    L. pneumophila is an intracellular pathogen that replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV). We previously observed that the polyamine spermidine, produced by host cells or added exogenously, enhances the intracellular growth of L. pneumophila. To study this enhancing effect and determine whether polyamines are used as nutrients, we deleted potD from L. pneumophila strain JR32. The gene potD encodes a spermidine-binding protein that in other bacteria is essential for the function of the PotABCD polyamine transporter. Deletion of potD did not affect L. pneumophila growth in vitro in the presence or absence of spermidine and putrescine, suggesting that PotD plays a redundant or no role in polyamine uptake. However, deletion of potD resulted in a puzzlingly complex phenotype that included defects in L. pneumophila's ability to form filaments, tolerate Na(+), associate with macrophages and amoeba, recruit host vesicles to the LCV, and initiate intracellular growth. Moreover, the ΔpotD mutant was completely unable to grow in L929 cells treated with a pharmacological inhibitor of spermidine synthesis. These complex and disparate effects suggest that the L. pneumophila potD encodes either: (i) a multifunctional protein, (ii) a protein that interacts with, or regulates a, multifunctional protein, or (iii) a protein that contributes (directly or indirectly) to a regulatory network. Protein function studies with the L. pneumophila PotD protein are thus warranted. PMID:24928741

  10. The yrpAB operon of Yersinia ruckeri encoding two putative U32 peptidases is involved in virulence and induced under microaerobic conditions.

    PubMed

    Navais, Roberto; Méndez, Jessica; Pérez-Pascual, David; Cascales, Desirée; Guijarro, José A

    2014-07-01

    In an attempt to dissect the virulence mechanisms of Yersinia ruckeri two adjacent genes, yrpA and yrpB, encoding putative peptidases belonging to the U32 family, were analyzed. Similar genes, with the same genetic organization were identified in genomic analysis of human-pathogenic yersiniae. RT-PCR studies indicated that these genes form an operon in Y. ruckeri. Transcriptional studies using an yrpB::lacZY fusion showed high levels of expression of these genes in the presence of peptone in the culture medium, as well as under oxygen-limited conditions. These two factors had a synergic effect on gene induction when both were present simultaneously during bacterial incubation, which indicates the important role that environmental conditions in the fish gut can play in the regulation of specific genes. LD 50 experiments using an yrpA insertional mutant strain demonstrated the participation of this gene in the virulence of Y. ruckeri. PMID:24865652

  11. The yeast Saccharomyces cerevisiae YDL112w ORF encodes the putative 2'-O-ribose methyltransferase catalyzing the formation of Gm18 in tRNAs.

    PubMed Central

    Cavaillé, J; Chetouani, F; Bachellerie, J P

    1999-01-01

    The protein sequences of three known RNA 2'-O-ribose methylases were used as probes for detecting putative homologs through iterative searches of genomic databases. We have identified 45 new positive Open Reading Frames (ORFs), mostly in prokaryotic genomes. Five complete eukaryotic ORFs were also detected, among which was a single ORF (YDL112w) in the yeast Saccharomyces cerevisiae genome. After genetic depletion of YDL112w, we observed a specific defect in tRNA ribose methylation, with the complete disappearance of Gm18 in all tRNAs that naturally contain this modification, whereas other tRNA ribose methylations and the complex pattern of rRNA ribose methylations were not affected. The tRNA G18 methylation defect was suppressed by transformation of the disrupted strain with a plasmid allowing expression of YDL112wp. The formation of Gm18 on an in vitro transcript of a yeast tRNASer naturally containing this methylation, which was efficiently catalyzed by cell-free extracts from the wild-type yeast strain, did not occur with extracts from the disrupted strain. The protein encoded by the YDL112w ORF, termed Trm3 (tRNA methylation), is therefore likely to be the tRNA (Gm18) ribose methylase. In in vitro assays, its activity is strongly dependent on tRNA architecture. Trm3p, the first putative tRNA ribose methylase identified in an eukaryotic organism, is considerably larger than its Escherichia coli functional homolog spoU (1,436 amino acids vs. 229 amino acids), or any known or putative prokaryotic RNA ribose methyltransferase. Homologs found in human (TRP-185 protein), Caenorhabditis elegans and Arabidopsis thaliana also exhibit a very long N-terminal extension not related to any protein sequence in databases. PMID:9917067

  12. Gene amplification at a locus encoding a putative Na+/H+ antiporter confers sodium and lithium tolerance in fission yeast.

    PubMed Central

    Jia, Z P; McCullough, N; Martel, R; Hemmingsen, S; Young, P G

    1992-01-01

    We have identified a new locus, sodium 2 (sod2) based on selection for increased LiCl tolerance in fission yeast, Schizosaccharomyces pombe. Tolerant strains have enhanced pH-dependent Na+ export capacity and sodium transport experiments suggest that the gene encodes an Na+/H+ antiport. The predicted sod2 gene product can be placed in the broad class of transporters which possess 12 hydrophobic transmembrane domains. The protein shows some sequence similarity to the human and bacterial Na+/H+ antiporters. Overexpression of sod2 increased Na+ export capacity and conferred sodium tolerance. Osmotolerance was not affected and sod2 cells were unaffected for growth in K+. In a sod2 disruption strain cells were incapable of exporting sodium. They were hypersensitive to Na+ or Li+ and could not grow under conditions that approximate pH7. The sod2 gene amplification could be selected stepwise and the degree of such amplification correlated with the level of Na+ or Li+ tolerance. Images PMID:1314171

  13. Two putative subunits of a peptide pump encoded in the human major histocompatability complex class 2 region

    SciTech Connect

    Bahram, S.; Arnold, D.; Bresnahan, M.; Strominger, J.L.; Spies, T. )

    1991-11-15

    The class 2 region of the human major histocompatibility complex (MHC) may encode several genes controlling the processing of endogenous antigen and the presentation of peptide epitopes by MHC class 1 molecules to cytotoxic T lymphocytes. A previously described peptide supply factor (PSF1) is a member of the multidrug-resistance family of transporters and may pump cytosolic peptides into the membrane-bound compartment where class 1 molecules assemble. A second transporter gene, PSF2, was identified 10 kilobases (kb) from PSF1, near the class 2 DOB gene. The complete sequences of PSF1 and PSF2 were determined from cDNA clones. The translation products are closely related in sequence and predicted secondary structure. Both contain a highly conserved ATP-binding fold and share 25% homology in a hydrophobic domain with a tentative number of eight membrane-spanning segments. Based on the principle dimeric organization of these two domains in other transporters, PSF1 and PSF2 may function as complementary subunits, independently as homodimers, or both. Taken together with previous genetic evidence, the coregulation of PSF1 and PSF2 by {gamma} interferon and the to-some-degree coordinate transcription of these genes suggest a common role in peptide-loading of class 1 molecules, although a distinct function of PSF2 cannot be ruled out.

  14. Gravistimulation changes expression of genes encoding putative carrier proteins of auxin polar transport in etiolated pea epicotyls

    NASA Astrophysics Data System (ADS)

    Hoshino, T.; Hitotsubashi, R.; Miyamoto, K.; Tanimoto, E.; Ueda, J.

    STS-95 space experiment has showed that auxin polar transport in etiolated epicotyls of pea (Pisum sativum L. cv. Alaska) seedlings is controlled by gravistimulation. In Arabidopsis thaliana auxin polar transport has considered to be regulated by efflux and influx carrier proteins in plasma membranes, AtPIN1 and AtAUX1, respectively. In order to know how gravistimuli control auxin polar transport in etiolated pea epicotyls at molecular levels, strenuous efforts have been made, resulting in successful isolation of full-length cDNAs of a putative auxin efflux and influx carriers, PsPIN2 and PsAUX1, respectively. Significantly high levels in homology were found on nucleotide and deduced amino acid sequences among PsPIN2, PsPIN1 (accession no. AY222857, Chawla and DeMason, 2003) and AtPINs, and also among PsAUX1, AtAUX1 and their related genes. Phylogenetic analyses based on the deduced amino acid sequences revealed that PsPIN2 belonged to a subclade including AtPIN3, AtPIN4 relating to lateral transport of auxin, while PsPIN1 belonged to the same clade as AtPIN1 relating to auxin polar transport. In the present study, we examined the effects of gravistimuli on the expression of PsPINs and PsAUX1 in etiolated pea seedlings by northern blot analysis. Expression of PsPIN1, PsPIN2 and PsAUX1 in hook region of 3.5-d-old etiolated pea seedlings grown under simulated microgravity conditions on a 3-D clinostat increased as compared with that of the seedlings grown under 1 g conditions. On the other hand, that of PsPIN1 and PsAUX1 in the 1st internode region under simulated microgravity conditions on a 3-D clinostat also increased, while that of PsPIN2 was affected little. These results suggest that expression of PsPIN1, PsPIN2 and PsAUX1 regulating polar/lateral transport of auxin is substantially under the control of gravity. A possible role of PsPINs and PsAUX1 of auxin polar transport in etiolated pea seedlings will also be discussed.

  15. ZNF1 Encodes a Putative C2H2 Zinc-Finger Protein Essential for Appressorium Differentiation by the Rice Blast Fungus Magnaporthe oryzae.

    PubMed

    Yue, Xiaofeng; Que, Yawei; Xu, Lin; Deng, Shuzhen; Peng, Youliang; Talbot, Nicholas J; Wang, Zhengyi

    2016-01-01

    The rice blast fungus Magnaporthe oryzae forms specialized infection structures called appressoria which are essential for gaining entry to plant tissue. Here, we report the identification of a novel nonpathogenic T-DNA-tagged mutant XF696 of M. oryzae with a single insertion in the promoter of ZNF1, which encodes a putative transcription factor (TF). Targeted gene deletion mutants of ZNF1 are nonpathogenic and unable to develop appressoria. However, Δznf1 mutants still respond to exogenous cyclic AMP on hydrophilic surfaces and can sense hydrophobic surfaces, initiating the differentiation of germ tubes. Interestingly, Δznf1 mutants also produce significantly more conidia compared with the isogenic wild-type strain. Quantitative reverse-transcription polymerase chain reaction analysis and green fluorescent protein fusion experiments revealed that expression of ZNF1 was highly induced during germination and appressorium development in M. oryzae and potentially regulated by the Pmk1 mitogen-activated protein kinase pathway. We observed that Δznf1 mutants are affected in mitosis and impaired in mobilization and degradation of lipid droplets and glycogen reserves during appressorium differentiation. Site-directed mutagenesis confirmed that three of the four C2H2 zinc-finger domains are essential for the function of Znf1. Taken together, we conclude that a C2H2 zinc-finger TF encoded by ZNF1 is essential for appressorium development by the rice blast fungus. PMID:26441322

  16. A Semidwarf Phenotype of Barley uzu Results from a Nucleotide Substitution in the Gene Encoding a Putative Brassinosteroid Receptor

    PubMed Central

    Chono, Makiko; Honda, Ichiro; Zeniya, Haruko; Yoneyama, Koichi; Saisho, Daisuke; Takeda, Kazuyoshi; Takatsuto, Suguru; Hoshino, Tsuguhiro; Watanabe, Yoshiaki

    2003-01-01

    Brassinosteroids (BRs) play important roles throughout plant growth and development. Despite the importance of clarifying the mechanism of BR-related growth regulation in cereal crops, BR-related cereal mutants have been identified only in rice (Oryza sativa). We previously found that semidwarf barley (Hordeum vulgare) accessions carrying the “uzu” gene, called “uzu” barley in Japan, are non-responding for brassinolide (BL). We then performed chemical and molecular analyses to clarify the mechanisms of uzu dwarfism using isogenic line pairs of uzu gene. The response of the uzu line to BL was significantly lower than that of its corresponding normal line. Measurement of BRs showed that the uzu line accumulates BRs, similar to known BR-insensitive mutants. The marker synteny of rice and barley chromosomes suggests that the uzu gene may be homologous to rice D61, a rice homolog of Arabidopsis BR-insensitive 1 (BRI1), encoding a BR-receptor protein. A barley homolog of BRI1, HvBRI1, was isolated by using degenerate primers. A comparison of HvBRI1 sequences in uzu and normal barley varieties showed that the uzu phenotype is correlated with a single nucleotide substitution. This substitution results in an amino acid change at a highly conserved residue in the kinase domain of the BR-receptor protein. These results may indicate that uzu dwarfism is caused by the missense mutation in HvBRI1. The uzu gene is being introduced into all hull-less barley cultivars in Japan as an effective dwarf gene for practical use, and this is the first report about an agronomically important mutation related to BRs. PMID:14551335

  17. Characterization of gprK Encoding a Putative Hybrid G-Protein-Coupled Receptor in Aspergillus fumigatus.

    PubMed

    Jung, Mun-Gu; Kim, Sung Su; Yu, Jae-Hyuk; Shin, Kwang-Soo

    2016-01-01

    The G-protein-coupled receptor (GPCR) family represents the largest and most varied collection of membrane embedded proteins that are sensitized by ligand binding and interact with heterotrimeric G proteins. Despite their presumed critical roles in fungal biology, the functions of the GPCR family members in the opportunistic human pathogen Aspergillus fumigatus are largely unknown, as only two (GprC and GprD) of the 15 predicted GPCRs have been studied. Here, we characterize the gprK gene, which is predicted to encode a hybrid GPCR with both 7-transmembrane and regulator of G-protein signaling (RGS) domains. The deletion of gprK causes severely impaired asexual development coupled with reduced expression of key developmental activators. Moreover, ΔgprK results in hyper-activation of germination even in the absence of carbon source, and elevated expression and activity of the protein kinase A PkaC1. Furthermore, proliferation of the ΔgprK mutant is restricted on the medium when pentose is the sole carbon source, suggesting that GprK may function in external carbon source sensing. Notably, the absence of gprK results in reduced tolerance to oxidative stress and significantly lowered mRNA levels of the stress-response associated genes sakA and atfA. Activities of catalases and SODs are severely decreased in the ΔgprK mutant, indicating that GprK may function in proper activation of general stress response. The ΔgprK mutant is also defective in gliotoxin (GT) production and slightly less virulent toward the greater wax moth, Galleria mellonella. Transcriptomic studies reveal that a majority of transporters are down-regulated by ΔgprK. In summary, GprK is necessary for proper development, GT production, and oxidative stress response, and functions in down-regulating the PKA-germination pathway. PMID:27584150

  18. Evidence for a methyl-accepting chemotaxis protein gene (mcp1) that encodes a putative sensory transducer in virulent Treponema pallidum.

    PubMed Central

    Hagman, K E; Porcella, S F; Popova, T G; Norgard, M V

    1997-01-01

    The clinical and histopathological manifestations of syphilis and the invasive behavior of Treponema pallidum in tissue culture systems reflect the propensity for treponemes to migrate through skin, hematogenously disseminate, and invade targeted tissues. Treponemal motility is believed to be essential to this process and thereby an important facet of syphilis pathogenesis. By analogy with other bacterial pathogens, it is plausible that treponemal motility and tissue invasion are modulated by sensory transduction events associated with chemotactic responses. Recent studies have demonstrated the existence in T. pallidum of accessory molecules typically associated with sensory transduction events involving methyl-accepting chemotaxis proteins (MCPs). Intrinsic radiolabeling of T. pallidum in vitro with L-[methyl-3H] methionine revealed one methylated treponemal polypeptide with an apparent molecular mass of 64 kDa. A degenerate oligonucleotide probe corresponding to a highly conserved C-terminal domain within Bacillus subtilis and Escherichia coli MCPs was used in Southern blotting of T. pallidum DNA to identify and subsequently clone a putative T. pallidum MCP gene (mcp1). Computer analyses predicted a near-consensus promoter upstream of mcp1, and primer extension analysis employing T. pallidum RNA revealed a transcriptional initiation site. T. pallidum mcp1 encoded a 579-amino-acid (64.6-kDa) polypeptide which was highly homologous to at least 69 other known or putative sensory transducer proteins, with the highest degrees of homology existing between the C terminus of mcp1 and the C-terminal (signaling) domains of the other bacterial MCPs. Other salient features of Mcp1 included (i) six potential membrane-spanning domains at the N terminus, (ii) two predicted alpha-helical coiled coil regions containing at least three putative methylation sites, and (iii) homologies with two ligand-binding domains (LI-1 and LI-2) of the E. coli MCPs Trg and Tar. This study is the

  19. Klebsiella pneumoniae Asparagine tDNAs Are Integration Hotspots for Different Genomic Islands Encoding Microcin E492 Production Determinants and Other Putative Virulence Factors Present in Hypervirulent Strains

    PubMed Central

    Marcoleta, Andrés E.; Berríos-Pastén, Camilo; Nuñez, Gonzalo; Monasterio, Octavio; Lagos, Rosalba

    2016-01-01

    Due to the developing of multi-resistant and invasive hypervirulent strains, Klebsiella pneumoniae has become one of the most urgent bacterial pathogen threats in the last years. Genomic comparison of a growing number of sequenced isolates has allowed the identification of putative virulence factors, proposed to be acquirable mainly through horizontal gene transfer. In particular, those related with synthesizing the antibacterial peptide microcin E492 (MccE492) and salmochelin siderophores were found to be highly prevalent among hypervirulent strains. The determinants for the production of both molecules were first reported as part of a 13-kbp segment of K. pneumoniae RYC492 chromosome, and were cloned and characterized in E. coli. However, the genomic context of this segment in K. pneumoniae remained uncharacterized. In this work, we provided experimental and bioinformatics evidence indicating that the MccE492 cluster is part of a highly conserved 23-kbp genomic island (GI) named GIE492, that was integrated in a specific asparagine-tRNA gene (asn-tDNA) and was found in a high proportion of isolates from liver abscesses sampled around the world. This element resulted to be unstable and its excision frequency increased after treating bacteria with mitomycin C and upon the overexpression of the island-encoded integrase. Besides the MccE492 genetic cluster, it invariably included an integrase-coding gene, at least seven protein-coding genes of unknown function, and a putative transfer origin that possibly allows this GI to be mobilized through conjugation. In addition, we analyzed the asn-tDNA loci of all the available K. pneumoniae assembled chromosomes to evaluate them as GI-integration sites. Remarkably, 73% of the strains harbored at least one GI integrated in one of the four asn-tDNA present in this species, confirming them as integration hotspots. Each of these tDNAs was occupied with different frequencies, although they were 100% identical. Also, we identified

  20. Molecular Cloning and Characterization of cDNA Encoding a Putative Stress-Induced Heat-Shock Protein from Camelus dromedarius

    PubMed Central

    Elrobh, Mohamed S.; Alanazi, Mohammad S.; Khan, Wajahatullah; Abduljaleel, Zainularifeen; Al-Amri, Abdullah; Bazzi, Mohammad D.

    2011-01-01

    Heat shock proteins are ubiquitous, induced under a number of environmental and metabolic stresses, with highly conserved DNA sequences among mammalian species. Camelus dromedaries (the Arabian camel) domesticated under semi-desert environments, is well adapted to tolerate and survive against severe drought and high temperatures for extended periods. This is the first report of molecular cloning and characterization of full length cDNA of encoding a putative stress-induced heat shock HSPA6 protein (also called HSP70B′) from Arabian camel. A full-length cDNA (2417 bp) was obtained by rapid amplification of cDNA ends (RACE) and cloned in pET-b expression vector. The sequence analysis of HSPA6 gene showed 1932 bp-long open reading frame encoding 643 amino acids. The complete cDNA sequence of the Arabian camel HSPA6 gene was submitted to NCBI GeneBank (accession number HQ214118.1). The BLAST analysis indicated that C. dromedaries HSPA6 gene nucleotides shared high similarity (77–91%) with heat shock gene nucleotide of other mammals. The deduced 643 amino acid sequences (accession number ADO12067.1) showed that the predicted protein has an estimated molecular weight of 70.5 kDa with a predicted isoelectric point (pI) of 6.0. The comparative analyses of camel HSPA6 protein sequences with other mammalian heat shock proteins (HSPs) showed high identity (80–94%). Predicted camel HSPA6 protein structure using Protein 3D structural analysis high similarities with human and mouse HSPs. Taken together, this study indicates that the cDNA sequences of HSPA6 gene and its amino acid and protein structure from the Arabian camel are highly conserved and have similarities with other mammalian species. PMID:21845074

  1. Transcriptional Organization and Regulatory Elements of a Pseudomonas sp. Strain ADP Operon Encoding a LysR-Type Regulator and a Putative Solute Transport System

    PubMed Central

    Platero, Ana Isabel; García-Jaramillo, Manuel; Santero, Eduardo

    2012-01-01

    The atzS-atzT-atzU-atzV-atzW gene cluster of the Pseudomonas sp. strain ADP atrazine-degradative plasmid pADP-1, which carries genes for an outer membrane protein and the components of a putative ABC-type solute transporter, is located downstream from atzR, which encodes the LysR-type transcriptional regulator of the cyanuric acid-degradative operon atzDEF. Here we describe the transcriptional organization of these genes. Our results show that all six genes are cotranscribed from the PatzR promoter to form the atzRSTUVW operon. A second, stronger promoter, PatzT, is found within atzS and directs transcription of the four distal genes. PatzT is σN dependent, activated by NtrC in response to nitrogen limitation with the aid of IHF, and repressed by AtzR. A combination of in vivo mutational analysis and primer extension allowed us to locate the PatzT promoter and map the transcriptional start site. Similarly, we used deletion and point mutation analyses, along with in vivo expression studies and in vitro binding assays, to locate the NtrC, IHF, and AtzR binding sites and address their functionality. Our results suggest a regulatory model in which NtrC activates PatzT transcription via DNA looping, while AtzR acts as an antiactivator that diminishes expression by interfering with the activation process. PMID:23042989

  2. Overexpression of the OsChI1 gene, encoding a putative laccase precursor, increases tolerance to drought and salinity stress in transgenic Arabidopsis.

    PubMed

    Cho, Hyun Yong; Lee, Chanhui; Hwang, Sun-Goo; Park, Yong Chan; Lim, Hye Lee; Jang, Cheol Seong

    2014-11-15

    In a previous study, we identified a number of genes induced by chilling using a microarray approach. In order to investigate the molecular mechanism underlying chilling tolerance and possible crosstalk with other abiotic stresses, we selected a rice gene, OsChI1 (Os01g61160), for further analysis. The OsChI1 gene encodes a putative laccase precursor protein. In accordance with our previous results, its transcript is highly accumulated during a 12-day period of chilling treatment. Higher expression of the OsChI1 gene was also detected in roots and tissues at the vegetative and productive stages. In addition, we also observed increased transcript levels of the OsChI1 gene during dehydration and high salinity conditions. Transient expression of OsChI1 proteins tagged with fluorescence protein in rice protoplasts revealed that OsChI1 is localized in the plasma membrane. The Arabidopsis transgenic plants overexpressing OsChI1-EGFP resulted in an increased tolerance to drought and salinity stress. In silico analysis of OsChI1 suggests that several genes coexpressed with OsChI1 in the root during various abiotic stresses, such as chilling, drought and salt stress, may play an important role in the ROS signaling pathway. Potential roles of OsChI1 in response to abiotic stresses are discussed. PMID:25218040

  3. LMOh7858_0369, a gene encoding a putative leucine-rich repeat-containing protein, is required for virulence of Listeria monocytogenes.

    PubMed

    Zhang, Ting; Bae, Dongryeoul; Wang, Chinling

    2016-05-01

    Listeria monocytogenes possesses the highest number of leucine-rich repeat (LRR)-containing proteins among all Gram-positive bacteria; these LRR-containing molecules are known as the 'internalin' family. To understand the functions of largely uncharacterized LRR-containing molecules, we constructed seven deletion mutants in the L. monocytogenes H7858 strain targeting genes in this family and tested their virulence. Among the seven mutants, the ΔLMOh7858_0369 strain and the ΔLMOh7858_2546 strain showed significantly impaired invasiveness of HepG2 cells. We further tested the virulence of these two strains in the intravascular sepsis model using BALB/c mice. Interestingly, the ΔLMOh7858_0369 strain showed significant reduction in organ colonization, bacteremia and invasion of the brain compared with the parental wild-type strain. Host immune responses to listerial intravascular infection were measured at 24 and 72 h post-infection. Transcript levels of several proinflammatory cytokines and chemokines were significantly lower when induced by the ΔlmOh7858_0369 strain than when induced by the wild type. These results suggest that the putative LRR-containing protein encoded by LMOh7858_0369 might be a novel virulence factor of the L. monocytogenes H7858 strain. PMID:26976852

  4. EFO1 and EFO2, encoding putative WD-domain proteins, have overlapping and distinct roles in the regulation of vegetative development and flowering of Arabidopsis.

    PubMed

    Wang, Wuyi; Yang, Dennis; Feldmann, Kenneth A

    2011-01-01

    From screening a population of Arabidopsis overexpression lines, two Arabidopsis genes were identified, EFO1 (early flowering by overexpression 1) and EFO2, that confer early flowering when overexpressed. The two genes encode putative WD-domain proteins which share high sequence similarity and constitute a small subfamily. Interestingly, the efo2-1 loss-of-function mutant also flowered earlier in short days and slightly earlier in long days than the wild type, while no flowering-time or morphological differences were observed in efo1-1 relative to the wild type. In addition, the efo2-1 mutation perturbed hypocotyl elongation, leaf expansion and formation, and stem elongation. EFO1 and EFO2 are both regulated by the circadian clock. Expression and genetic analyses revealed that EFO2 suppresses flowering largely through the action of CONSTANS (CO) and flowering locus T (FT), suggesting that EFO2 is a negative regulator of photoperiodic flowering. The growth defects in efo2-1 were augmented in efo1 efo2, but the induction of FT in the double mutant was comparable to that in efo2-1. Thus, while EFO2 acts as a floral repressor, EFO1 may not be directly involved in flowering, but the two genes do have overlapping roles in regulating other developmental processes. EFO1 and EFO2 may function collectively to serve as one of the converging points where the signals of growth and flowering intersect. PMID:21242318

  5. Molecular cloning and characterization of a novel repeat-containing Leishmania major gene, ppg1, that encodes a membrane-associated form of proteophosphoglycan with a putative glycosylphosphatidylinositol anchor.

    PubMed

    Ilg, T; Montgomery, J; Stierhof, Y D; Handman, E

    1999-10-29

    Leishmania parasites secrete a variety of proteins that are modified by phosphoglycan chains structurally similar to those of the cell surface glycolipid lipophosphoglycan. These proteins are collectively called proteophosphoglycans. We report here the cloning and sequencing of a novel Leishmania major proteophosphoglycan gene, ppg1. It encodes a large polypeptide of approximately 2300 amino acids. The N-terminal domain of approximately 70 kDa exhibits 11 imperfect amino acid repeats that show some homology to promastigote surface glycoproteins of the psa2/gp46 complex. The large central domain apparently consists exclusively of approximately 100 repetitive peptides of the sequence APSASSSSA(P/S)SSSSS(+/-S). Gene fusion experiments demonstrate that these peptide repeats are the targets of phosphoglycosylation in Leishmania and that they form extended filamentous structures reminiscent of mammalian mucins. The C-terminal domain contains a functional glycosylphosphatidylinositol anchor addition signal sequence, which confers cell surface localization to a normally secreted Leishmania acid phosphatase, when fused to its C terminus. Antibody binding studies show that the ppg1 gene product is phosphoglycosylated by phosphoglycan repeats and cap oligosaccharides. In contrast to previously characterized proteophosphoglycans, the ppg1 gene product is predominantly membrane-associated and it is expressed on the promastigote cell surface. Therefore this membrane-bound proteophosphoglycan may be important for direct host-parasite interactions. PMID:10531342

  6. TCFL4: a gene at 17q21.1 encoding a putative basic helix-loop-helix leucine-zipper transcription factor.

    PubMed

    Bjerknes, M; Cheng, H

    1996-11-28

    TCFL4 (transcription factor like 4) is the HGMW-approved symbol for the gene of a widely expressed putative basic helix-loop-helix leucine-zipper (bHLH-zip) transcription factor which is located 3' to HSD17B1 (17-beta-hydroxysteroid dehydrogenase gene) at 17q21.1, centromeric to the BRCA1 (a gene implicated in familial breast cancer) locus. We report the human gene structure and the murine cDNA sequence of two variants, about 1.5 and 2.2 kb in size. The deduced protein is highly conserved between mouse and man. PMID:8973301

  7. Analysis of the Corynebacterium diphtheriae DtxR regulon: identification of a putative siderophore synthesis and transport system that is similar to the Yersinia high-pathogenicity island-encoded yersiniabactin synthesis and uptake system.

    PubMed

    Kunkle, Carey A; Schmitt, Michael P

    2003-12-01

    The diphtheria toxin repressor, DtxR, is a global iron-dependent regulatory protein in Corynebacterium diphtheriae that controls gene expression by binding to 19-bp operator sequences. To further define the DtxR regulon in C. diphtheriae, a DtxR repressor titration assay (DRTA) was developed and used to identify 10 previously unknown DtxR binding sites. Open reading frames downstream from seven of the newly identified DtxR binding sites are predicted to encode proteins associated with iron or heme transport. Electrophoretic mobility shift assays indicated that DtxR was able to bind to DNA fragments carrying the 19-bp operator regions, and transcriptional analysis of putative promoter elements adjacent to the binding site sequences revealed that most of these regions displayed iron- and DtxR-regulated activity. A putative siderophore biosynthesis and transport operon located downstream from one of the DtxR binding sites, designated sid, is similar to the yersiniabactin synthesis and uptake genes encoded on the Yersinia pestis high pathogenicity island. The siderophore biosynthetic genes in the sid operon contained a large deletion in the C. diphtheriae C7 strain, but the sid genes were unaffected in four clinical isolates that are representative of the dominant strains from the recent diphtheria epidemic in the former Soviet Union. Mutations in the siderophore biosynthetic genes in a clinical strain had no effect on siderophore synthesis or growth in low-iron conditions; however, a mutation in one of the putative transport proteins, cdtP, resulted in reduced growth in iron-depleted media, which suggests that this system may have a role in iron uptake. The findings from this study indicate that C. diphtheriae contains at least 18 DtxR binding sites and that DtxR may affect the expression of as many as 40 genes. PMID:14617647

  8. The ken and barbie gene encoding a putative transcription factor with a BTB domain and three zinc finger motifs functions in terminalia development of Drosophila.

    PubMed

    Lukacsovich, Tamas; Yuge, Kazuya; Awano, Wakae; Asztalos, Zoltan; Kondo, Shunzo; Juni, Naoto; Yamamoto, Daisuke

    2003-10-01

    Mutations in the ken and barbie locus are accompanied by the malformation of terminalia in adult Drosophila. Male and female genitalia often remain inside the body, and the same portions of genitalia and analia are missing in a fraction of homozygous flies. Rotated and/or duplicated terminalia are also observed. Terminalia phenotypes are enhanced by mutations in the gap gene tailless, the homeobox gene caudal, and the decapentaplegic gene that encodes a TGFbeta-like morphogen. The ken and barbie gene encodes a protein with three CCHH-type zinc finger motifs that are conserved in several transcription factors such as Krüppel and BCL-6. All defects in ken and barbie mutants are fully rescued by the expression of a wild-type genomic construct, which establishes the causality between phenotypes and the gene. PMID:14518006

  9. Phylogenetic Relationships and Functional Genes: Distribution of a Gene (mnxG) Encoding a Putative Manganese-Oxidizing Enzyme in Bacillus Species▿

    PubMed Central

    Mayhew, Lisa E.; Swanner, Elizabeth D.; Martin, Andy P.; Templeton, Alexis S.

    2008-01-01

    Several Bacillus and Paenibacillus species were isolated from Fe and Mn oxide minerals precipitating at a deep subsurface oxic-anoxic interface at Henderson Molybdenum Mine, Empire, CO. The isolates were investigated for their Mn(II)-oxidizing potential and interrogated for possession of the mnxG gene, a gene that codes for a putative Mn(II)-oxidizing enzyme in Bacillus species. Seven of eight Bacillus species were capable of Mn(II) oxidation; however, the mnxG gene was detected in only one isolate. Using sequences of known Bacillus species both with and without amplifiable mnxG genes and Henderson Mine isolates, the 16S rRNA and mnxG gene phylogenies were compared to determine if 16S rRNA sequences could be used to predict the presence or absence of an amplifiable mnxG gene within the genomes of the isolates. We discovered a strong correspondence between 16S rRNA sequence similarity and the presence/absence of an amplifiable mnxG gene in the isolates. The data revealed a complex phylogenetic distribution of the mnxG gene in which vertical inheritance and gene loss influence the distribution of the gene among the Bacillus species included in this study. Comparisons of 16S rRNA and functional gene phylogenies can be used as a tool to aid in unraveling the history and dispersal of the mnxG gene within the Bacillus clade. PMID:18849460

  10. Molecular characterization of two human autoantigens: unique cDNAs encoding 95- and 160-kD proteins of a putative family in the Golgi complex.

    PubMed

    Fritzler, M J; Hamel, J C; Ochs, R L; Chan, E K

    1993-07-01

    Serum autoantibodies from a patient with autoantibodies directed against the Golgi complex were used to screen clones from a HepG2 lambda Zap cDNA library. Three related clones, designated SY2, SY10, and SY11, encoding two distinct polypeptides were purified for further analysis. Antibodies affinity purified by adsorption to the lambda Zap-cloned recombinant proteins and antibodies from NZW rabbits immunized with purified recombinant proteins reproduced Golgi staining and bound two different proteins, 95 and 160 kD, from whole cell extracts. The SY11 protein was provisionally named golgin-95 and the SY2/SY10 protein was named golgin-160. The deduced amino acid sequence of the cDNA clone of SY2 and SY11 represented 58.7- and 70-kD proteins of 568 and 620 amino acids. The in vitro translation products of SY2 and SY11 cDNAs migrated in SDS-PAGE at 65 and 95 kD, respectively. The in vitro translated proteins were immunoprecipitated by human anti-Golgi serum or immune rabbit serum, but not by normal human serum or preimmune rabbit serum. Features of the cDNA suggested that SY11 was a full-length clone encoding golgin-95 but SY2 and SY10 together encoded a partial sequence of golgin-160. Analysis of the SY11 recombinant protein identified a leucine zipper spanning positions 419-455, a glutamic acid-rich tract spanning positions 322-333, and a proline-rich tract spanning positions 67-73. A search of the SwissProt data bank indicated sequence similarity of SY11 to human restin, the heavy chain of kinesin, and the heavy chain of myosin. SY2 shared sequence similarity with the heavy chain of myosin, the USO1 transport protein from yeast, and the 150-kD cytoplasmic dynein-associated polypeptide. Sequence analysis demonstrated that golgin-95 and golgin-160 share 43% sequence similarity and, therefore, may be functionally related proteins. PMID:8315394

  11. Cloning of a cDNA encoding a putative human very low density lipoprotein/Apolipoprotein E receptor and assignment of the gene to chromosome 9pter-p23[sup 6

    SciTech Connect

    Gafvels, M.E.; Strauss, J.F. III ); Caird, M.; Patterson, D. ); Britt, D.; Jackson, C.L. )

    1993-11-01

    The authors report the cloning of a 3656-bp cDNA encoding a putative human very low density lipoprotein (VLDL)/apolipoprotein E (ApoE) receptor. The gene encoding this protein was mapped to chromosome 9pter-p23. Northern analysis of human RNA identified cognate mRNAs of 6.0 and 3.8 kb with most abundant expression in heart and skeletal muscle, followed by kidney, placenta, pancreas, and brain. The pattern of expression generally paralleled that of lipoprotein lipase mRNA but differed from that of the low density lipoprotein (LDL) receptor and the low density lipoprotein receptor-related protein/[alpha][sub 2]-macroglobulin receptor (LRP), which are members of the same gene family. VLDL/ApoE receptor message was not detected in liver, whereas mRNAs for both LDL receptor and LRP were found in hepatic tissue. In mouse 3T3-L1 cells, VLDL/ApoE receptor mRNA was induced during the transformation of the cells into adipocytes. Expression was also detected in human choriocarcinoma cells, suggesting that at least part of the expression observed in placenta may be in trophoblasts, cells which would be exposed to maternal blood. Expression in brain may be related to high levels of ApoE expression in that organ, an observation of potential relevance to the recently hypothesized role for ApoE in late onset Alzheimer disease. The results suggest that the putative VLDL/ApoE receptor could play a role in the uptake of triglyceride-rich lipoprotein particles by specific organs including striated and cardiac muscle and adipose tissue and in the transport of maternal lipids across the placenta. The findings presented here, together with recent observations from other laboratories, bring up the possibility that a single gene, the VLDL/ApoE receptor, may play a role in the pathogenesis of certain forms of atherosclerosis, Alzheimer disease, and obesity.

  12. A mutation in the Cc.arp9 gene encoding a putative actin-related protein causes defects in fruiting initiation and asexual development in the agaricomycete Coprinopsis cinerea.

    PubMed

    Nakazawa, Takehito; Ando, Yuki; Hata, Takeshi; Nakahori, Kiyoshi

    2016-08-01

    Agaricomycetes exhibit a remarkable morphological differentiation from vegetative mycelia to huge fruiting bodies. To investigate the molecular mechanism underlying the fruiting body development, we have isolated and characterized many Coprinopsis cinerea mutant strains defective in fruiting initiation to date. Dikaryon formation in agaricomycetes, which is followed by fruiting development, is governed by the mating type loci, A and B. Recently, mutations in the Cc.snf5 gene, which encodes a putative component of the chromatin remodeling complex switch/sucrose non-fermentable (SWI/SNF), were shown to cause defects in A-regulated clamp cell morphogenesis, as well as in fruiting initiation. Here, we demonstrate that Cc.arp9, which encodes a putative actin-related protein associated with two chromatin remodeling complexes, SWI/SNF and remodels the structure of chromatin (RSC), is also essential for fruiting initiation. In contrast to Cc.snf5 mutants, Cc.arp9 mutants were not defective in clamp cell formation. The effects of mutations in Cc.arp9 and Cc.snf5 on oidia production and the transcriptional expression levels of clp1 and pcc1, which are under the control of the A gene, were also examined. These indicated that Cc.Snf5 is involved in A-regulated pathways, whereas Cc.Arp9 is not apparently. Cc.arp9/Cc.snf5 double-gene disruptants were generated and their phenotypes were analyzed, which suggested a complicated developmental regulation mechanism mediated by chromatin remodeling. PMID:26746642

  13. Isolation and fine mapping of 16 novel human zinc finger-encoding cDNAs identify putative candidate genes for developmental and malignant disorders

    SciTech Connect

    Tommerup, N.; Vissing, H.

    1995-05-20

    The authors have isolated and chromosomally fine-mapped 16 novel genes belonging to the human zinc finger Krueppel family (ZNF131-140, 142, 143, 148, 151, 154, and 155), including 1 of the GLI type (ZNF143) and 3 containing a KRAB (Krueppel-associated box) segment (ZNF133, 136, and 140). Based on their map position, several of these ZNF genes are putative candidate genes for both developmental and malignant disorders: ZNF138, ZNF139, and ZNF143 were localized to 7q11.2, 7q21.3-q22.1, and 11p15.3-p15.4, regions involved in deletions and/or translocations associated with Williams syndrome, split hand and foot disease (SHFD1), and Beckwith-Wiedemann syndrome, respectively. ZNF133 was localized to 20p11.2, close to, but probably distinct from, the region deleted in Alagille syndrome. Zinc finger genes mapping to regions commonly deleted in solid tumors included ZNF132, 134, 135, 137, 154, and 155, all located on 19q13 (thyroid adenoma), and ZNF151, at 1p36.1-p36.2 (neuroblastoma, colon cancer, and other tumors). In addition, several of the ZNFs mapped to regions implicated in recurrent chromosomal rearrangements in hematological malignancies (ZNF139, 7q21.3-q22.1; ZNF148, 3q21-q22; ZNF151, 1p36.1-p36.2). The study indicates that the number of ZNF genes in human is large and that systematic isolation and mapping of ZNF genes is a straightforward approach for the identification of novel candidate disease genes. 47 refs., 2 figs., 1 tab.

  14. The maize glossy13 gene, cloned via BSR-Seq and Seq-walking encodes a putative ABC transporter required for the normal accumulation of epicuticular waxes.

    PubMed

    Li, Li; Li, Delin; Liu, Sanzhen; Ma, Xiaoli; Dietrich, Charles R; Hu, Heng-Cheng; Zhang, Gaisheng; Liu, Zhiyong; Zheng, Jun; Wang, Guoying; Schnable, Patrick S

    2013-01-01

    Aerial plant surfaces are covered by epicuticular waxes that among other purposes serve to control water loss. Maize glossy mutants originally identified by their "glossy" phenotypes exhibit alterations in the accumulation of epicuticular waxes. By combining data from a BSR-Seq experiment and the newly developed Seq-Walking technology, GRMZM2G118243 was identified as a strong candidate for being the glossy13 gene. The finding that multiple EMS-induced alleles contain premature stop codons in GRMZM2G118243, and the one knockout allele of gl13, validates the hypothesis that gene GRMZM2G118243 is gl13. Consistent with this, GRMZM2G118243 is an ortholog of AtABCG32 (Arabidopsis thaliana), HvABCG31 (barley) and OsABCG31 (rice), which encode ABCG subfamily transporters involved in the trans-membrane transport of various secondary metabolites. We therefore hypothesize that gl13 is involved in the transport of epicuticular waxes onto the surfaces of seedling leaves. PMID:24324772

  15. Functional characterization of the gene FoOCH1 encoding a putative α-1,6-mannosyltransferase in Fusarium oxysporum f. sp. cubense.

    PubMed

    Li, Min-Hui; Xie, Xiao-Ling; Lin, Xian-Feng; Shi, Jin-Xiu; Ding, Zhao-Jian; Ling, Jin-Feng; Xi, Ping-Gen; Zhou, Jia-Nuan; Leng, Yueqiang; Zhong, Shaobin; Jiang, Zi-De

    2014-04-01

    Fusarium oxysporum f. sp. cubense (FOC) is the causal agent of banana Fusarium wilt and has become one of the most destructive pathogens threatening the banana production worldwide. However, few genes related to morphogenesis and pathogenicity of this fungal pathogen have been functionally characterized. In this study, we identified and characterized the disrupted gene in a T-DNA insertional mutant (L953) of FOC with significantly reduced virulence on banana plants. The gene disrupted by T-DNA insertion in L953 harbors an open reading frame, which encodes a protein with homology to α-1,6-mannosyltransferase (OCH1) in fungi. The deletion mutants (ΔFoOCH1) of the OCH1 orthologue (FoOCH1) in FOC were impaired in fungal growth, exhibited brighter staining with fluorescein isothiocyanate (FITC)-Concanavalin A, had less cell wall proteins and secreted more proteins into liquid media than the wild type. Furthermore, the mutation or deletion of FoOCH1 led to loss of ability to penetrate cellophane membrane and decline in hyphal attachment and colonization as well as virulence to the banana host. The mutant phenotypes were fully restored by complementation with the wild type FoOCH1 gene. Our data provide a first evidence for the critical role of FoOCH1 in maintenance of cell wall integrity and virulence of F. oxysporum f. sp. cubense. PMID:24503549

  16. p190RhoGAP can act to inhibit PDGF-induced gliomas in mice: a putative tumor suppressor encoded on human Chromosome 19q13.3

    PubMed Central

    Wolf, Rebecca M.; Draghi, Nicole; Liang, Xiquan; Dai, Chengkai; Uhrbom, Lene; Eklöf, Charlotta; Westermark, Bengt; Holland, Eric C.; Resh, Marilyn D.

    2003-01-01

    p190RhoGAP and Rho are key regulators of oligodendrocyte differentiation. The gene encoding p190RhoGAP is located at 19q13.3 of the human chromosome, a locus that is deleted in 50%–80% of oligodendrogliomas. Here we provide evidence that p190RhoGAP may suppress gliomagenesis by inducing a differentiated glial phenotype. Using a cell culture model of autocrine loop PDGF stimulation, we show that reduced Rho activity via p190RhoGAP overexpression or Rho kinase inhibition induced cellular process extension, a block in proliferation, and reduced expression of the neural precursor marker nestin. In vivo infection of mice with retrovirus expressing PDGF and the p190 GAP domain caused a decreased incidence of oligodendrogliomas compared with that observed with PDGF alone. Independent experiments revealed that the retroviral vector insertion site in 3 of 50 PDGF-induced gliomas was within the p190RhoGAP gene. This evidence strongly suggests that p190 regulates critical components of PDGF oncogenesis and can act as a tumor suppressor in PDGF-induced gliomas by down-regulating Rho activity. PMID:12600941

  17. Human TRMU encoding the mitochondrial 5-methylaminomethyl-2-thiouridylate-methyltransferase is a putative nuclear modifier gene for the phenotypic expression of the deafness-associated 12S rRNA mutations

    SciTech Connect

    Yan Qingfeng; Bykhovskaya, Yelena; Li Ronghua; Mengesha, Emebet; Shohat, Mordechai; Estivill, Xavier; Fischel-Ghodsian, Nathan; Guan Minxin . E-mail: min-xin.guan@chmcc.org

    2006-04-21

    Nuclear modifier genes have been proposed to modulate the phenotypic manifestation of human mitochondrial 12S rRNA A1491G mutation associated with deafness in many families world-wide. Here we identified and characterized the putative nuclear modifier gene TRMU encoding a highly conserved mitochondrial protein related to tRNA modification. A 1937 bp TRMU cDNA has been isolated and the genomic organization of TRMU has been elucidated. The human TRMU gene containing 11 exons encodes a 421 residue protein with a strong homology to the TRMU-like proteins of bacteria and other homologs. TRMU is ubiquitously expressed in various tissues, but abundantly in tissues with high metabolic rates including heart, liver, kidney, and brain. Immunofluorescence analysis of human 143B cells expressing TRMU-GFP fusion protein demonstrated that the human Trmu localizes and functions in mitochondrion. Furthermore, we show that in families with the deafness-associated 12S rRNA A1491G mutation there is highly suggestive linkage and linkage disequilibrium between microsatellite markers adjacent to TRMU and the presence of deafness. These observations suggest that human TRMU may modulate the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA mutations.

  18. Ectopic Expression in Arabidopsis thaliana of an NB-ARC Encoding Putative Disease Resistance Gene from Wild Chinese Vitis pseudoreticulata Enhances Resistance to Phytopathogenic Fungi and Bacteria

    PubMed Central

    Wen, Zhifeng; Yao, Liping; Wan, Ran; Li, Zhi; Liu, Chonghuai; Wang, Xiping

    2015-01-01

    Plant resistance proteins mediate pathogen recognition and activate innate immune responses to restrict pathogen proliferation. One common feature of these proteins is an NB-ARC domain. In this study, we characterized a gene encoding a protein with an NB-ARC domain from wild Chinese grapevine Vitis pseudoreticulata accession “Baihe-35-1,” which was identified in a transcriptome analysis of the leaves following inoculation with Erysiphe necator (Schw.), a causal agent of powdery mildew. Transcript levels of this gene, designated VpCN (GenBank accession number KT265084), increased strongly after challenge of grapevine leaves with E. necator. The deduced amino acid sequence was predicted to contain an NB-ARC domain in the C-terminus and an RxCC-like domain similar to CC domain of Rx protein in the N-terminus. Ectopic expression of VpCN in Arabidopsis thaliana resulted in either a wild-type phenotype or a dwarf phenotype. The phenotypically normal transgenic A. thaliana showed enhance resistance to A. thaliana powdery mildew Golovinomyces cichoracearum, as well as to a virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Moreover, promoter::GUS (β-glucuronidase) analysis revealed that powdery mildew infection induced the promoter activity of VpCN in grapevine leaves. Finally, a promoter deletion analysis showed that TC rich repeat elements likely play an important role in the response to E. necator infection. Taken together, our results suggest that VpCN contribute to powdery mildew disease resistant in grapevine. PMID:26697041

  19. The SUD1 Gene Encodes a Putative E3 Ubiquitin Ligase and Is a Positive Regulator of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Arabidopsis[C][W

    PubMed Central

    Doblas, Verónica G.; Amorim-Silva, Vítor; Posé, David; Rosado, Abel; Esteban, Alicia; Arró, Montserrat; Azevedo, Herlander; Bombarely, Aureliano; Borsani, Omar; Valpuesta, Victoriano; Ferrer, Albert; Tavares, Rui M.; Botella, Miguel A.

    2013-01-01

    The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum–associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals. PMID:23404890

  20. Expression of PIN and AUX1 genes encoding putative carrier proteins for auxin polar transport in etiolated pea epicotyls [correction of epicotyles] under simulated microgravity conditions on a three-dimensional clinostat.

    PubMed

    Hoshino, Tomoki; Hitotsubashi, Reiko; Miyamoto, Kensuke; Tanimoto, Eiichi; Ueda, Junichi

    2003-10-01

    Etiolated pea (Pisum sativum L. cv. Alaska) seedlings grown under simulated microgravity conditions on a 3-dimensional clinostat showed automorphosis-like growth and development similar to that observed in true microgravity conditions in space. Application of inhibitors of auxin polar transport phenocopied automorphosis-like growth on 1 g conditions, suggesting that automorophosis is closely related to auxin polar transport. Strenuous efforts to know the relationships between automorphosis and auxin polar transport in pea seedlings at molecular bases resulted in successful identification of PsPIN2 and PsAUX1 encoding putative auxin efflux and influx carrier protein, respectively. Significantly high levels in homology were found on nucleotide and deduced amino acid sequences among PsPIN2, PsPIN1 and AtPINs, and between PsAUX1 and AtAUX1. Expression of PsPIN1 and PsAUX1 genes in etiolated pea seedlings grown on the clinostat were substantially affected, but that of PsPIN2 was not. Roles of these genes in auxin polar transport and automorphosis of etiolated pea seedlings are also described. PMID:14676360

  1. The putative imprinted locus D15S9 within the common deletion region for the Prader-Willi and Angelman syndromes encodes two overlapping mRNAs transcribed from opposite strands

    SciTech Connect

    Glenn, C.C.; Driscoll, D.J.; Saitoh, S.

    1994-09-01

    Prader-Willi syndrome is typically caused by a deletion of paternal 15q11-q13, or maternal uniparental disomy (UPD) of chromosome 15, while Angelman syndrome is caused by a maternal deletion or paternal UPD of the same region. Therefore, these two clinically distinct neurobehavioral syndromes result from differential expression of imprinted genes within 15q11-q13. A 3.1 kb cDNA, DN34, from the D15S9 locus within 15q11-q13 was isolated from a human fetal brain library. We showed previously that DN34 probe detects a DNA methylation imprint and therefore may represent a candidate imprinted gene. Isolation of genomic clones and DNA sequencing demonstrated that the gene segment encoding the partial cDNA DN34 was split by a 2 kb intron, but did not encode a substantial open reading frame (ORF). Preliminary analysis of expression by RT-PCR suggests that this gene is expressed in fetal but not in tested tissue types from the adult, and thus its imprinting status has not been possible to assess at present. Surprisingly, we found an ORF on the antisense strand of the DN34 cDNA. This ORF encodes a putative polypeptide of 505 amino acid residues containing a RING C{sub 3}HC{sub 4} zinc-finger motif and other features of nuclear proteins. Subsequent characterization of this gene, ZNF127, and a mouse homolog, demonstrated expression of 3.2 kb transcript from all tested fetal and adult tissues. Transcripts initiate from within a CpG-island, shown to be differentially methylated on parental alleles in the human. Interestingly, functional imprinting of the mouse homolog was subsequently demonstrated in an F{sub 1} cross by analyzing a VNTR polymorphism in the mRNA. The ZNF127 gene is intronless, has significant overlap with the DN34 gene on the antisense strand, and a 1 kb 3{prime} end within the 2 kb DN34 intron.

  2. Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins.

    PubMed

    Neeper, M; Schmidt, A M; Brett, J; Yan, S D; Wang, F; Pan, Y C; Elliston, K; Stern, D; Shaw, A

    1992-07-25

    Advanced glycosylation end products of proteins (AGEs) are nonenzymatically glycosylated proteins which accumulate in vascular tissue in aging and at an accelerated rate in diabetes. A approximately 35-kDa polypeptide with a unique NH2-terminal sequence has been isolated from bovine lung and found to be present on the surface of endothelial cells where it mediates the binding of AGEs (receptor for advanced glycosylation end product or RAGE). Using an oligonucleotide probe based on the amino-terminal sequence of RAGE, an apparently full-length cDNA of 1.5 kilobases was isolated from a bovine lung cDNA library. This cDNA encoded a 394 amino acid mature protein comprised of the following putative domains: an extracellular domain of 332 amino acids, a single hydrophobic membrane spanning domain of 19 amino acids, and a carboxyl-terminal domain of 43 amino acids. A partial clone encoding the human counterpart of RAGE, isolated from a human lung library, was found to be approximately 90% homologous to the bovine molecule. Based on computer analysis of the amino acid sequence of RAGE and comparison with databases, RAGE is a new member of the immunoglobulin superfamily of cell surface molecules and shares significant homology with MUC 18, NCAM, and the cytoplasmic domain of CD20. Expression of the RAGE cDNA in 293 cells allowed them to bind 125I-AGE-albumin in a saturable and dose-dependent manner (Kd approximately 100 nM), blocked by antibody to RAGE. Western blots of 293 cells transfected with RAGE cDNA probed with anti-RAGE IgG demonstrated expression of immunoreactive protein compared to its absence in mock-transfected cells. These results suggest that RAGE functions as a cell surface receptor for AGEs, which could potentially mediate cellular effects of this class of glycosylated proteins. PMID:1378843

  3. An update on cell surface proteins containing extensin-motifs.

    PubMed

    Borassi, Cecilia; Sede, Ana R; Mecchia, Martin A; Salgado Salter, Juan D; Marzol, Eliana; Muschietti, Jorge P; Estevez, Jose M

    2016-01-01

    In recent years it has become clear that there are several molecular links that interconnect the plant cell surface continuum, which is highly important in many biological processes such as plant growth, development, and interaction with the environment. The plant cell surface continuum can be defined as the space that contains and interlinks the cell wall, plasma membrane and cytoskeleton compartments. In this review, we provide an updated view of cell surface proteins that include modular domains with an extensin (EXT)-motif followed by a cytoplasmic kinase-like domain, known as PERKs (for proline-rich extensin-like receptor kinases); with an EXT-motif and an actin binding domain, known as formins; and with extracellular hybrid-EXTs. We focus our attention on the EXT-motifs with the short sequence Ser-Pro(3-5), which is found in several different protein contexts within the same extracellular space, highlighting a putative conserved structural and functional role. A closer understanding of the dynamic regulation of plant cell surface continuum and its relationship with the downstream signalling cascade is a crucial forthcoming challenge. PMID:26475923

  4. ENCODE data at the ENCODE portal.

    PubMed

    Sloan, Cricket A; Chan, Esther T; Davidson, Jean M; Malladi, Venkat S; Strattan, J Seth; Hitz, Benjamin C; Gabdank, Idan; Narayanan, Aditi K; Ho, Marcus; Lee, Brian T; Rowe, Laurence D; Dreszer, Timothy R; Roe, Greg; Podduturi, Nikhil R; Tanaka, Forrest; Hong, Eurie L; Cherry, J Michael

    2016-01-01

    The Encyclopedia of DNA Elements (ENCODE) Project is in its third phase of creating a comprehensive catalog of functional elements in the human genome. This phase of the project includes an expansion of assays that measure diverse RNA populations, identify proteins that interact with RNA and DNA, probe regions of DNA hypersensitivity, and measure levels of DNA methylation in a wide range of cell and tissue types to identify putative regulatory elements. To date, results for almost 5000 experiments have been released for use by the scientific community. These data are available for searching, visualization and download at the new ENCODE Portal (www.encodeproject.org). The revamped ENCODE Portal provides new ways to browse and search the ENCODE data based on the metadata that describe the assays as well as summaries of the assays that focus on data provenance. In addition, it is a flexible platform that allows integration of genomic data from multiple projects. The portal experience was designed to improve access to ENCODE data by relying on metadata that allow reusability and reproducibility of the experiments. PMID:26527727

  5. ENCODE data at the ENCODE portal

    PubMed Central

    Sloan, Cricket A.; Chan, Esther T.; Davidson, Jean M.; Malladi, Venkat S.; Strattan, J. Seth; Hitz, Benjamin C.; Gabdank, Idan; Narayanan, Aditi K.; Ho, Marcus; Lee, Brian T.; Rowe, Laurence D.; Dreszer, Timothy R.; Roe, Greg; Podduturi, Nikhil R.; Tanaka, Forrest; Hong, Eurie L.; Cherry, J. Michael

    2016-01-01

    The Encyclopedia of DNA Elements (ENCODE) Project is in its third phase of creating a comprehensive catalog of functional elements in the human genome. This phase of the project includes an expansion of assays that measure diverse RNA populations, identify proteins that interact with RNA and DNA, probe regions of DNA hypersensitivity, and measure levels of DNA methylation in a wide range of cell and tissue types to identify putative regulatory elements. To date, results for almost 5000 experiments have been released for use by the scientific community. These data are available for searching, visualization and download at the new ENCODE Portal (www.encodeproject.org). The revamped ENCODE Portal provides new ways to browse and search the ENCODE data based on the metadata that describe the assays as well as summaries of the assays that focus on data provenance. In addition, it is a flexible platform that allows integration of genomic data from multiple projects. The portal experience was designed to improve access to ENCODE data by relying on metadata that allow reusability and reproducibility of the experiments. PMID:26527727

  6. Diffusing colloidal probes of cell surfaces.

    PubMed

    Duncan, Gregg A; Fairbrother, D Howard; Bevan, Michael A

    2016-05-25

    Measurements and analyses are reported to quantify dynamic and equilibrium interactions between colloidal particles and live cell surfaces using dark field video microscopy. Two-dimensional trajectories of micron-sized polyethylene glycol (PEG)-coated silica colloids relative to adherent epithelial breast cancer cell perimeters are determined allowing measurement of position dependent diffusivities and interaction potentials. PEG was chosen as the material system of interest to assess non-specific interactions with cell surfaces and establishes a basis for investigation of specific interactions in future studies. Analysis of measured potential energies on cell surfaces reveals the spatial dependence in cell topography. With the measured cell topography and models for particle-cell surface hydrodynamic interactions, excellent agreement is obtained between theoretical and measured colloidal transport on cell surfaces. Quantitative analyses of association lifetimes showed that PEG coatings act to stabilize colloids above the cell surface through net repulsive, steric interactions. Our results demonstrate a self-consistent analysis of diffusing colloidal probe interactions due to conservative and non-conservative forces to characterize biophysical cell surface properties. PMID:27117575

  7. The molecular cloning and characterization of BM1P1 and BM1P2 proteins, putative positive transcription factors involved in barbiturate-mediated induction of the genes encoding cytochrome P450BM-1 of Bacillus megaterium.

    PubMed

    He, J S; Liang, Q; Fulco, A J

    1995-08-01

    Analysis of a 1.3-kilobase segment of 5'-flanking DNA from the barbiturate-inducible P450BM-1 gene (CYP106) of Bacillus megaterium revealed two open reading frames. One, BM1P1, encodes 98 amino acids and is located 267 base pairs upstream from the sequence encoding cytochrome P450BM-1 but in the opposite orientation. The second, BM1P2 (88 amino acids), is 892 base pairs upstream from the P450BM-1 coding sequence and in the same coding strand. The expression of BM1P1 and BM1P2 was strongly stimulated in cells grown in the presence of pentobarbital, and the BM1P1 gene product exerted positive control on expression of P450BM-1. When a 177-base pair fragment encompassing the overlapping promoter regions of the P450BM-1 and BM1P1 genes was used as a probe in DNA binding assays, the BM1P1 and BM1P2 gene products and Bm3R1 (the repressor protein regulating the barbiturate-mediated expression of P450BM-3) could bind individually, but the addition of BM1P1 or BM1P2 to a binding mixture containing Bm3R1 completely prevented the appearance of a Bm3R1 binding band. When a 208-base pair fragment containing a Barbie box sequence and located upstream of the 177-base pair fragment was used as a probe, only a Bm3R1 binding band was detected. Although neither BM1P1 and BM1P2 appeared to bind to this 208-base pair fragment, their presence strongly inhibited the binding of Bm3R1 to the same probe. The evidence suggests that BM1P1 and BM1P2 may, in part, act as positive regulatory proteins involved in the expression of the P450BM-1 gene by interfering with the binding of the repressor protein, Bm3R1, to the regulatory regions of P450BM-1. PMID:7629192

  8. Characterization of the lacticin 481 operon: the Lactococcus lactis genes lctF, lctE, and lctG encode a putative ABC transporter involved in bacteriocin immunity.

    PubMed Central

    Rincé, A; Dufour, A; Uguen, P; Le Pennec, J P; Haras, D

    1997-01-01

    The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis strains. The genetic determinants of lacticin 481 production are organized as an operon encoded by a 70-kb plasmid. We previously reported the first three genes of this operon, lctA, lctM, and lctT, which are involved in the bacteriocin biosynthesis and export (A. Rincé, A. Dufour, S. Le Pogam, D. Thuault, C. M. Bourgeois, and J.-P. Le Pennec, Appl. Environ. Microbiol. 60:1652-1657, 1994). The operon contains three additional open reading frames: lctF, lctE, and lctG. The hydrophobicity profiles and sequence similarities strongly suggest that the three gene products associate to form an ABC transporter. When the three genes were coexpressed into a lacticin 481-sensitive L. lactis strain, the strain became resistant to the bacteriocin. This protection could not be obtained when any of the three genes was deleted, confirming that lctF, lctE, and lctG are all necessary to provide immunity to lacticin 481. The quantification of the levels of immunity showed that lctF, lctE, and lctG could account for at least 6% and up to 100% of the immunity of the wild-type lacticin 481 producer strain, depending on the gene expression regulation. The lacticin 481 biosynthesis and immunity systems are discussed and compared to other lantibiotic systems. PMID:9361411

  9. The glucose metabolite methylglyoxal inhibits expression of the glucose transporter genes by inactivating the cell surface glucose sensors Rgt2 and Snf3 in yeast

    PubMed Central

    Roy, Adhiraj; Hashmi, Salman; Li, Zerui; Dement, Angela D.; Hong Cho, Kyu; Kim, Jeong-Ho

    2016-01-01

    Methylglyoxal (MG) is a cytotoxic by-product of glycolysis. MG has inhibitory effect on the growth of cells ranging from microorganisms to higher eukaryotes, but its molecular targets are largely unknown. The yeast cell-surface glucose sensors Rgt2 and Snf3 function as glucose receptors that sense extracellular glucose and generate a signal for induction of expression of genes encoding glucose transporters (HXTs). Here we provide evidence that these glucose sensors are primary targets of MG in yeast. MG inhibits the growth of glucose-fermenting yeast cells by inducing endocytosis and degradation of the glucose sensors. However, the glucose sensors with mutations at their putative ubiquitin-acceptor lysine residues are resistant to MG-induced degradation. These results suggest that the glucose sensors are inactivated through ubiquitin-mediated endocytosis and degraded in the presence of MG. In addition, the inhibitory effect of MG on the glucose sensors is greatly enhanced in cells lacking Glo1, a key component of the MG detoxification system. Thus the stability of these glucose sensors seems to be critically regulated by intracellular MG levels. Taken together, these findings suggest that MG attenuates glycolysis by promoting degradation of the cell-surface glucose sensors and thus identify MG as a potential glycolytic inhibitor. PMID:26764094

  10. Cell surface localization and processing of the ComG proteins, required for DNA binding during transformation of Bacillus subtilis.

    PubMed

    Chung, Y S; Breidt, F; Dubnau, D

    1998-08-01

    The comG operon of Bacillus subtilis encodes seven proteins essential for the binding of transforming DNA to the competent cell surface. We have explored the processing of the ComG proteins and the cellular localization of six of them. All of the proteins were found to be membrane associated. The four proteins with N-terminal sequence motifs typical of type 4 pre-pilins (ComGC, GD, GE and GG) are processed by a pathway that requires the product of comC, also an essential competence gene. The unprocessed forms of ComGC and GD behave like integral membrane proteins. Pre-ComGG differs from pre-ComGC and pre-ComGD, in that it is accessible to proteolysis only from the cytoplasmic face of the membrane and at least a portion of it behaves like a peripheral membrane protein. The mature forms of these proteins are translocated to the outer face of the membrane and are liberated when peptidoglycan is hydrolysed by lysozyme or mutanolysin. ComGG exists in part as a disulphide-cross-linked homodimer in vivo. ComGC was found to possess an intramolecular disulphide bond. The previously identified homodimer form of this protein is not stabilized by disulphide bond formation. ComGF behaves as an integral membrane protein, while ComGA, a putative ATPase, is located on the inner face of the membrane as a peripheral membrane protein. Possible roles of the ComG proteins in DNA binding to the competent cell surface are discussed in the light of these and other results. PMID:9723928

  11. Messenger RNAs encoding the beta subunits of guinea pig (Cavia porcellus) luteinizing hormone (gpLH) and putative chorionic gonadotropin (gpCG) are transcribed from a single-copy gpLH/CGbeta gene.

    PubMed

    Sherman, G B; Heilman, D F; Hoss, A J; Bunick, D; Lund, L A

    2001-06-01

    Neither gene locus nor gene sequence characterizations have been reported for the beta subunits of guinea pig (gp) LH and putative gp chorionic gonadotropin (CG). Descriptions of this locus would allow comparison with functionally relevant molecular genetic features of other species' homologous loci including the single-copy equid LH/CGbeta gene and the primate LHbeta-CGbeta gene cluster locus. Contiguous cDNA and genomic DNA fragments spanning the entire mature coding sequence of gpLHbeta mRNA, gpCGbeta mRNA and a homologous gpLH/CGbeta gene were amplified using PCR methodologies. With the exception of one silent mutation, the two cDNA and the genomic sequences were identical where they overlapped. Comparison of guinea pig coding sequence with LHbeta, CGbeta and LH/CGbeta sequences of other vertebrate species revealed the following order of similarity expressed as per cent coding sequence identity: rhinoceros LHbeta (83.6%)>pig LHbeta (81.8%)>donkey LH/CGbeta=bovine LHbeta (81.5%)> horse LH/CGbeta (80.6%)>dog LHbeta (79.7%)>human LHbeta (78.2%)>rat LHbeta (77.9%)>human CGbeta (75.8%)>turkey LHbeta (52.7%); values that are generally consistent with recently postulated phylogenetic relationships. Like the consensus mammalian LHbeta gene, the 5'-flanking region of the gpLH/CGbeta gene contains a single TATA sequence 37 bp upstream of the translation start codon. The first in-frame stop codon occurred at codon position +122 which is consistent with the 121 amino acid residue length of the consensus mammalian mature LHbeta peptide. To estimate gene copy number, full-length gpLHbeta cDNA was radiolabeled and hybridized to Southern blots of guinea pig genomic DNA digested with a panel of six restriction endonucleases. The resulting simple hybridization pattern strongly suggested that there is a single-copy gpLH/CGbeta gene. Northern analysis of total pituitary RNA using the same probe indicated that gpLHbeta transcript size is indistinguishable from that of consensus

  12. A testis-specific gene, TPTE, encodes a putative transmembrane tyrosine phosphatase and maps to the pericentromeric region of human chromosomes 21 and 13, and to chromosomes 15, 22, and Y.

    PubMed

    Chen, H; Rossier, C; Morris, M A; Scott, H S; Gos, A; Bairoch, A; Antonarakis, S E

    1999-11-01

    To contribute to the creation of a transcription map of human chromosome 21 (HC21) and to the identification of genes that may be involved in the pathogenesis of Down syndrome, exon trapping was performed from HC21-specific cosmids covering the entire chromosome. More than 700 exons have been identified to date. One such exon, hmc01a06, maps to YAC 831B6 which contains marker D21Z1 (alphoid repeats) and had previously been localized to the pericentromeric region of HC21. Northern-blot analysis revealed a 2.5-kb mRNA species strongly and exclusively expressed in the testis. We cloned the corresponding full-length cDNA, which encodes a predicted polypeptide of 551 amino acids with at least two potential transmembrane domains and a tyrosine phosphatase motif. The cDNA has sequence homology to chicken tensin, bovine auxilin and rat cyclin-G associated kinase (GAK). The entire polypeptide sequence also has significant homology to tumor suppressor PTEN/MMAC1 protein. We termed this novel gene/protein TPTE (transmembrane phosphatase with tensin homology). Polymerase chain reaction amplification, fluorescent in situ hybridization, Southern-blot and sequence analysis using monochromosomal somatic cell hybrids showed that this gene has highly homologous copies on HC13, 15, 22, and Y, in addition to its HC21 copy or copies. The estimated minimum number of copies of the TPTE gene in the haploid human genome is 7 in male and 6 in female. Zoo-blot analysis showed that TPTE is conserved between humans and other species. The biological function of the TPTE gene is presently unknown; however, its expression pattern, sequence homologies, and the presence of a potential tyrosine phosphatase domain suggest that it may be involved in signal transduction pathways of the endocrine or spermatogenetic function of the testis. It is also unknown whether all copies of TPTE are functional or whether some are pseudogenes. TPTE is, to our knowledge, the gene located closest to the human

  13. A lectin chromatography/mass spectrometry discovery workflow identifies putative biomarkers of aggressive breast cancers

    PubMed Central

    Drake, Penelope M.; Schilling, Birgit; Niles, Richard K.; Prakobphol, Akraporn; Li, Bensheng; Jung, Kwanyoung; Cho, Wonryeon; Braten, Miles; Inerowicz, Halina D.; Williams, Katherine; Albertolle, Matthew; Held, Jason M.; Iacovides, Demetris; Sorensen, Dylan J.; Griffith, Obi L.; Johansen, Eric; Zawadzka, Anna M.; Cusack, Michael P.; Allen, Simon; Gormley, Matthew; Hall, Steven C.; Witkowska, H. Ewa; Gray, Joe W.; Regnier, Fred; Gibson, Bradford W.; Fisher, Susan J.

    2012-01-01

    We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n = 5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, ~90% of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNAseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were upregulated in triple negative vs. luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype. PMID:22309216

  14. Cell Surface Relocalization of the Endoplasmic Reticulum Chaperone and Unfolded Protein Response Regulator GRP78/BiP*

    PubMed Central

    Zhang, Yi; Liu, Ren; Ni, Min; Gill, Parkash; Lee, Amy S.

    2010-01-01

    The recent discovery that GRP78/BiP, a typical endoplasmic reticulum (ER) lumenal chaperone, can be expressed on the cell surface, interacting with an increasing repertoire of surface proteins and acting as receptor in signaling pathways, represents a paradigm shift in its biological function. However, the mechanism of GRP78 trafficking from the ER to the cell surface is not well understood. Using a combination of cellular, biochemical, and mutational approaches, we tested multiple hypotheses. Here we report that ER stress actively promotes GRP78 localization on the cell surface, whereas ectopic expression of GRP78 is also able to cause cell surface relocation in the absence of ER stress. Moreover, deletion of the C-terminal ER retention motif in GRP78 alters its cell surface presentation in a dose-dependent manner; however, mutation of the putative O-linked glycosylation site Thr648 of human GRP78 is without effect. We also identified the exposure of multiple domains of GRP78 on the cell surface and determined that binding of extracellular GRP78 to the cell surface is unlikely. A new topology model for cell surface GRP78 is presented. PMID:20208072

  15. Bacteriophage-encoded depolymerases: their diversity and biotechnological applications.

    PubMed

    Pires, Diana P; Oliveira, Hugo; Melo, Luís D R; Sillankorva, Sanna; Azeredo, Joana

    2016-03-01

    Bacteriophages (phages), natural enemies of bacteria, can encode enzymes able to degrade polymeric substances. These substances can be found in the bacterial cell surface, such as polysaccharides, or are produced by bacteria when they are living in biofilm communities, the most common bacterial lifestyle. Consequently, phages with depolymerase activity have a facilitated access to the host receptors, by degrading the capsular polysaccharides, and are believed to have a better performance against bacterial biofilms, since the degradation of extracellular polymeric substances by depolymerases might facilitate the access of phages to the cells within different biofilm layers. Since the diversity of phage depolymerases is not yet fully explored, this is the first review gathering information about all the depolymerases encoded by fully sequenced phages. Overall, in this study, 160 putative depolymerases, including sialidases, levanases, xylosidases, dextranases, hyaluronidases, peptidases as well as pectate/pectin lyases, were found in 143 phages (43 Myoviridae, 47 Siphoviridae, 37 Podoviridae, and 16 unclassified) infecting 24 genera of bacteria. We further provide information about the main applications of phage depolymerases, which can comprise areas as diverse as medical, chemical, or food-processing industry. PMID:26767986

  16. Exposure of phosphatidylserine on the cell surface.

    PubMed

    Nagata, S; Suzuki, J; Segawa, K; Fujii, T

    2016-06-01

    Phosphatidylserine (PtdSer) is a phospholipid that is abundant in eukaryotic plasma membranes. An ATP-dependent enzyme called flippase normally keeps PtdSer inside the cell, but PtdSer is exposed by the action of scramblase on the cell's surface in biological processes such as apoptosis and platelet activation. Once exposed to the cell surface, PtdSer acts as an 'eat me' signal on dead cells, and creates a scaffold for blood-clotting factors on activated platelets. The molecular identities of the flippase and scramblase that work at plasma membranes have long eluded researchers. Indeed, their identity as well as the mechanism of the PtdSer exposure to the cell surface has only recently been revealed. Here, we describe how PtdSer is exposed in apoptotic cells and in activated platelets, and discuss PtdSer exposure in other biological processes. PMID:26891692

  17. Vesicle trafficking and cell surface membrane patchiness.

    PubMed

    Tang, Q; Edidin, M

    2001-07-01

    Membrane proteins and lipids often appear to be distributed in patches on the cell surface. These patches are often assumed to be membrane domains, arising from specific molecular associations. However, a computer simulation (Gheber and Edidin, 1999) shows that membrane patchiness may result from a combination of vesicle trafficking and dynamic barriers to lateral mobility. The simulation predicts that the steady-state patches of proteins and lipids seen on the cell surface will decay if vesicle trafficking is inhibited. To test this prediction, we compared the apparent sizes and intensities of patches of class I HLA molecules, integral membrane proteins, before and after inhibiting endocytic vesicle traffic from the cell surface, either by incubation in hypertonic medium or by expression of a dominant-negative mutant dynamin. As predicted by the simulation, the apparent sizes of HLA patches increased, whereas their intensities decreased after endocytosis and vesicle trafficking were inhibited. PMID:11423406

  18. Functional dynamics of cell surface membrane proteins

    NASA Astrophysics Data System (ADS)

    Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

    2014-04-01

    Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

  19. The yeast SNF3 gene encodes a glucose transporter homologous to the mammalian protein.

    PubMed Central

    Celenza, J L; Marshall-Carlson, L; Carlson, M

    1988-01-01

    The SNF3 gene is required for high-affinity glucose transport in the yeast Saccharomyces cerevisiae and has also been implicated in control of gene expression by glucose repression. We report here the nucleotide sequence of the cloned SNF3 gene. The predicted amino acid sequence shows that SNF3 encodes a 97-kilodalton protein that is homologous to mammalian glucose transporters and has 12 putative membrane-spanning regions. We also show that a functional SNF3-lacZ gene-fusion product cofractionates with membrane proteins and is localized to the cell surface, as judged by indirect immunofluorescence microscopy. Expression of the fusion protein is regulated by glucose repression. Images PMID:3281163

  20. Structure and functions of fungal cell surfaces

    NASA Technical Reports Server (NTRS)

    Nozawa, Y.

    1984-01-01

    A review with 24 references on the biochemistry, molecular structure, and function of cell surfaces of fungi, especially dermatophytes: the chemistry and structure of the cell wall, the effect of polyene antibiotics on the morphology and function of cytoplasmic membranes, and the chemical structure and function of pigments produced by various fungi are discussed.

  1. Encoding Dictionaries.

    ERIC Educational Resources Information Center

    Ide, Nancy

    1995-01-01

    Describes problems in devising a Text Encoding Initiative (TEI) encoding format for dictionaries. Asserts that the high degree of structuring and compression of information are among the most complex text types treated in the TEI. Concludes that the source of some TEI problems lies in the design of Standard Generalized Markup Language (SGML). (CFR)

  2. Large Scale Identification of Genes Involved in Cell Surface Biosynthesis and Architecture in Saccharomyces Cerevisiae

    PubMed Central

    Lussier, M.; White, A. M.; Sheraton, J.; di-Paolo, T.; Treadwell, J.; Southard, S. B.; Horenstein, C. I.; Chen-Weiner, J.; Ram, AFJ.; Kapteyn, J. C.; Roemer, T. W.; Vo, D. H.; Bondoc, D. C.; Hall, J.; Wei Zhong, W.; Sdicu, A. M.; Davies, J.; Klis, F. M.; Robbins, P. W.; Bussey, H.

    1997-01-01

    The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype. PMID:9335584

  3. Phenotypic and functional changes in peripheral blood monocytes during progression of human immunodeficiency virus infection. Effects of soluble immune complexes, cytokines, subcellular particulates from apoptotic cells, and HIV-1-encoded proteins on monocytes phagocytic function, oxidative burst, transendothelial migration, and cell surface phenotype.

    PubMed Central

    Trial, J; Birdsall, H H; Hallum, J A; Crane, M L; Rodriguez-Barradas, M C; de Jong, A L; Krishnan, B; Lacke, C E; Figdor, C G; Rossen, R D

    1995-01-01

    We postulated that changes in the cell surface display of molecules that facilitate cell-cell and cell-matrix adhesions may reflect the changing immunosurveillance capacity of blood monocytes during progression of human immunodeficiency virus (HIV) infections. In Centers for Disease Control (CDC) stage A patients, whose monocytes' ability to phagocytose bacteria and generate reactive oxygen intermediates is often increased, the frequency of monocytes expressing CD49d, HLA-DP, HLA-DQ, and an activation epitope of CD11a/CD18 was increased and monocyte transendothelial migration was unimpaired. In CDC stage B/C patients, whose monocytes' ability to phagocytose bacteria and migrate across confluent endothelial monolayers was diminished, surface expression of CD49e and CD62L and the percentage of monocytes expressing CD18, CD11a, CD29, CD49e, CD54, CD58, CD31, and HLA-I were significantly decreased. Incubating normal donor monocytes with immune complexes in vitro reproduced the phenotypic and functional abnormalities seen in stage B/C patients. By contrast, in vitro stimulation with subcellular particulates released by apoptotic lymphocytes reproduced changes seen in stage A patients' monocytes. Although circulating monocytes appear to be activated at all stages, these data suggest that the high levels of circulating immune complexes, found predominantly in the later stages of HIV infection, may be particularly instrumental in reducing the monocyte's capacity to maintain surveillance against infection. Images PMID:7706478

  4. 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

  5. Cell Surface Analysis Techniques: What Do Cell Preparation Protocols Do to Cell Surface Properties?

    PubMed Central

    Pembrey, Richard S.; Marshall, Kevin C.; Schneider, René P.

    1999-01-01

    Cell surface analysis often requires manipulation of cells prior to examination. The most commonly employed procedures are centrifugation at different speeds, changes of media during washing or final resuspension, desiccation (either air drying for contact angle measurements or freeze-drying for sensitive spectroscopic analysis, such as X-ray photoelectron spectroscopy), and contact with hydrocarbon (hydrophobicity assays). The effects of these procedures on electrophoretic mobility, adhesion to solid substrata, affinity to a number of Sepharose columns, structural integrity, and cell viability were systematically investigated for a range of model organisms, including carbon- and nitrogen-limited Psychrobacter sp. strain SW8 (glycocalyx-bearing cells), Escherichia coli (gram-negative cells without a glycocalyx), and Staphylococcus epidermidis (gram-positive cells without a glycocalyx). All of the cell manipulation procedures severely modified the physicochemical properties of cells, but with each procedure some organisms were more susceptible than others. Considerable disruption of cell surfaces occurred when organisms were placed in contact with a hydrocarbon (hexadecane). The majority of cells became nonculturable after air drying and freeze-drying. Centrifugation at a high speed (15,000 × g) modified many cell surface parameters significantly, although cell viability was considerably affected only in E. coli. The type of washing or resuspension medium had a strong influence on the values of cell surface parameters, particularly when high-salt solutions were compared with low-salt buffers. The values for parameters obtained with different methods that allegedly measure similar cell surface properties did not correlate for most cells. These results demonstrate that the methods used to prepare cells for cell surface analysis need to be critically investigated for each microorganism so that the final results obtained reflect the nature of the in situ microbial cell

  6. Multilocus analysis of extracellular putative virulence proteins made by group A Streptococcus: population genetics, human serologic response, and gene transcription.

    PubMed

    Reid, S D; Green, N M; Buss, J K; Lei, B; Musser, J M

    2001-06-19

    Species of pathogenic microbes are composed of an array of evolutionarily distinct chromosomal genotypes characterized by diversity in gene content and sequence (allelic variation). The occurrence of substantial genetic diversity has hindered progress in developing a comprehensive understanding of the molecular basis of virulence and new therapeutics such as vaccines. To provide new information that bears on these issues, 11 genes encoding extracellular proteins in the human bacterial pathogen group A Streptococcus identified by analysis of four genomes were studied. Eight of the 11 genes encode proteins with a LPXTG(L) motif that covalently links Gram-positive virulence factors to the bacterial cell surface. Sequence analysis of the 11 genes in 37 geographically and phylogenetically diverse group A Streptococcus strains cultured from patients with different infection types found that recent horizontal gene transfer has contributed substantially to chromosomal diversity. Regions of the inferred proteins likely to interact with the host were identified by molecular population genetic analysis, and Western immunoblot analysis with sera from infected patients confirmed that they were antigenic. Real-time reverse transcriptase-PCR (TaqMan) assays found that transcription of six of the 11 genes was substantially up-regulated in the stationary phase. In addition, transcription of many genes was influenced by the covR and mga trans-acting gene regulatory loci. Multilocus investigation of putative virulence genes by the integrated approach described herein provides an important strategy to aid microbial pathogenesis research and rapidly identify new targets for therapeutics research. PMID:11416223

  7. Adhesion properties of a putative polymorphic fimbrial subunit protein from Bifidobacterium longum subsp. longum

    PubMed Central

    SUZUKI, Kenta; NISHIYAMA, Keita; MIYAJIMA, Hiroki; OSAWA, Ro; YAMAMOTO, Yuji; MUKAI, Takao

    2015-01-01

    In our previous study, we found that the open reading frame bl0675 in the genome of Bifidobacterium longum subsp. longum isolated from human feces encoded a novel putative fimbrial protein, was highly polymorphic, and had five variants (A, B, C, D, and E types). The aim of this study was to evaluate the affinity of these variants to porcine colonic mucins (PCMs). Protein-binding properties were examined using the recombinant BL0675 protein containing a C-terminal 6 × His tag (His-BL0675). Surface plasmon resonance analysis demonstrated that the His-BL0675 A type had strong affinity to PCMs (KD = 9.82 × 10−8 M), whereas the B, C, D, and E types exhibited little or no binding. In a competitive enzyme-linked immunosorbent assay, His-BL0675 A type binding was reduced by addition of mucin oligosaccharides, suggesting that the binding occurs via carbohydrate chains of PCMs. The localization of BL0675 to the B. longum subsp. longum cell surface was confirmed by western blot analysis using A type polyclonal antibodies. Bacterial adhesion of B. longum subsp. longum to PCMs was also blocked by A type-specific antibodies; however, its adhesion properties were strain specific. Our results suggest that the BL0675 variants significantly contribute to the adhesion of B. longum subsp. longum strains. The expression and the adhesive properties of this protein are affected by genetic polymorphisms and are specific for B. longum subsp. longum strains. However, further studies are required on the properties of binding of these putative fimbrial proteins to the human gastrointestinal tract. PMID:26858927

  8. Adhesion properties of a putative polymorphic fimbrial subunit protein from Bifidobacterium longum subsp. longum.

    PubMed

    Suzuki, Kenta; Nishiyama, Keita; Miyajima, Hiroki; Osawa, Ro; Yamamoto, Yuji; Mukai, Takao

    2016-01-01

    In our previous study, we found that the open reading frame bl0675 in the genome of Bifidobacterium longum subsp. longum isolated from human feces encoded a novel putative fimbrial protein, was highly polymorphic, and had five variants (A, B, C, D, and E types). The aim of this study was to evaluate the affinity of these variants to porcine colonic mucins (PCMs). Protein-binding properties were examined using the recombinant BL0675 protein containing a C-terminal 6 × His tag (His-BL0675). Surface plasmon resonance analysis demonstrated that the His-BL0675 A type had strong affinity to PCMs (KD = 9.82 × 10(-8) M), whereas the B, C, D, and E types exhibited little or no binding. In a competitive enzyme-linked immunosorbent assay, His-BL0675 A type binding was reduced by addition of mucin oligosaccharides, suggesting that the binding occurs via carbohydrate chains of PCMs. The localization of BL0675 to the B. longum subsp. longum cell surface was confirmed by western blot analysis using A type polyclonal antibodies. Bacterial adhesion of B. longum subsp. longum to PCMs was also blocked by A type-specific antibodies; however, its adhesion properties were strain specific. Our results suggest that the BL0675 variants significantly contribute to the adhesion of B. longum subsp. longum strains. The expression and the adhesive properties of this protein are affected by genetic polymorphisms and are specific for B. longum subsp. longum strains. However, further studies are required on the properties of binding of these putative fimbrial proteins to the human gastrointestinal tract. PMID:26858927

  9. Deciphering ENCODE.

    PubMed

    Diehl, Adam G; Boyle, Alan P

    2016-04-01

    The ENCODE project represents a major leap from merely describing and comparing genomic sequences to surveying them for direct indicators of function. The astounding quantity of data produced by the ENCODE consortium can serve as a map to locate specific landmarks, guide hypothesis generation, and lead us to principles and mechanisms underlying genome biology. Despite its broad appeal, the size and complexity of the repository can be intimidating to prospective users. We present here some background about the ENCODE data, survey the resources available for accessing them, and describe a few simple principles to help prospective users choose the data type(s) that best suit their needs, where to get them, and how to use them to their best advantage. PMID:26962025

  10. Probes for anionic cell surface detection

    DOEpatents

    Smith, Bradley D.

    2013-03-05

    Embodiments of the present invention are generally directed to compositions comprising a class of molecular probes for detecting the presence of anionic cell surfaces. Embodiments include compositions that are enriched for these compositions and preparations, particularly preparations suitable for use as laboratory/clinical reagents and diagnostic indicators, either alone or as part of a kit. An embodiment of the invention provides for a highly selective agent useful in the discernment and identification of dead or dying cells, such as apoptotic cells, in a relatively calcium-free environment. An embodiment of the invention provides a selective agent for the identification of bacteria in a mixed population of bacterial cells and nonbacterial cells.

  11. Cell surface receptors for CCN proteins.

    PubMed

    Lau, Lester F

    2016-06-01

    The CCN family (CYR61; CTGF; NOV; CCN1-6; WISP1-3) of matricellular proteins in mammals is comprised of six homologous members that play important roles in development, inflammation, tissue repair, and a broad range of pathological processes including fibrosis and cancer. Despite considerable effort to search for a high affinity CCN-specific receptor akin to growth factor receptors, no such receptor has been found. Rather, CCNs bind several groups of multi-ligand receptors as characteristic of other matricellular proteins. The most extensively documented among CCN-binding receptors are integrins, including αvβ3, αvβ5, α5β1, α6β1, αIIbβ3, αMβ2, and αDβ2, which mediate diverse CCN functions in various cell types. CCNs also bind cell surface heparan sulfate proteoglycans (HSPGs), low density liproprotein receptor-related proteins (LRPs), and the cation-independent mannose-6-phosphate (M6P) receptor, which are endocytic receptors that may also serve as co-receptors in cooperation with other cell surface receptors. CCNs have also been reported to bind FGFR-2, Notch, RANK, and TrkA, potentially altering the affinities of these receptors for their ligands. The ability of CCNs to bind a multitude of receptors in various cell types may account for the remarkable versatility of their functions, and underscore the diverse signaling pathways that mediate their activities. PMID:27098435

  12. A secreted form of the human lymphocyte cell surface molecule CD8 arises from alternative splicing

    SciTech Connect

    Giblin, P.; Kavathas, P. ); Ledbetter, J.A. )

    1989-02-01

    The human lymphocyte differentiation antigen CD8 is encoded by a single gene that gives rise to a 33- to 34-kDa glycoprotein expressed on the cell surface as a dimer and in higher molecular mass forms. The authors demonstrate that the mRNA is alternatively spliced so that an exon encoding a transmembrane domain is deleted. This gives rise to a 30-kDa molecule that is secreted and exists primarily as a monomer. mRNA corresponding to both forms is present in peripheral blood lymphocytes, Con A-activated peripheral blood lymphocytes, and three CD8{sup +} T-cell lines, with the membrane form being the major species. However, differences in the ratio of mRNA for membrane CD8 and secreted CD8 exist. In addition, the splicing pattern observed differs from the pattern found for the mouse CD8 gene. This mRNA is also alternatively spliced, but an exon encoding a cytoplasmic region is deleted, giving rise to a cell surface molecule that differs in its cytoplasmic tail from the protein encoded by the longer mRNA. Neither protein is secreted. This is one of the first examples of a different splicing pattern between two homologous mouse and human genes giving rise to very different proteins. This represents one mechanism of generating diversity during speciation.

  13. Biomolecular strategies for cell surface engineering

    NASA Astrophysics Data System (ADS)

    Wilson, John Tanner

    Islet transplantation has emerged as a promising cell-based therapy for the treatment of diabetes, but its clinical efficacy remains limited by deleterious host responses that underlie islet destruction. In this dissertation, we describe the assembly of ultrathin conformal coatings that confer molecular-level control over the composition and biophysicochemical properties of the islet surface with implications for improving islet engraftment. Significantly, this work provides novel biomolecular strategies for cell surface engineering with broad biomedical and biotechnological applications in cell-based therapeutics and beyond. Encapsulation of cells and tissue offers a rational approach for attenuating deleterious host responses towards transplanted cells, but a need exists to develop cell encapsulation strategies that minimize transplant volume. Towards this end, we endeavored to generate nanothin films of diverse architecture with tunable properties on the extracellular surface of individual pancreatic islets through a process of layer-by-layer (LbL) self assembly. We first describe the formation of poly(ethylene glycol) (PEG)-rich conformal coatings on islets via LbL self assembly of poly(L-lysine)-g-PEG(biotin) and streptavidin. Multilayer thin films conformed to the geometrically and chemically heterogeneous islet surface, and could be assembled without loss of islet viability or function. Significantly, coated islets performed comparably to untreated controls in a murine model of allogenic intraportal islet transplantation, and, to our knowledge, this is the first study to report in vivo survival and function of nanoencapsulated cells or cell aggregates. Based on these findings, we next postulated that structurally similar PLL-g-PEG copolymers comprised of shorter PEG grafts might be used to initiate and propagate the assembly of polyelectrolyte multilayer (PEM) films on pancreatic islets, while simultaneously preserving islet viability. Through control of PLL

  14. Specificity of human galectins on cell surfaces.

    PubMed

    Rapoport, E M; Bovin, N V

    2015-07-01

    Galectins are β-galactoside-binding proteins sharing homology in amino acid sequence of their carbohydrate-recognition domain. Their carbohydrate specificity outside cells has been studied previously. The main conclusion of these studies was that several levels of glycan ligand recognition exist for galectins: (i) disaccharide Galβ1-4GlcNAc (LN, N-acetyllactosamine) binds stronger than β-galactopyranose; (ii) substitution at O-2 and O-3 of galactose residue as well as core fragments ("right" from GlcNAc) provides significant increase in affinity; (iii) similarly glycosylated proteins can differ significantly in affinity to galectins. Information about the natural cellular receptors of galectins is limited. Until recently, it was impossible to study specificity of cell-bound galectins. A model based on controlled incorporation of a single protein into glycocalyx of cells and subsequent interaction of loaded cells with synthetic glycoprobes measured by flow cytometry made this possible recently. In this review, data about glycan specificity of proto-, chimera-, and tandem-repeat type galectins on the cell surface are systematized, and comparative analysis of the results with data on specificity of galectins in artificial systems was performed. The following conclusions from these studies were made: (i) cellular galectins have practically no ability to bind disaccharide LNn, but display affinity to 3'-substituted oligolactosamines and oligomers LNn; (ii) tandem-repeat type galectins recognize another disaccharide, namely Galβ1-3GlcNAc (Le(c)); (iii) on the cell surface, tandem-repeat type galectins conserve the ability to display high affinity to blood group antigens of ABH system; (iv) in general, when galectins are immersed into glycocalyx, they are more selective regarding glycan interactions. Thus, we conclude that competitive interaction of galectins with cell microenvironment (endogenous cell glycans) is the main factor providing selectivity of galectins in

  15. TgaA, a VirB1-Like Component Belonging to a Putative Type IV Secretion System of Bifidobacterium bifidum MIMBb75

    PubMed Central

    Balzaretti, Silvia; Taverniti, Valentina; Miriani, Matteo; Milani, Christian; Scarafoni, Alessio; Corona, Silvia; Ciranna, Alessandro; Arioli, Stefania; Santala, Ville; Iametti, Stefania; Bonomi, Francesco; Ventura, Marco; Mora, Diego; Karp, Matti

    2014-01-01

    Bifidobacterium bifidum MIMBb75 is a human intestinal isolate demonstrated to be interactive with the host and efficacious as a probiotic. However, the molecular biology of this microorganism is yet largely unknown. For this reason, we undertook whole-genome sequencing of B. bifidum MIMBb75 to identify potential genetic factors that would explain the metabolic and probiotic attributes of this bacterium. Comparative genomic analysis revealed a 45-kb chromosomal region that comprises 19 putative genes coding for a potential type IV secretion system (T4SS). Thus, we undertook the initial characterization of this genetic region by studying the putative virB1-like gene, named tgaA. Gene tgaA encodes a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT, cd00254.3) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP, pfam05257.4). By means of several in vitro assays, we experimentally confirmed that protein TgaA, consistent with its computationally assigned role, has peptidoglycan lytic activity, which is principally associated to the LT domain. Furthermore, immunofluorescence and immunogold labeling showed that the protein TgaA is abundantly expressed on the cell surface of B. bifidum MIMBb75. According to the literature, the T4SSs, which have not been characterized before in bifidobacteria, can have important implications for bacterial cell-to-cell communication as well as cross talk with host cells, justifying the interest for further studies aimed at the investigation of this genetic region. PMID:24951779

  16. Cell Surface Markers in Colorectal Cancer Prognosis

    PubMed Central

    Belov, Larissa; Zhou, Jerry; Christopherson, Richard I.

    2011-01-01

    The classification of colorectal cancers (CRC) is currently based largely on histologically determined tumour characteristics, such as differentiation status and tumour stage, i.e., depth of tumour invasion, involvement of regional lymph nodes and the occurrence of metastatic spread to other organs. These are the conventional prognostic factors for patient survival and often determine the requirement for adjuvant therapy after surgical resection of the primary tumour. However, patients with the same CRC stage can have very different disease-related outcomes. For some, surgical removal of early-stage tumours leads to full recovery, while for others, disease recurrence and metastasis may occur regardless of adjuvant therapy. It is therefore important to understand the molecular processes that lead to disease progression and metastasis and to find more reliable prognostic markers and novel targets for therapy. This review focuses on cell surface proteins that correlate with tumour progression, metastasis and patient outcome, and discusses some of the challenges in finding prognostic protein markers in CRC. PMID:21339979

  17. Glycopeptide Capture for Cell Surface Proteomics

    PubMed Central

    Lee, M. C. Gilbert; Sun, Bingyun

    2014-01-01

    Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins. PMID:24836557

  18. Unveiling Cell Surface and Type IV Secretion Proteins Responsible for Archaeal Rudivirus Entry

    PubMed Central

    Deng, Ling; He, Fei; Bhoobalan-Chitty, Yuvaraj; Martinez-Alvarez, Laura; Guo, Yang

    2014-01-01

    Sulfolobus mutants resistant to archaeal lytic virus Sulfolobus islandicus rod-shaped virus 2 (SIRV2) were isolated, and mutations were identified in two gene clusters, cluster sso3138 to sso3141 and cluster sso2386 and sso2387, encoding cell surface and type IV secretion proteins, respectively. The involvement of the mutations in the resistance was confirmed by genetic complementation. Blocking of virus entry into the mutants was demonstrated by the lack of early gene transcription, strongly supporting the idea of a role of the proteins in SIRV2 entry. PMID:24965447

  19. Yeast cell-surface expression of chitosanase from Paenibacillus fukuinensis.

    PubMed

    Fukuda, Takeshi; Isogawa, Danya; Takagi, Madoka; Kato-Murai, Michiko; Kimoto, Hisashi; Kusaoke, Hideo; Ueda, Mitsuyoshi; Suye, Shin-Ichiro

    2007-11-01

    To produce chitoorigosaccharides using chitosan, we attempted to construct Paenibacillus fukuinensis chitosanase-displaying yeast cells as a whole-cell biocatalyst through yeast cell-surface engineering. The localization of the chitosanase on the yeast cell surface was confirmed by immunofluorescence labeling of cells. The chitosanase activity of the constructed yeast was investigated by halo assay and the dinitrosalicylic acid method. PMID:17986777

  20. Cell Surface-based Sensing with Metallic Nanoparticles

    PubMed Central

    Jiang, Ziwen; Rotello, Vincent M.

    2015-01-01

    Metallic nanoparticles provide versatile scaffolds for biosensing applications. In this review, we focus on the use of metallic nanoparticles for cell surface sensings. Examples of the use of both specific recognition and array-based “chemical nose” approaches to cell surface sensing will be discussed. PMID:25853985

  1. Rare TREM2 variants associated with Alzheimer's disease display reduced cell surface expression.

    PubMed

    Sirkis, Daniel W; Bonham, Luke W; Aparicio, Renan E; Geier, Ethan G; Ramos, Eliana Marisa; Wang, Qing; Karydas, Anna; Miller, Zachary A; Miller, Bruce L; Coppola, Giovanni; Yokoyama, Jennifer S

    2016-01-01

    Rare variation in TREM2 has been associated with greater risk for Alzheimer's disease (AD). TREM2 encodes a cell surface receptor expressed on microglia and related cells, and the R47H variant associated with AD appears to affect the ability of TREM2 to bind extracellular ligands. In addition, other rare TREM2 mutations causing early-onset neurodegeneration are thought to impair cell surface expression. Using a sequence kernel association (SKAT) analysis in two independent AD cohorts, we found significant enrichment of rare TREM2 variants not previously characterized at the protein level. Heterologous expression of the identified variants showed that novel variants S31F and R47C displayed significantly reduced cell surface expression. In addition, we identified rare variant R136Q in a patient with language-predominant AD that also showed impaired surface expression. The results suggest rare TREM2 variants enriched in AD may be associated with altered TREM2 function and that AD risk may be conferred, in part, from altered TREM2 surface expression. PMID:27589997

  2. Improving genetic immobilization of a cellulase on yeast cell surface for bioethanol production using cellulose.

    PubMed

    Yang, Jinying; Dang, Hongyue; Lu, Jian Ren

    2013-04-01

    In this study, Saccharomyces cerevisiae was genetically engineered to harbor the capability of utilizing celluloses for bioethanol production by displaying active cellulolytic enzymes on the cell surface. An endo-1,4-β-glucanase gene egX was cloned from Bacillus pumilus C-9 and its expression products, the EGX cellulases, were displayed on the cell surface of S. cerevisiae by fusing egX with aga2 that encodes the binding subunit of the S. cerevisiae cell wall protein α-agglutinin. To achieve high gene copies and stability, multicopy integration was obtained by integrating the fusion aga2-egX gene into the rDNA region of the S. cerevisiae chromosome. To achieve high expression and surface display efficiency, the aga2-egX gene was expressed under the control of a strong promoter. The presence of the enzymatically active cellulase fusion proteins on the S. cerevisiae cell surface was verified by carboxymethyl cellulase activity assay and immunofluorescence microscopy. This work presented a promising strategy to genetically engineer yeasts to perform efficient fermentation of cellulosic materials for bioethanol production. PMID:22915066

  3. Illumination of growth, division and secretion by metabolic labeling of the bacterial cell surface

    PubMed Central

    Siegrist, M. Sloan; Swarts, Benjamin M.; Fox, Douglas M.; Lim, Shion An; Bertozzi, Carolyn R.

    2015-01-01

    The cell surface is the essential interface between a bacterium and its surroundings. Composed primarily of molecules that are not directly genetically encoded, this highly dynamic structure accommodates the basic cellular processes of growth and division as well as the transport of molecules between the cytoplasm and the extracellular milieu. In this review, we describe aspects of bacterial growth, division and secretion that have recently been uncovered by metabolic labeling of the cell envelope. Metabolite derivatives can be used to label a variety of macromolecules, from proteins to non-genetically-encoded glycans and lipids. The embedded metabolite enables precise tracking in time and space, and the versatility of newer chemoselective detection methods offers the ability to execute multiple experiments concurrently. In addition to reviewing the discoveries enabled by metabolic labeling of the bacterial cell envelope, we also discuss the potential of these techniques for translational applications. Finally, we offer some guidelines for implementing this emerging technology. PMID:25725012

  4. Calreticulin: Roles in Cell-Surface Protein Expression

    PubMed Central

    Jiang, Yue; Dey, Sandeepa; Matsunami, Hiroaki

    2014-01-01

    In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins. PMID:25230046

  5. Cell-surface MHC density profiling reveals instability of autoimmunity-associated HLA

    PubMed Central

    Miyadera, Hiroko; Ohashi, Jun; Lernmark, Åke; Kitamura, Toshio; Tokunaga, Katsushi

    2014-01-01

    Polymorphisms within HLA gene loci are strongly associated with susceptibility to autoimmune disorders; however, it is not clear how genetic variations in these loci confer a disease risk. Here, we devised a cell-surface MHC expression assay to detect allelic differences in the intrinsic stability of HLA-DQ proteins. We found extreme variation in cell-surface MHC density among HLA-DQ alleles, indicating a dynamic allelic hierarchy in the intrinsic stability of HLA-DQ proteins. Using the case-control data for type 1 diabetes (T1D) for the Swedish and Japanese populations, we determined that T1D risk–associated HLA-DQ haplotypes, which also increase risk for autoimmune endocrinopathies and other autoimmune disorders, encode unstable proteins, whereas the T1D–protective haplotypes encode the most stable HLA-DQ proteins. Among the amino acid variants of HLA-DQ, alterations in 47α, the residue that is located on the outside of the peptide-binding groove and acts as a key stability regulator, showed strong association with T1D. Evolutionary analysis suggested that 47α variants have been the target of positive diversifying selection. Our study demonstrates a steep allelic hierarchy in the intrinsic stability of HLA-DQ that is associated with T1D risk and protection, suggesting that HLA instability mediates the development of autoimmune disorders. PMID:25485681

  6. Construction of a starch-utilizing yeast by cell surface engineering.

    PubMed Central

    Murai, T; Ueda, M; Yamamura, M; Atomi, H; Shibasaki, Y; Kamasawa, N; Osumi, M; Amachi, T; Tanaka, A

    1997-01-01

    We have engineered the cell surface of the yeast Saccharomyces cerevisiae by anchoring active glucoamylase protein on the cell wall, and we have endowed the yeast cells with the ability to utilize starch directly as the sole carbon source. The gene encoding Rhizopus oryzae glucoamylase with its secretion signal peptide was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast alpha-agglutinin, a protein involved in mating and covalently anchored to the cell wall. The constructed plasmid containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The glucoamylase activity as not detected in the culture medium, but it was detected in the cell pellet fraction. The glucoamylase protein transferred to the soluble fraction from the cell wall fraction after glucanase treatment but not after sodium dodecyl sulfate treatment, indicating the covalent binding of the fusion protein to the cell wall. Display of the fused protein was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. The transformant cells could surely grow on starch as the sole carbon source. These results showed that the glucoamylase was anchored on the cell wall and displayed as its active form. This is the first example of an application of cell surface engineering to utilize and improve the metabolic ability of cells. PMID:9097432

  7. A Putative Cell Surface Receptor for White Spot Syndrome Virus Is a Member of a Transporter Superfamily

    PubMed Central

    Huang, Huai-Ting; Leu, Jiann-Horng; Huang, Po-Yu; Chen, Li-Li

    2012-01-01

    White spot syndrome virus (WSSV), a large enveloped DNA virus, can cause the most serious viral disease in shrimp and has a wide host range among crustaceans. In this study, we identified a surface protein, named glucose transporter 1 (Glut1), which could also interact with WSSV envelope protein, VP53A. Sequence analysis revealed that Glut1 is a member of a large superfamily of transporters and that it is most closely related to evolutionary branches of this superfamily, branches that function to transport this sugar. Tissue tropism analysis showed that Glut1 was constitutive and highly expressed in almost all organs. Glut1's localization in shrimp cells was further verified and so was its interaction with Penaeus monodon chitin-binding protein (PmCBP), which was itself identified to interact with an envelope protein complex formed by 11 WSSV envelope proteins. In vitro and in vivo neutralization experiments using synthetic peptide contained WSSV binding domain (WBD) showed that the WBD peptide could inhibit WSSV infection in primary cultured hemocytes and delay the mortality in shrimps challenged with WSSV. These findings have important implications for our understanding of WSSV entry. PMID:22427993

  8. Cell surface expression and turnover of the alpha-subunit of the epithelial sodium channel.

    PubMed

    Kleyman, T R; Zuckerman, J B; Middleton, P; McNulty, K A; Hu, B; Su, X; An, B; Eaton, D C; Smith, P R

    2001-08-01

    The renal epithelial cell line A6, derived from Xenopus laevis, expresses epithelial Na(+) channels (ENaCs) and serves as a model system to study hormonal regulation and turnover of ENaCs. Our previous studies suggest that the alpha-subunit of Xenopus ENaC (alpha-xENaC) is detectable as 150- and 180-kDa polypeptides, putative immature and mature alpha-subunit heterodimers. The 150- and 180-kDa alpha-xENaC were present in distinct fractions after sedimentation of A6 cell lysate through a sucrose density gradient. Two anti-alpha-xENaC antibodies directed against distinct domains demonstrated that only 180-kDa alpha-xENaC was expressed at the apical cell surface. The half-life of cell surface-expressed alpha-xENaC was 24-30 h, suggesting that once ENaC matures and is expressed at the plasma membrane, its turnover is similar to that reported for mature cystic fibrosis transmembrane conductance regulator. No significant changes in apical surface expression of alpha-xENaC were observed after treatment of A6 cells with aldosterone for 24 h, despite a 5.3-fold increase in short-circuit current. This lack of change in surface expression is consistent with previous observations in A6 cells and suggests that aldosterone regulates ENaC gating and increases channel open probability. PMID:11457713

  9. A Mass Spectrometric-Derived Cell Surface Protein Atlas

    PubMed Central

    Bausch-Fluck, Damaris; Hofmann, Andreas; Bock, Thomas; Frei, Andreas P.; Cerciello, Ferdinando; Jacobs, Andrea; Moest, Hansjoerg; Omasits, Ulrich; Gundry, Rebekah L.; Yoon, Charles; Schiess, Ralph; Schmidt, Alexander; Mirkowska, Paulina; Härtlová, Anetta; Van Eyk, Jennifer E.; Bourquin, Jean-Pierre; Aebersold, Ruedi; Boheler, Kenneth R.; Zandstra, Peter; Wollscheid, Bernd

    2015-01-01

    Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome) of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC) technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA) providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa). The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments. PMID:25894527

  10. Beyond the cell surface: new mechanisms of receptor function.

    PubMed

    Ibáñez, Carlos F

    2010-05-21

    The text book view of cell surface receptors depicts them at the top of a vertical chain of command that starts with ligand binding and proceeds in a lineal fashion towards the cell nucleus. Although pedagogically useful, this view is incomplete and recent findings suggest that the extracellular domain of cell surface receptors can be a transmitter as much as a receiver in intercellular communication. GFRalpha1 is a GPI-anchored receptor for GDNF (glial cell line-derived neurotrophic factor), a neuronal growth factor with widespread functions in the developing and adult nervous system. GFRalpha1 partners with transmembrane proteins, such as the receptor tyrosine kinase RET or the cell adhesion molecule NCAM, for intracellular transmission of the GDNF signal. In addition to this canonical role, GFRalpha1 can also engage in horizontal interactions and thereby modify the function of other cell surface components. GFRalpha1 can also function as a ligand-induced adhesion cell molecule, mediating homophilic cell-cell interactions in response to GDNF. Finally, GFRalpha1 can also be released from the cell surface and act at a distance as a soluble factor together with its ligand. This plethora of unconventional mechanisms is likely to be a feature common to several other receptors and considerably expands our view of cell surface receptor function. PMID:20494105

  11. Expanding the diversity of unnatural cell surface sialic acids

    SciTech Connect

    Luchansky, Sarah J.; Goon, Scarlett; Bertozzi, Carolyn R.

    2003-10-30

    Novel chemical reactivity can be introduced onto cell surfaces through metabolic oligosaccharide engineering. This technique exploits the substrate promiscuity of cellular biosynthetic enzymes to deliver unnatural monosaccharides bearing bioorthogonal functional groups into cellular glycans. For example, derivatives of N-acetylmannosamine (ManNAc) are converted by the cellular biosynthetic machinery into the corresponding sialic acids and subsequently delivered to the cell surface in the form of sialoglycoconjugates. Analogs of N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) are also metabolized and incorporated into cell surface glycans, likely through the sialic acid and GalNAc salvage pathways, respectively. Furthermore, GlcNAc analogs can be incorporated into nucleocytoplasmic proteins in place of {beta}-O-GlcNAc residues. These pathways have been exploited to integrate unique electrophiles such as ketones and azides into the target glycoconjugate class. These functional groups can be further elaborated in a chemoselective fashion by condensation with hydrazides and by Staudinger ligation, respectively, thereby introducing detectable probes onto the cell. In conclusion, sialic acid derivatives are efficient vehicles for delivery of bulky functional groups to cell surfaces and masking of their hydroxyl groups improves their cellular uptake and utilization. Furthermore, the successful introduction of photoactivatable aryl azides into cell surface glycans opens up new avenues for studying sialic acid-binding proteins and elucidating the role of sialic acid in essential processes such as signaling and cell adhesion.

  12. Recent Insights into Cell Surface Heparan Sulphate Proteoglycans and Cancer.

    PubMed

    Couchman, John R; Multhaupt, Hinke; Sanderson, Ralph D

    2016-01-01

    A small group of cell surface receptors are proteoglycans, possessing a core protein with one or more covalently attached glycosaminoglycan chains. They are virtually ubiquitous and their chains are major sites at which protein ligands of many types interact. These proteoglycans can signal and regulate important cell processes, such as adhesion, migration, proliferation, and differentiation. Since many protein ligands, such as growth factors, morphogens, and cytokines, are also implicated in tumour progression, it is increasingly apparent that cell surface proteoglycans impact tumour cell behaviour. Here, we review some recent advances, emphasising that many tumour-related functions of proteoglycans are revealed only after their modification in processes subsequent to synthesis and export to the cell surface. These include enzymes that modify heparan sulphate structure, recycling of whole or fragmented proteoglycans into exosomes that can be paracrine effectors or biomarkers, and lateral interactions between some proteoglycans and calcium channels that impact the actin cytoskeleton. PMID:27408707

  13. Recent Insights into Cell Surface Heparan Sulphate Proteoglycans and Cancer

    PubMed Central

    Couchman, John R; Multhaupt, Hinke; Sanderson, Ralph D.

    2016-01-01

    A small group of cell surface receptors are proteoglycans, possessing a core protein with one or more covalently attached glycosaminoglycan chains. They are virtually ubiquitous and their chains are major sites at which protein ligands of many types interact. These proteoglycans can signal and regulate important cell processes, such as adhesion, migration, proliferation, and differentiation. Since many protein ligands, such as growth factors, morphogens, and cytokines, are also implicated in tumour progression, it is increasingly apparent that cell surface proteoglycans impact tumour cell behaviour. Here, we review some recent advances, emphasising that many tumour-related functions of proteoglycans are revealed only after their modification in processes subsequent to synthesis and export to the cell surface. These include enzymes that modify heparan sulphate structure, recycling of whole or fragmented proteoglycans into exosomes that can be paracrine effectors or biomarkers, and lateral interactions between some proteoglycans and calcium channels that impact the actin cytoskeleton. PMID:27408707

  14. A Rapid Method for Refolding Cell Surface Receptors and Ligands

    PubMed Central

    Zhai, Lu; Wu, Ling; Li, Feng; Burnham, Robert S.; Pizarro, Juan C.; Xu, Bin

    2016-01-01

    Production of membrane-associated cell surface receptors and their ligands is often a cumbersome, expensive, and time-consuming process that limits detailed structural and functional characterization of this important class of proteins. Here we report a rapid method for refolding inclusion-body-based, recombinant cell surface receptors and ligands in one day, a speed equivalent to that of soluble protein production. This method efficiently couples modular on-column immobilized metal ion affinity purification and solid-phase protein refolding. We demonstrated the general utility of this method for producing multiple functionally active immunoreceptors, ligands, and viral decoys, including challenging cell surface proteins that cannot be produced using typical dialysis- or dilution-based refolding approaches. PMID:27215173

  15. Atomic force and super-resolution microscopy support a role for LapA as a cell-surface biofilm adhesin of Pseudomonas fluorescens

    PubMed Central

    Ivanov, Ivan E.; Boyd, Chelsea D.; Newell, Peter D.; Schwartz, Mary E.; Turnbull, Lynne; Johnson, Michael S.; Whitchurch, Cynthia B.; O’Toole, George A.; Camesano, Terri A.

    2012-01-01

    Pseudomonas fluorescence Pf0-1 requires the large repeat protein LapA for stable surface attachment. This study presents direct evidence that LapA is a cell-surface-localized adhesin. Atomic force microscopy (AFM) revealed a significant twofold reduction in adhesion force for mutants lacking the LapA protein on the cell surface compared to the wild-type strain. Deletion of lapG, a gene encoding a periplasmic cysteine protease that functions to release LapA from the cell surface, resulted in a twofold increase in the force of adhesion. Three-dimensional structured illumination microscopy (3D-SIM) revealed the presence of the LapA protein on the cell surface, consistent with its role as an adhesin. The protein is only visualized in the cytoplasm for a mutant of the ABC transporter responsible for translocating LapA to the cell surface. Together, these data highlight the power of combining the use of AFM and 3D-SIM with genetic studies to demonstrate that LapA, a member of a large group of RTX-like repeat proteins, is a cell-surface adhesin. PMID:23064158

  16. 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.

  17. An in-house multiplex pcr method to detect of putative virulence factors in aeromonas species

    PubMed Central

    Aguilera-Arreola, Ma. Guadalupe; Martínez, Alma Aidee Carmona; Castro-Escarpulli, Graciela

    2011-01-01

    A pentaplex PCR was developed and optimised to detect the genes that encode the five most important putative virulence factors in Aeromonas isolates. It seems to be more efficient than previously reported techniques and promises to be a powerful tool for more accurate risk assessments and for monitoring pathogenic strains. PMID:24031758

  18. Identification of Cell Surface Targets through Meta-analysis of Microarray Data1

    PubMed Central

    Haeberle, Henry; Dudley, Joel T; Liu, Jonathan TC; Butte, Atul J; Contag, Christopher H

    2012-01-01

    High-resolution image guidance for resection of residual tumor cells would enable more precise and complete excision for more effective treatment of cancers, such as medulloblastoma, the most common pediatric brain cancer. Numerous studies have shown that brain tumor patient outcomes correlate with the precision of resection. To enable guided resection with molecular specificity and cellular resolution, molecular probes that effectively delineate brain tumor boundaries are essential. Therefore, we developed a bioinformatics approach to analyze micro-array datasets for the identification of transcripts that encode candidate cell surface biomarkers that are highly enriched in medulloblastoma. The results identified 380 genes with greater than a two-fold increase in the expression in the medulloblastoma compared with that in the normal cerebellum. To enrich for targets with accessibility for extracellular molecular probes, we further refined this list by filtering it with gene ontology to identify genes with protein localization on, or within, the plasma membrane. To validate this meta-analysis, the top 10 candidates were evaluated with immunohistochemistry. We identified two targets, fibrillin 2 and EphA3, which specifically stain medulloblastoma. These results demonstrate a novel bioinformatics approach that successfully identified cell surface and extracellular candidate markers enriched in medulloblastoma versus adjacent cerebellum. These two proteins are high-value targets for the development of tumor-specific probes in medulloblastoma. This bioinformatics method has broad utility for the identification of accessible molecular targets in a variety of cancers and will enable probe development for guided resection. PMID:22904683

  19. Complete genome sequences of a putative new alphapartitivirus detected in Rosa spp.

    PubMed

    Phelan, James; James, Delano

    2016-09-01

    A putative new alphapartitivirus was detected by next-generation sequencing (NGS) in Rosa spp. and identified as rose partitivirus isolate Phyllis Bide (RoPV-PB). The virus is bipartite with a dsRNA1 fragment (1937 bp) encoding a putative RdRp and a dsRNA2 fragment (1811 bp) encoding the putative CP subunit of the virus. dsRNA1 of RoPV-BP is closely related to Vicia faba partitivirus 1, with identities of 67 % and 72 % for the nucleotide (nt) and deduced amino acid (aa) sequences, respectively. In NGS analysis of RoPV-BP, coverage was uneven across both dsRNA fragments, with GC/AT content appearing to be a major determinant of depth of coverage. PMID:27368993

  20. Las1 Is an Essential Nuclear Protein Involved in Cell Morphogenesis and Cell Surface Growth

    PubMed Central

    Doseff, A. I.; Arndt, K. T.

    1995-01-01

    Saccharomyces cerevisiae mutations that cause a requirement for SSD1-v for viability were isolated, yielding one new gene, LAS1, and three previously identified genes, SIT4, BCK1/SLK1, and SMP3. Three of these genes, LAS1, SIT4, and BCK1/SLK1, encode proteins that have roles in bud formation or morphogenesis. LAS1 is essential and loss of LAS1 function causes the cells to arrest as 80% unbudded cells and 20% large budded cells that accumulate many vesicles at the mother-daughter neck. Overexpression of LAS1 results in extra cell surface projections in the mother cell, alterations in actin and SPA2 localization, and the accumulation of electron-dense structures along the periphery of both the mother cell and the bud. The nuclear localization of LAS1 suggests a role of LAS1 for regulating bud formation and morphogenesis via the expression of components that function directly in these processes. PMID:8582632

  1. M135R is a novel cell surface virulence factor of myxoma virus.

    PubMed

    Barrett, John W; Sypula, Joanna; Wang, Fuan; Alston, Lindsay R; Shao, Zhuhong; Gao, Xiujuan; Irvine, Timothy S; McFadden, Grant

    2007-01-01

    Myxoma virus (MV) encodes a cell surface protein (M135R) that is predicted to mimic the host alpha/beta interferon receptor (IFN-alpha/beta-R) and thus prevent IFN-alpha/beta from triggering a host antiviral response. This prediction is based on sequence similarity to B18R, the viral IFN-alpha/beta-R from vaccinia virus (VV), which has been demonstrated to bind and inhibit type I interferons. However, M135R is only half the size of VV B18R. All other poxvirus-encoded IFN-alpha/beta-R homologs align only to the amino-terminal half of M135R. Peptide antibodies raised against M135R were used for immunoblotting and immunofluorescence and indicate that M135R is expressed as an early gene and that the product is a cell surface N-linked glycoprotein that is not secreted. In contrast to the predicted properties of M135R as an inhibitor of type I interferon, all binding and inhibition assays designed to demonstrate whether M135R can interact with IFN-alpha/beta have been negative. However, pathogenesis studies with a targeted M135-knockout MV construct (vMyx135KO) indicate that the deletion of M135R severely attenuates MV pathogenesis in the European rabbit. We propose that M135R is an important immunomodulatory virulence factor for myxomatosis but that the target immune ligand is not from the predicted type I interferon family and remains to be identified. PMID:17065210

  2. Structure of a bacterial cell surface decaheme electron conduit

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves decaheme cytochromes that are located on the bacterial cell surface at the termini of trans-outer-membrane electron transfer conduits...

  3. The cell surface environment for pathogen recognition and entry

    PubMed Central

    Stow, Jennifer L; Condon, Nicholas D

    2016-01-01

    The surface of mammalian cells offers an interface between the cell interior and its surrounding milieu. As part of the innate immune system, macrophages have cell surface features optimised for probing and sampling as they patrol our tissues for pathogens, debris or dead cells. Their highly dynamic and constantly moving cell surface has extensions such as lamellipodia, filopodia and dorsal ruffles that help detect pathogens. Dorsal ruffles give rise to macropinosomes for rapid, high volume non-selective fluid sampling, receptor internalisation and plasma membrane turnover. Ruffles can also generate phagocytic cups for the receptor-mediated uptake of pathogens or particles. The membrane lipids, actin cytoskeleton, receptors and signalling proteins that constitute these cell surface domains are discussed. Although the cell surface is designed to counteract pathogens, many bacteria, viruses and other pathogens have evolved to circumvent or hijack these cell structures and their underlying machinery for entry and survival. Nevertheless, these features offer important potential for developing vaccines, drugs and preventative measures to help fight infection. PMID:27195114

  4. Transcript Abundance of Putative Lipid Phosphate Phosphatases During Development of Trypanosoma brucei in the Tsetse Fly.

    PubMed

    Alves e Silva, Thiago Luiz; Savage, Amy F; Aksoy, Serap

    2016-04-01

    African trypanosomes (Trypanosoma brucei spp.) cause devastating diseases in sub-Saharan Africa. Trypanosomes differentiate repeatedly during development in tsetse flies before gaining mammalian infectivity in fly salivary glands. Lipid phosphate phosphatases (LPPs) are involved in diverse biological processes, such as cell differentiation and cell migration. Gene sequences encoding two putative T. brucei LPP proteins were used to search the T. brucei genome, revealing two additional putative family members. Putative structural features and transcript abundance during parasite development in tsetse fly were characterized. Three of the four LPP proteins are predicted to have six transmembrane domains, while the fourth shows only one. Semiquantitative gene expression revealed differential regulation of LPPs during parasite development. Transcript abundance for three of the four putative LPP genes was elevated in parasites infecting salivary glands, but not mammalian-infective metacyclic cells in fly saliva, indicating a potential role of this family in parasite establishment in tsetse salivary glands. PMID:26856918

  5. Hundreds of putatively functional small open reading frames in Drosophila

    PubMed Central

    2011-01-01

    Background The relationship between DNA sequence and encoded information is still an unsolved puzzle. The number of protein-coding genes in higher eukaryotes identified by genome projects is lower than was expected, while a considerable amount of putatively non-coding transcription has been detected. Functional small open reading frames (smORFs) are known to exist in several organisms. However, coding sequence detection methods are biased against detecting such very short open reading frames. Thus, a substantial number of non-canonical coding regions encoding short peptides might await characterization. Results Using bio-informatics methods, we have searched for smORFs of less than 100 amino acids in the putatively non-coding euchromatic DNA of Drosophila melanogaster, and initially identified nearly 600,000 of them. We have studied the pattern of conservation of these smORFs as coding entities between D. melanogaster and Drosophila pseudoobscura, their presence in syntenic and in transcribed regions of the genome, and their ratio of conservative versus non-conservative nucleotide changes. For negative controls, we compared the results with those obtained using random short sequences, while a positive control was provided by smORFs validated by proteomics data. Conclusions The combination of these analyses led us to postulate the existence of at least 401 functional smORFs in Drosophila, with the possibility that as many as 4,561 such functional smORFs may exist. PMID:22118156

  6. 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

  7. 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

  8. KSHV attachment and entry are dependent on αVβ3 integrin localized to specific cell surface microdomains and do not correlate with the presence of heparan sulfate

    PubMed Central

    Garrigues, H. Jacques; DeMaster, Laura K.; Rubinchikova, Yelena E.; Rose, Timothy M.

    2014-01-01

    Cellular receptors for KSHV attachment and entry were characterized using tyramide signal amplification (TSA)-enhanced confocal microscopy. Integrins αVβ3, αVβ5 and α3β1 were detected on essentially all the actin-based cell surface microdomains that initially bind KSHV, while the presence of CD98 and heparan sulfate (HS), the putative attachment receptor, was more variable. KSHV bound to the same cell surface microdomains with and without HS indicating that initial attachment of KSHV is not dependent on HS and that receptors other than HS can mediate attachment. A human salivary gland (HSG) epithelial line was identified, which lacks αVβ3 but expresses high levels of HS, α3β1 and other putative KSHV receptors. These cells were resistant to KSHV-binding and infection. Reconstitution of cell surface αVβ3 rendered HSG cells highly susceptible to KSHV infection, demonstrating a critical role for αVβ3 in the binding and entry of KSHV that is not shared with other proposed receptors. PMID:25063885

  9. Cell surface engineering of microorganisms towards adsorption of heavy metals.

    PubMed

    Li, Peng-Song; Tao, Hu-Chun

    2015-06-01

    Heavy metal contamination has become a worldwide environmental concern due to its toxicity, non-degradability and food-chain bioaccumulation. Conventional physical and chemical treatment methods for heavy metal removal have disadvantages such as cost-intensiveness, incomplete removal, secondary pollution and the lack of metal specificity. Microbial biomass-based biosorption is one of the approaches gaining increasing attention because it is effective, cheap, and environmental friendly and can work well at low concentrations. To enhance the adsorption properties of microbial cells to heavy metal ions, the cell surface display of various metal-binding proteins/peptides have been performed using a cell surface engineering approach. The surface engineering of Gram-negative bacteria, Gram-positive bacteria and yeast towards the adsorption of heavy metals are reviewed in this article. The problems and future perspectives of this technology are discussed. PMID:23915280

  10. Cell surface recycling in yeast: mechanisms and machineries.

    PubMed

    MacDonald, Chris; Piper, Robert C

    2016-04-15

    Sorting internalized proteins and lipids back to the cell surface controls the supply of molecules throughout the cell and regulates integral membrane protein activity at the surface. One central process in mammalian cells is the transit of cargo from endosomes back to the plasma membrane (PM) directly, along a route that bypasses retrograde movement to the Golgi. Despite recognition of this pathway for decades we are only beginning to understand the machinery controlling this overall process. The budding yeastSaccharomyces cerevisiae, a stalwart genetic system, has been routinely used to identify fundamental proteins and their modes of action in conserved trafficking pathways. However, the study of cell surface recycling from endosomes in yeast is hampered by difficulties that obscure visualization of the pathway. Here we briefly discuss how recycling is likely a more prevalent process in yeast than is widely appreciated and how tools might be built to better study the pathway. PMID:27068957

  11. A Generalizable Platform for the Photoactivation of Cell Surface Receptors.

    PubMed

    Duc, Thinh Nguyen; Huse, Morgan

    2015-11-20

    Polarized signal transduction from cell surface receptors plays a central role in the development and homeostasis of multicellular organisms, and it also contributes to cellular dysfunction in many disease states. Understanding the molecular and cellular bases of polarized signaling requires experimental methods that provide precise spatiotemporal control of receptor activation. However, we currently lack strategies for inducing both sustained and spatially constrained signal transduction. In the present study, we combined synthetic and cell biological tools to develop a generalizable photoactivation approach for the stimulation of cell surface receptors. Our system, which is based upon the local decaging of a "universal" peptide ligand, is particularly well suited for the live imaging of single cells. We anticipate that it will greatly facilitate future mechanistic analyses of polarized signal transduction in a variety of cell types. PMID:26295186

  12. Cell Surface Changes Associated with Cellular Immune Reactions in Drosophila

    NASA Astrophysics Data System (ADS)

    Nappi, Anthony J.; Silvers, Michael

    1984-09-01

    In Drosophila melanogaster a temperature-induced change in immune competence accompanies cell surface alterations that cause its blood cells to adhere and to encapsulate a parasite. At 29 degrees C the blood cells of the tumorous-lethal (Tuml) mutant show a high degree of immune competence and encapsulate the eggs of the parasitic wasp Leptopilina heterotoma. At 21 degrees C the blood cells are essentially immune incompetent. High percentages of lectin binding cells were found under conditions which potentiated cellular encapsulation responses. Some immune reactive blood cells did not bind lectin. The low percentages of lectin binding cells in susceptible hosts suggest that developing parasites alter the cell surface of the blood cells of immune reactive hosts.

  13. Cell surface changes associated with cellular immune reactions in Drosophila.

    PubMed

    Nappi, A J; Silvers, M

    1984-09-14

    In Drosophila melanogaster a temperature-induced change in immune competence accompanies cell surface alterations that cause its blood cells to adhere and to encapsulate a parasite. At 29 degrees C the blood cells of the tumorous-lethal (Tuml) mutant show a high degree of immune competence and encapsulate the eggs of the parasitic wasp Leptopilina heterotoma. At 21 degrees C the blood cells are essentially immune incompetent. High percentages of lectin binding cells were found under conditions which potentiated cellular encapsulation responses. Some immune reactive blood cells did not bind lectin. The low percentages of lectin binding cells in susceptible hosts suggest that developing parasites alter the cell surface of the blood cells of immune reactive hosts. PMID:6433482

  14. 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

  15. Cell-surface markers for colon adenoma and adenocarcinoma

    PubMed Central

    Sewda, Kamini; Coppola, Domenico; Enkemann, Steven; Yue, Binglin; Kim, Jongphil; Lopez, Alexis S.; Wojtkowiak, Jonathan W.; Stark, Valerie E.; Morse, Brian; Shibata, David; Vignesh, Shivakumar; Morse, David L.

    2016-01-01

    Early detection of colorectal cancer (CRC) is crucial for effective treatment. Among CRC screening techniques, optical colonoscopy is widely considered the gold standard. However, it is a costly and invasive procedure with a low rate of compliance. Our long-term goal is to develop molecular imaging agents for the non-invasive detection of CRC by molecular imaging-based colonoscopy using CT, MRI or fluorescence. To achieve this, cell surface targets must be identified and validated. Here, we report the discovery of cell-surface markers that distinguish CRC from surrounding tissues that could be used as molecular imaging targets. Profiling of mRNA expression microarray data from patient tissues including adenoma, adenocarcinoma, and normal gastrointestinal tissues was used to identify potential CRC specific cell-surface markers. Of the identified markers, six were selected for further validation (CLDN1, GPR56, GRM8, LY6G6D/F, SLCO1B3 and TLR4). Protein expression was confirmed by immunohistochemistry of patient tissues. Except for SLCO1B3, diffuse and low expression was observed for each marker in normal colon tissues. The three markers with the greatest protein overexpression were CLDN1, LY6G6D/F and TLR4, where at least one of these markers was overexpressed in 97% of the CRC samples. GPR56, LY6G6D/F and SLCO1B3 protein expression was significantly correlated with the proximal tumor location and with expression of mismatch repair genes. Marker expression was further validated in CRC cell lines. Hence, three cell-surface markers were discovered that distinguish CRC from surrounding normal tissues. These markers can be used to develop imaging or therapeutic agents targeted to the luminal surface of CRC. PMID:26894861

  16. Structure of a Bacterial Cell Surface Decaheme Electron Conduit

    SciTech Connect

    Clarke, Thomas A.; Edwards, Marcus; Gates, Andrew J.; Hall, Andrea; White, Gaye; Bradley, Justin; Reardon, Catherine L.; Shi, Liang; Beliaev, Alex S.; Marshall, Matthew J.; Wang, Zheming; Watmough, Nicholas; Fredrickson, Jim K.; Zachara, John M.; Butt, Julea N.; Richardson, David J.

    2011-05-23

    Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves deca-heme cytochromes that are located on the bacterial cell surface at the termini of trans-outermembrane (OM) electron transfer conduits. The cell surface cytochromes can potentially play multiple roles in mediating electron transfer directly to insoluble electron sinks, catalyzing electron exchange with flavin electron shuttles or participating in extracellular inter-cytochrome electron exchange along ‘nanowire’ appendages. We present a 3.2 Å crystal structure of one of these deca-heme cytochromes, MtrF, that allows the spatial organization of the ten hemes to be visualized for the first time. The hemes are organized across four domains in a unique crossed conformation, in which a staggered 65 Å octa-heme chain transects the length of the protein and is bisected by a planar 45 Å tetra-heme chain that connects two extended Greek key split β-barrel domains. The structure provides molecular insight into how reduction of insoluble substrate (e.g. minerals), soluble substrates (e.g. flavins) and cytochrome redox partners might be possible in tandem at different termini of a trifurcated electron transport chain on the cell surface.

  17. Cell surface expression and biosynthesis of epithelial Na+ channels.

    PubMed Central

    Prince, L S; Welsh, M J

    1998-01-01

    The epithelial Na+ channel (ENaC) complex is composed of three homologous subunits: alpha, beta and gamma. Mutations in ENaC subunits can increase the number of channels on the cell surface, causing a hereditary form of hypertension called Liddle's syndrome, or can decrease channel activity, causing pseudohypoaldosteronism type I, a salt-wasting disease of infancy. To investigate surface expression, we studied ENaC subunits expressed in COS-7 and HEK293 cells. Using surface biotinylation and protease sensitivity, we found that when individual ENaC subunits are expressed alone, they traffic to the cell surface. The subunits are glycosylated with high-mannose oligosaccharides, but seem to have the carbohydrate removed before they reach the cell surface. Moreover, subunits form a complex that cannot be disrupted by several non-ionic detergents. The pattern of glycosylation and detergent solubility/insolubility persists when the N-teminal and C-terminal cytoplasmic regions of ENaC are removed. With co-expression of all three ENaC subunits, the insoluble complex is the predominant species. These results show that ENaC and its family members are unique in their trafficking, biochemical characteristics and post-translational modifications. PMID:9841884

  18. Engineering novel cell surface chemistry for selective tumor cell targeting

    SciTech Connect

    Bertozzi, C.R. |

    1997-12-31

    A common feature of many different cancers is the high expression level of the two monosaccharides sialic acid and fucose within the context of cell-surface associated glycoconjugates. A correlation has been made between hypersialylation and/or hyperfucosylation and the highly metastatic phenotype. Thus, a targeting strategy based on sialic acid or fucose expression would be a powerful tool for the development of new cancer cell-selective therapies and diagnostic agents. We have discovered that ketone groups can be incorporated metabolically into cell-surface associated sialic acids. The ketone is can be covalently ligated with hydrazide functionalized proteins or small molecules under physiological conditions. Thus, we have discovered a mechanism to selectively target hydrazide conjugates to highly sialylated cells such as cancer cells. Applications of this technology to the generation of novel cancer cell-selective toxins and MRI contrast reagents will be discussed, in addition to progress towards the use of cell surface fucose residues as vehicles for ketone expression.

  19. Establishment of cell surface engineering and its development.

    PubMed

    Ueda, Mitsuyoshi

    2016-07-01

    Cell surface display of proteins/peptides has been established based on mechanisms of localizing proteins to the cell surface. In contrast to conventional intracellular and extracellular (secretion) expression systems, this method, generally called an arming technology, is particularly effective when using yeasts as a host, because the control of protein folding that is often required for the preparation of proteins can be natural. This technology can be employed for basic and applied research purposes. In this review, I describe various strategies for the construction of engineered yeasts and provide an outline of the diverse applications of this technology to industrial processes such as the production of biofuels and chemicals, as well as bioremediation and health-related processes. Furthermore, this technology is suitable for novel protein engineering and directed evolution through high-throughput screening, because proteins/peptides displayed on the cell surface can be directly analyzed using intact cells without concentration and purification. Functional proteins/peptides with improved or novel functions can be created using this beneficial, powerful, and promising technique. PMID:27305282

  20. Actomyosin contractility controls cell surface area of oligodendrocytes

    PubMed Central

    Kippert, Angelika; Fitzner, Dirk; Helenius, Jonne; Simons, Mikael

    2009-01-01

    Background To form myelin oligodendrocytes expand and wrap their plasma membrane multiple times around an axon. How is this expansion controlled? Results Here we show that cell surface area depends on actomyosin contractility and is regulated by physical properties of the supporting matrix. Moreover, we find that chondroitin sulfate proteoglycans (CSPG), molecules associated with non-permissive growth properties within the central nervous system (CNS), block cell surface spreading. Most importantly, the inhibitory effects of CSPG on plasma membrane extension were completely prevented by treatment with inhibitors of actomyosin contractility and by RNAi mediated knockdown of myosin II. In addition, we found that reductions of plasma membrane area were accompanied by changes in the rate of fluid-phase endocytosis. Conclusion In summary, our results establish a novel connection between endocytosis, cell surface extension and actomyosin contractility. These findings open up new possibilities of how to promote the morphological differentiation of oligodendrocytes in a non-permissive growth environment. See related minireview by Bauer and ffrench-Constant: PMID:19781079

  1. Cell Surface Vimentin Is an Attachment Receptor for Enterovirus 71

    PubMed Central

    Du, Ning; Cong, Haolong; Tian, Hongchao; Zhang, Hua; Zhang, Wenliang; Song, Lei

    2014-01-01

    ABSTRACT Enterovirus 71 (EV71) is a highly transmissible pathogenic agent that causes severe central nervous system diseases in infected infants and young children. Here, we reported that EV71 VP1 protein could bind to vimentin intermediate filaments expressed on the host cell surface. Soluble vimentin or an antibody against vimentin could inhibit the binding of EV71 to host cells. Accompanied with the reduction of vimentin expression on the cell surface, the binding of EV71 to cells was remarkably decreased. Further evidence showed that the N terminus of vimentin is responsible for the interaction between EV71 and vimentin. These results indicated that vimentin on the host cell surface may serve as an attachment site that mediated the initial binding and subsequently increased the infectivity of EV71. IMPORTANCE This study delivers important findings on the roles of vimentin filaments in relation to EV71 infection and provides information that not only improves our understanding of EV71 pathogenesis but also presents us with potentially new strategies for the treatment of diseases caused by EV71 infections. PMID:24623428

  2. 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.

  3. Cloning of a DNA fragment encoding a heme-repressible hemoglobin-binding outer membrane protein from Haemophilus influenzae.

    PubMed Central

    Jin, H; Ren, Z; Pozsgay, J M; Elkins, C; Whitby, P W; Morton, D J; Stull, T L

    1996-01-01

    Haemophilus influenzae is able to use hemoglobin as a sole source of heme, and heme-repressible hemoglobin binding to the cell surface has been demonstrated. Using an affinity purification methodology, a hemoglobin-binding protein of approximately 120 kDa was isolated from H. influenzae type b strain HI689 grown in heme-restricted but not in heme-replete conditions. The isolated protein was subjected to N-terminal amino acid sequencing, and the derived amino acid sequence was used to design corresponding oligonucleotides. The oligonucleotides were used to probe a Southern blot of EcoRI-digested HI689 genomic DNA. A hybridizing band of approximately 4.2 kb was successfully cloned into pUC19. Using a 1.9-kb internal BglII fragment of the 4.2-kb clone as a probe, hybridization was seen in both typeable and nontypeable H. influenzae but not in other bacterial species tested. Following partial nucleotide sequencing of the 4.2-kb insert, a putative open reading frame was subcloned into an expression vector. The host Escherichia coli strain in which the cloned fragment was expressed bound biotinylated human hemoglobin, whereas binding of hemoglobin was not detected in E. coli with the vector alone. In conclusion, we hypothesize that the DNA fragment encoding an approximately 120-kDa heme-repressible hemoglobin-binding protein mediates one step in the acquisition of hemoglobin by H. influenzae in vivo. PMID:8757844

  4. Targeting vaccinia virus-expressed secretory beta subunit of human chorionic gonadotropin to the cell surface induces antibodies.

    PubMed Central

    Srinivasan, J; Singh, O; Chakrabarti, S; Talwar, G P

    1995-01-01

    We carried out experiments designed to study the effect of a protein's localization on its immunogenicity. A novel cell-surface protein was generated from a small, glycosylated secretory protein. The DNA sequence encoding the entire precursor of the human chorionic gonadotropin beta (beta hCG) subunit was fused in the correct reading frame to the DNA sequence encoding the transmembrane and cytoplasmic domains of vesicular stomatitis virus glycoprotein. This chimeric gene was introduced into the vaccinia virus genome to generate a recombinant virus. The recombinant virus, when used to infect animal cells, expressed a 135-amino-acid beta hCG subunit anchored in cellular membranes by the 48 carboxy-terminal amino acids of vesicular stomatitis virus glycoprotein. The immunogenicity of this recombinant virus with respect to its ability to generate anti-hCG antibodies was compared with that of a second recombinant vaccinia virus expressing the native secretory form of beta hCG. All animals immunized with the vaccinia virus expressing beta hCG on the cell surface elicited high titers of anti-hCG antibodies. Even after a single immunization with the recombinant vaccinia virus, the anti-hCG antibody titers persisted for a long period of time (more than 6 months). None of the animals immunized with vaccinia virus expressing the native secretory form of beta hCG showed any hCG-specific antibody response. PMID:7591154

  5. neu protooncogene fused to an immunoglobulin heavy chain gene requires immunoglobulin light chain for cell surface expression and oncogenic transformation.

    PubMed Central

    Flanagan, J G; Leder, P

    1988-01-01

    The protein encoded by the neu protooncogene (human gene symbol NGL for neuro/glioblastoma-derived) is a member of the surface receptor/tyrosine kinase family. Though its structure suggests that it can transduce a transmembrane signal, neither its extracellular ligand nor its critical intracellular substrates are known. To explore the functional properties of the protein encoded by neu, we created a fusion gene that joins the cytoplasmic domain of neu to the extracellular portion of an immunoglobulin heavy chain. The localization of the fusion polypeptide can then be controlled by coexpression with immunoglobulin light chain. In the absence of light chain, the heavy chain-neu polypeptide is expressed intracellularly and has no transforming activity. By contrast, in the presence of light chain the fusion polypeptide is expressed at the cell surface and produces tumorigenic foci. Thus, transformation apparently requires expression at the cell surface, where the neu intracellular domain can interact with components that are localized to the plasma membrane. The fusion protein is active in cellular transformation when the transmembrane domain is derived either from neu or from immunoglobulin, indicating that the neu transmembrane domain is not specifically required for transformation, although neu activation in tumors is known to result from a point mutation in this region. The extracellular immunoglobulin heavy and light chain domains of the fusion protein form a functional binding site that allows antigen to modulate its activity, reversing the transforming effect. Images PMID:2903500

  6. Characterization of a Cell Surface Protein of Clostridium difficile with Adhesive Properties

    PubMed Central

    Waligora, Anne-Judith; Hennequin, Claire; Mullany, Peter; Bourlioux, Pierre; Collignon, Anne; Karjalainen, Tuomo

    2001-01-01

    Our laboratory has previously shown that Clostridium difficile adherence to cultured cells is enhanced after heat shock at 60°C and that it is mediated by a proteinaceous surface component. The present study was undertaken to identify the surface molecules of this bacterium that could play a role in its adherence to the intestine. The cwp66 gene, encoding a cell surface-associated protein of C. difficile 79-685, was isolated by immunoscreening of a C. difficile gene library with polyclonal antibodies against C. difficile heated at 60°C. The Cwp66 protein (66 kDa) contains two domains, each carrying three imperfect repeats and one presenting homologies to the autolysin CwlB of Bacillus subtilis. A survey of 36 strains of C. difficile representing 11 serogroups showed that the 3′ portion of the cwp66 gene is variable; this was confirmed by sequencing of cwp66 from another strain, C-253. Two recombinant protein fragments corresponding to the two domains of Cwp66 were expressed in fusion with glutathione S-transferase in Escherichia coli and purified by affinity chromatography using gluthatione-Sepharose 4B. Antibodies raised against the two domains recognized Cwp66 in bacterial surface extracts. By immunoelectron microscopy, the C-terminal domain was found to be cell surface exposed. When used as inhibitors in cell binding studies, the antibodies and protein fragments partially inhibited adherence of C. difficile to cultured cells, confirming that Cwp66 is an adhesin, the first to be identified in clostridia. PMID:11254569

  7. Sialylation of cell surface glycoconjugates is essential for osteoclastogenesis.

    PubMed

    Takahata, Masahiko; Iwasaki, Norimasa; Nakagawa, Hiroaki; Abe, Yuichiro; Watanabe, Takuya; Ito, Manabu; Majima, Tokifumi; Minami, Akio

    2007-07-01

    Sialic acid, which is located at the end of the carbohydrate moiety of cell surface glycoconjugates, is involved in many biologic responses, such as intercellular reactions and virus-cell fusion, especially in hematopoietic cells. Here we provide experimental evidence that the sialic acid of cell surface glycoconjugates has a role in osteoclast differentiation. Lectin histochemical study demonstrated the existence of both alpha (2,3)-linked-sialic acid and alpha (2,6)-linked-sialic acid in mouse bone marrow-derived macrophages and in the RAW264.7 macrophage cell line, which are osteoclast precursors. Flow cytometric analysis of surface lectin staining revealed the kinetics of these sialic acids during osteoclastogenesis: alpha (2,3)-linked-sialic acid was abundantly expressed throughout osteoclastogenesis, whereas alpha (2,6)-linked-sialic acid levels declined at the terminal stage of osteoclast differentiation. To investigate the role of sialic acid in osteoclast differentiation, we performed an osteoclastogenesis assay with or without exogenous sialidase treatment. Desialylated cells formed TRAP-positive mononuclear cells, but did not become multinuclear cells despite the normal expression of osteoclast markers such as cathepsin K, integrin beta3, and nuclear factor-ATc1. Flow cytometric analysis also demonstrated that exogenous sialidase effectively removed alpha (2,6)-linked-sialic acid, but only slightly changed the alpha (2,3)-linked-sialic acid content, suggesting that alpha (2,6)-linked-sialic acid might be involved in osteoclast differentiation. Findings from knockdown analysis using small interfering RNA oligonucleotides against alpha 2,6-sialyltransferase support this idea: alpha (2,6)-linked-sialic acid-deficient cells markedly inhibit the formation of multinuclear osteoclasts. Our findings suggest that alpha (2,6)-linked-sialic acid of cell surface glycoconjugates has a role in osteoclast differentiation, possibly via its role in the cell-cell fusion

  8. Cell-surface prion protein interacts with glycosaminoglycans.

    PubMed Central

    Pan, Tao; Wong, Boon-Seng; Liu, Tong; Li, Ruliang; Petersen, Robert B; Sy, Man-Sun

    2002-01-01

    We used ELISA and flow cytometry to study the binding of prion protein PrP to glycosaminoglycans (GAGs). We found that recombinant human PrP (rPrP) binds GAGs including chondroitin sulphate A, chondroitin sulphate B, hyaluronic acid, and heparin. rPrP binding to GAGs occurs via the N-terminus, a region known to bind divalent cations. Additionally, rPrP binding to GAGs is enhanced in the presence of Cu2+ and Zn2+, but not Ca2+ and Mn2+. rPrP binds heparin strongest, and the binding is inhibited by certain heparin analogues, including heparin disaccharide and sulphate-containing monosaccharides, but not by acetylated heparin. Full-length normal cellular prion protein (PrPC), but not N-terminally truncated PrPC species, from human brain bind GAGs in a similar Cu2+/Zn2+-enhanced fashion. We found that GAGs specifically bind to a synthetic peptide corresponding to amino acid residues 23-35 in the N-terminus of rPrP. We further demonstrated that while both wild-type PrPC and an octapeptide-repeat-deleted mutant PrP produced by transfected cells bound heparin at the cell surface, the PrP N-terminal deletion mutant and non-transfectant control failed to bind heparin. Binding of heparin to wild-type PrPC on the cell surface results in a reduction of the level of cell-surface PrPC. These results provide strong evidence that PrPC is a surface receptor for GAGs. PMID:12186633

  9. Cell Surface Nucleolin Facilitates Enterovirus 71 Binding and Infection

    PubMed Central

    Su, Pei-Yi; Wang, Ya-Fang; Huang, Sheng-Wen; Lo, Yu-Chih; Wang, Ya-Hui; Wu, Shang-Rung; Shieh, Dar-Bin; Wang, Jen-Ren; Lai, Ming-Der

    2015-01-01

    ABSTRACT Because the pathogenesis of enterovirus 71 (EV71) remains mostly ambiguous, identifying the factors that mediate viral binding and entry to host cells is indispensable to ultimately uncover the mechanisms that underlie virus infection and pathogenesis. Despite the identification of several receptors/attachment molecules for EV71, the binding, entry, and infection mechanisms of EV71 remain unclear. Herein, we employed glycoproteomic approaches to identify human nucleolin as a novel binding receptor for EV71. Glycoproteins purified by lectin chromatography from the membrane extraction of human cells were treated with sialidase, followed by immunoprecipitation with EV71 particles. Among the 16 proteins identified by tandem mass spectrometry analysis, cell surface nucleolin attracted our attention. We found that EV71 interacted directly with nucleolin via the VP1 capsid protein and that an antinucleolin antibody reduced the binding of EV71 to human cells. In addition, the knockdown of cell surface nucleolin decreased EV71 binding, infection, and production in human cells. Furthermore, the expression of human nucleolin on the cell surface of a mouse cell line increased EV71 binding and conferred EV71 infection and production in the cells. These results strongly indicate that human nucleolin can mediate EV71 binding to and infection of cells. Our findings also demonstrate that the use of glycoproteomic approaches is a reliable methodology to discover novel receptors for pathogens. IMPORTANCE Outbreaks of EV71 have been reported in Asia-Pacific countries and have caused thousands of deaths in young children during the last 2 decades. The discovery of new EV71-interacting molecules to understand the infection mechanism has become an emergent issue. Hence, this study uses glycoproteomic approaches to comprehensively investigate the EV71-interacting glycoproteins. Several EV71-interacting glycoproteins are identified, and the role of cell surface nucleolin in

  10. Putative Genes Involved in Saikosaponin Biosynthesis in Bupleurum Species

    PubMed Central

    Lin, Tsai-Yun; Chiou, Chung-Yi; Chiou, Shu-Jiau

    2013-01-01

    Alternative medicinal agents, such as the herb Bupleurum, are increasingly used in modern medicine to supplement synthetic drugs. First, we present a review of the currently known effects of triterpene saponins-saikosaponins of Bupleurum species. The putative biosynthetic pathway of saikosaponins in Bupleurum species is summarized, followed by discussions on identification and characterization of genes involved in the biosynthesis of saikosaponins. The purpose is to provide a brief review of gene extraction, functional characterization of isolated genes and assessment of expression patterns of genes encoding enzymes in the process of saikosaponin production in Bupleurum species, mainly B. kaoi. We focus on the effects of MeJA on saikosaponin production, transcription patterns of genes involved in biosynthesis and on functional depiction. PMID:23783277

  11. Mycobacteriophage putative GTPase-activating protein can potentiate antibiotics.

    PubMed

    Yan, Shuangquan; Xu, Mengmeng; Duan, Xiangke; Yu, Zhaoxiao; Li, Qiming; Xie, Longxiang; Fan, Xiangyu; Xie, Jianping

    2016-09-01

    The soaring incidences of infection by antimicrobial resistant (AR) pathogens and shortage of effective antibiotics with new mechanisms of action have renewed interest in phage therapy. This scenario is exemplified by resistant tuberculosis (TB), caused by resistant Mycobacterium tuberculosis. Mycobacteriophage SWU1 A321_gp67 encodes a putative GTPase-activating protein. Mycobacterium smegmatis with gp67 overexpression showed changed colony formation and biofilm morphology and supports the efficacy of streptomycin and capreomycin against Mycobacterium. gp67 down-regulated the transcription of genes involved in cell wall and biofilm development. To our knowledge, this is the first report to show that phage protein in addition to lysin or recombination components can synergize with existing antibiotics. Phage components might represent a promising new clue for better antibiotic potentiators. PMID:27345061

  12. A Human Pluripotent Stem Cell Surface N-Glycoproteome Resource Reveals Markers, Extracellular Epitopes, and Drug Targets

    PubMed Central

    Boheler, Kenneth R.; Bhattacharya, Subarna; Kropp, Erin M.; Chuppa, Sandra; Riordon, Daniel R.; Bausch-Fluck, Damaris; Burridge, Paul W.; Wu, Joseph C.; Wersto, Robert P.; Chan, Godfrey Chi Fung; Rao, Sridhar; Wollscheid, Bernd; Gundry, Rebekah L.

    2014-01-01

    Summary Detailed knowledge of cell-surface proteins for isolating well-defined populations of human pluripotent stem cells (hPSCs) would significantly enhance their characterization and translational potential. Through a chemoproteomic approach, we developed a cell-surface proteome inventory containing 496 N-linked glycoproteins on human embryonic (hESCs) and induced PSCs (hiPSCs). Against a backdrop of human fibroblasts and 50 other cell types, >100 surface proteins of interest for hPSCs were revealed. The >30 positive and negative markers verified here by orthogonal approaches provide experimental justification for the rational selection of pluripotency and lineage markers, epitopes for cell isolation, and reagents for the characterization of putative hiPSC lines. Comparative differences between the chemoproteomic-defined surfaceome and the transcriptome-predicted surfaceome directly led to the discovery that STF-31, a reported GLUT-1 inhibitor, is toxic to hPSCs and efficient for selective elimination of hPSCs from mixed cultures. PMID:25068131

  13. Expression analysis of secreted and cell surface genes of five transformed human cell lines and derivative xenograft tumors

    PubMed Central

    Stull, Robert A; Tavassoli, Roya; Kennedy, Scot; Osborn, Steve; Harte, Rachel; Lu, Yan; Napier, Cheryl; Abo, Arie; Chin, Daniel J

    2005-01-01

    Background Since the early stages of tumorigenesis involve adhesion, escape from immune surveillance, vascularization and angiogenesis, we devised a strategy to study the expression profiles of all publicly known and putative secreted and cell surface genes. We designed a custom oligonucleotide microarray containing probes for 3531 secreted and cell surface genes to study 5 diverse human transformed cell lines and their derivative xenograft tumors. The origins of these human cell lines were lung (A549), breast (MDA MB-231), colon (HCT-116), ovarian (SK-OV-3) and prostate (PC3) carcinomas. Results Three different analyses were performed: (1) A PCA-based linear discriminant analysis identified a 54 gene profile characteristic of all tumors, (2) Application of MANOVA (Pcorr < .05) to tumor data revealed a larger set of 149 differentially expressed genes. (3) After MANOVA was performed on data from individual tumors, a comparison of differential genes amongst all tumor types revealed 12 common differential genes. Seven of the 12 genes were identified by all three analytical methods. These included late angiogenic, morphogenic and extracellular matrix genes such as ANGPTL4, COL1A1, GP2, GPR57, LAMB3, PCDHB9 and PTGER3. The differential expression of ANGPTL4 and COL1A1 and other genes was confirmed by quantitative PCR. Conclusion Overall, a comparison of the three analyses revealed an expression pattern indicative of late angiogenic processes. These results show that a xenograft model using multiple cell lines of diverse tissue origin can identify common tumorigenic cell surface or secreted molecules that may be important biomarker and therapeutic discoveries. PMID:15836779

  14. Functional analysis of putative adhesion factors in Lactobacillus acidophilus NCFM.

    PubMed

    Buck, B Logan; Altermann, Eric; Svingerud, Tina; Klaenhammer, Todd R

    2005-12-01

    Lactobacilli are major inhabitants of the normal microflora of the gastrointestinal tract, and some select species have been used extensively as probiotic cultures. One potentially important property of these organisms is their ability to interact with epithelial cells in the intestinal tract, which may promote retention and host-bacterial communication. However, the mechanisms by which they attach to intestinal epithelial cells are unknown. The objective of this study was to investigate cell surface proteins in Lactobacillus acidophilus that may promote attachment to intestinal tissues. Using genome sequence data, predicted open reading frames were searched against known protein and protein motif databases to identify four proteins potentially involved in adhesion to epithelial cells. Homologous recombination was used to construct isogenic mutations in genes encoding a mucin-binding protein, a fibronectin-binding protein, a surface layer protein, and two streptococcal R28 homologs. The abilities of the mutants to adhere to intestinal epithelial cells were then evaluated in vitro. Each strain was screened on Caco-2 cells, which differentiate and express markers characteristic of normal small-intestine cells. A significant decrease in adhesion was observed in the fibronectin-binding protein mutant (76%) and the mucin-binding protein mutant (65%). A surface layer protein mutant also showed reduction in adhesion ability (84%), but the effect of this mutation is likely due to the loss of multiple surface proteins that may be embedded in the S-layer. This study demonstrated that multiple cell surface proteins in L. acidophilus NCFM can individually contribute to the organism's ability to attach to intestinal cells in vitro. PMID:16332821

  15. Methods To Identify Aptamers against Cell Surface Biomarkers

    PubMed Central

    Cibiel, Agnes; Dupont, Daniel Miotto; Ducongé, Frédéric

    2011-01-01

    Aptamers are nucleic acid-based ligands identified through a process of molecular evolution named SELEX (Systematic Evolution of Ligands by Exponential enrichment). During the last 10-15 years, numerous aptamers have been developed specifically against targets present on or associated with the surface of human cells or infectious pathogens such as viruses, bacteria, fungi or parasites. Several of the aptamers have been described as potent probes, rivalling antibodies, for use in flow cytometry or microscopy. Some have also been used as drugs by inhibiting or activating functions of their targets in a manner similar to neutralizing or agonistic antibodies. Additionally, it is straightforward to conjugate aptamers to other agents without losing their affinity and they have successfully been used in vitro and in vivo to deliver drugs, siRNA, nanoparticles or contrast agents to target cells. Hence, aptamers identified against cell surface biomarkers represent a promising class of ligands. This review presents the different strategies of SELEX that have been developed to identify aptamers for cell surface-associated proteins as well as some of the methods that are used to study their binding on living cells.

  16. Interactions of macromolecules with the mammalian cell surface.

    PubMed

    Wall, J; Ayoub, F; O'Shea, P

    1995-07-01

    The characterisation of fluoresceinphosphatidylethanolamine (FPE) as a real-time indicator of the electrostatic nature of the cell membrane surface is described. The conditions appropriate for the labelling of cell membranes and the implementation of FPE as a tool to monitor the interactions of various proteins and peptides with membranes are outlined. Some complications attributed to the erythrocyte glycocalyx are examined. In addition it is shown using neuraminidase as an example, that some types of enzyme-catalysed reactions on the cell surface may be monitored in real time. It is also shown that information concerning the binding of several proteins such as serum albumin and monoclonal antibodies are accessible with this technique. The albumin in particular is shown to exhibit a saturation of binding, the analysis of which indicates that the dissociation constant for erythrocytes was determined to be 8 microM and for lymphocytes to be almost 3 microM. On the basis of this comparison together with artificial membranes, the membrane protein components of the lymphocyte surface are implicated in the binding of albumin or the erythrocyte membrane proteins reduce the affinity of the cell surface for albumin. PMID:7593308

  17. Cell surface morphology in epithelial malignancy and its precursor lesions.

    PubMed

    Kenemans, P; Davina, J H; de Haan, R W; van der Zanden, P; Vooys, G P; Stolk, J G; Stadhouders, A M

    1981-01-01

    The cell surface organization of cancer cells is of potentially great significance, as it may not only allow (early) diagnosis, but as it may also harbour markers for refined prognosis (degree of oncogenetic and metastatic potential), and targets for selective cancer (chemo- and immuno) therapy. With these aspects in mind, the present review deals with SEM work done on (pre-) malignant cells, both in vivo and in vitro, and in animal models. Attention, however, is focused on human cancer cells. Cancer cells in vitro may lose many of their original malignant characteristics, and show adaptations to culture conditions. Many other factors have been shown to influence cell surface morphology, such as cell cycle, cell contacts, and preparations technique. Cancer cells differ in their surface morphology from normal cells, and have an extra ordinary amount of surface activity. Human malignant epithelial cells show abundant long. pleomorphic microvilli, especially those present in effusions. In squamous epithelium (bladder, cervix) microridge system present on normal superficial cells are progressively replaced by microvilli which increase in number and degree of pleomorphism during experimental and clinical oncogenesis. The question of whether or not the appearance of long. Pleomorphic microvilli reflects an irreversible alteration of the epithelium, and thus provides an early marker of irreversible neoplastic transformation is considered and assessed on the basis of our work with (pre-) malignant cells of the human uterine cervix. Although SEM has contributed significantly to the description of oncogenesis, up to now it has no early diagnostic, prognostic or therapeutic significance. PMID:7199203

  18. Bacterial Cell Surface Adsorption of Rare Earth Elements

    NASA Astrophysics Data System (ADS)

    Jiao, Y.; Park, D.; Reed, D.; Fujita, Y.; Yung, M.; Anderko, A.; Eslamimanesh, A.

    2015-12-01

    Rare earth elements (REE) play a critical role in many emerging clean energy technologies, including high-power magnets, wind turbines, solar panels, hybrid/electric vehicle batteries and lamp phosphors. In order to sustain demand for such technologies given current domestic REE shortages, there is a need to develop new approaches for ore processing/refining and recycling of REE-containing materials. To this end, we have developed a microbially-mediated bioadsorption strategy with application towards enrichment of REE from complex mixtures. Specifically, the bacterium Caulobacter crescentus was genetically engineered to display lanthanide binding tags (LBTs), short peptides that possess high affinity and specificity for rare earth elements, on its cell surface S-layer protein. Under optimal conditions, LBT-displayed cells adsorbed greater than 5-fold more REE than control cells lacking LBTs. Competition binding experiments with a selection of REEs demonstrated that our engineered cells could facilitate separation of light- from heavy- REE. Importantly, binding of REE onto our engineered strains was much more favorable compared to non-REE metals. Finally, REE bound to the cell surface could be stripped off using citrate, providing an effective and non-toxic REE recovery method. Together, this data highlights the potential of our approach for selective REE enrichment from REE containing mixtures.

  19. Cdon, a cell surface protein, mediates oligodendrocyte differentiation and myelination.

    PubMed

    Wang, Li-Chun; Almazan, Guillermina

    2016-06-01

    During central nervous system development, oligodendrocyte progenitors (OLPs) establish multiple branched processes and axonal contacts to initiate myelination. A complete understanding of the molecular signals implicated in cell surface interaction to initiate myelination/remyelination is currently lacking. The objective of our study was to assess whether Cdon, a cell surface protein that was shown to participate in muscle and neuron cell development, is involved in oligodendrocyte (OLG) differentiation and myelination. Here, we demonstrate that endogenous Cdon protein is expressed in OLPs, increasing in the early differentiation stages and decreasing in mature OLGs. Immunocytochemistry of endogenous Cdon showed localization on both OLG cell membranes and cellular processes exhibiting puncta- or varicosity-like structures. Cdon knockdown with siRNA decreased protein levels by 62% as well as two myelin-specific proteins, MBP and MAG. Conversely, overexpression of full-length rat Cdon increased myelin proteins in OLGs. The complexity of OLGs branching and contact point numbers with axons were also increased in Cdon overexpressing cells growing alone or in coculture with dorsal root ganglion neurons (DRGNs). Furthermore, myelination of DRGNs was decreased when OLPs were transfected with Cdon siRNA. Altogether, our results suggest that Cdon participates in OLG differentiation and myelination, most likely in the initial stages of development. GLIA 2016;64:1021-1033. PMID:26988125

  20. Equine herpesvirus type 4 UL56 and UL49.5 proteins downregulate cell surface major histocompatibility complex class I expression independently of each other.

    PubMed

    Said, Abdelrahman; Azab, Walid; Damiani, Armando; Osterrieder, Nikolaus

    2012-08-01

    Major histocompatibility complex class I (MHC-I) molecules are critically important in the host defense against various pathogens through presentation of viral peptides to cytotoxic T lymphocytes (CTLs), a process resulting in the destruction of virus-infected cells. Herpesviruses interfere with CTL-mediated elimination of infected cells by various mechanisms, including inhibition of peptide transport and loading, perturbation of MHC-I trafficking, and rerouting and proteolysis of cell surface MHC-I. In this study, we show that equine herpesvirus type 4 (EHV-4) modulates MHC-I cell surface expression through two different mechanisms. First, EHV-4 can lead to a significant downregulation of MHC-I expression at the cell surface through the product of ORF1, a protein expressed with early kinetics from a gene that is homologous to herpes simplex virus 1 UL56. The EHV-4 UL56 protein reduces cell surface MHC-I as early as 4 h after infection. Second, EHV-4 can interfere with MHC-I antigen presentation, starting at 6 h after infection, by inhibition of the transporter associated with antigen processing (TAP) through its UL49.5 protein. Although pUL49.5 has no immediate effect on overall surface MHC-I levels in infected cells, it blocks the supply of antigenic peptides to the endoplasmic reticulum (ER) and transport of peptide-loaded MHC-I to the cell surface. Taken together, our results show that EHV-4 encodes at least two viral immune evasion proteins: pUL56 reduces MHC-I molecules on the cell surface at early times after infection, and pUL49.5 interferes with MHC-I antigen presentation by blocking peptide transport in the ER. PMID:22623773

  1. Exceptional error minimization in putative primordial genetic codes

    PubMed Central

    2009-01-01

    Background The standard genetic code is redundant and has a highly non-random structure. Codons for the same amino acids typically differ only by the nucleotide in the third position, whereas similar amino acids are encoded, mostly, by codon series that differ by a single base substitution in the third or the first position. As a result, the code is highly albeit not optimally robust to errors of translation, a property that has been interpreted either as a product of selection directed at the minimization of errors or as a non-adaptive by-product of evolution of the code driven by other forces. Results We investigated the error-minimization properties of putative primordial codes that consisted of 16 supercodons, with the third base being completely redundant, using a previously derived cost function and the error minimization percentage as the measure of a code's robustness to mistranslation. It is shown that, when the 16-supercodon table is populated with 10 putative primordial amino acids, inferred from the results of abiotic synthesis experiments and other evidence independent of the code's evolution, and with minimal assumptions used to assign the remaining supercodons, the resulting 2-letter codes are nearly optimal in terms of the error minimization level. Conclusion The results of the computational experiments with putative primordial genetic codes that contained only two meaningful letters in all codons and encoded 10 to 16 amino acids indicate that such codes are likely to have been nearly optimal with respect to the minimization of translation errors. This near-optimality could be the outcome of extensive early selection during the co-evolution of the code with the primordial, error-prone translation system, or a result of a unique, accidental event. Under this hypothesis, the subsequent expansion of the code resulted in a decrease of the error minimization level that became sustainable owing to the evolution of a high-fidelity translation system

  2. The Putative Son's Attractiveness Alters the Perceived Attractiveness of the Putative Father.

    PubMed

    Prokop, Pavol

    2015-08-01

    A body of literature has investigated female mate choice in the pre-mating context (pre-mating sexual selection). Humans, however, are long-living mammals forming pair-bonds which sequentially produce offspring. Post-mating evaluations of a partner's attractiveness may thus significantly influence the reproductive success of men and women. I tested herein the theory that the attractiveness of putative sons provides extra information about the genetic quality of fathers, thereby influencing fathers' attractiveness across three studies. As predicted, facially attractive boys were more frequently attributed to attractive putative fathers and vice versa (Study 1). Furthermore, priming with an attractive putative son increased the attractiveness of the putative father with the reverse being true for unattractive putative sons. When putative fathers were presented as stepfathers, the effect of the boy's attractiveness on the stepfather's attractiveness was lower and less consistent (Study 2). This suggests that the presence of an attractive boy has the strongest effect on the perceived attractiveness of putative fathers rather than on non-fathers. The generalized effect of priming with beautiful non-human objects also exists, but its effect is much weaker compared with the effects of putative biological sons (Study 3). Overall, this study highlighted the importance of post-mating sexual selection in humans and suggests that the heritable attractive traits of men are also evaluated by females after mating and/or may be used by females in mate poaching. PMID:25731909

  3. Autonomous molecular cascades for evaluation of cell surfaces.

    PubMed

    Rudchenko, Maria; Taylor, Steven; Pallavi, Payal; Dechkovskaia, Alesia; Khan, Safana; Butler, Vincent P; Rudchenko, Sergei; Stojanovic, Milan N

    2013-08-01

    Molecular automata are mixtures of molecules that undergo precisely defined structural changes in response to sequential interactions with inputs. Previously studied nucleic acid-based automata include game-playing molecular devices (MAYA automata) and finite-state automata for the analysis of nucleic acids, with the latter inspiring circuits for the analysis of RNA species inside cells. Here, we describe automata based on strand-displacement cascades directed by antibodies that can analyse cells by using their surface markers as inputs. The final output of a molecular automaton that successfully completes its analysis is the presence of a unique molecular tag on the cell surface of a specific subpopulation of lymphocytes within human blood cells. PMID:23892986

  4. Cell Surface GRP78 Accelerated Breast Cancer Cell Proliferation and Migration by Activating STAT3.

    PubMed

    Yao, Xiaoli; Liu, Hua; Zhang, Xinghua; Zhang, Liang; Li, Xiang; Wang, Changhua; Sun, Shengrong

    2015-01-01

    High levels of cell surface glucose regulated protein 78 (sGRP78) have been implicated in cancer growth, survival, metastasis, and chemotherapy resistance. However, the underlying mechanism remains largely unknown. Here we report that the level of sGRP78 expression in human breast tumors gradually increases during cancer progression. Overexpression of GRP78 significantly enhanced its membrane distribution in human MCF-7 breast cancer cells, but had no effect on endoplasmic reticulum (ER) stress. High levels of sGRP78 facilitated cell proliferation and migration, as well as suppressed cell apoptosis. Neutralization of sGRP78 by a specific antibody against GRP78 alleviated sGRP78-induced cell growth and migration. Importantly, high phosphorylation levels of the signal transducer and activator of transcription 3 (STAT3) were found in human breast tumors that express sGRP78 and MCF-7 cells infected with adenovirus encoding human GRP78. Pretreatment with a GRP78 antibody suppressed STAT3 phosphorylation. Furthermore, genetic and pharmacological inhibition of STAT3 reversed the impacts of GRP78 on cell proliferation, apoptosis, and migration. These findings indicate that STAT3 mediates sGRP78-promoted breast cancer cell growth and migration. PMID:25973748

  5. Substrate recognition by the cell surface palmitoyl transferase DHHC5

    PubMed Central

    Howie, Jacqueline; Reilly, Louise; Fraser, Niall J.; Vlachaki Walker, Julia M.; Wypijewski, Krzysztof J.; Ashford, Michael L. J.; Calaghan, Sarah C.; McClafferty, Heather; Tian, Lijun; Shipston, Michael J.; Boguslavskyi, Andrii; Shattock, Michael J.; Fuller, William

    2014-01-01

    The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discs-large/ZO-1 homology (PDZ) binding motif, but requires a ∼120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme–substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipid interactions that inhibit the Na pump. PMID:25422474

  6. Cell-surface translational dynamics of nicotinic acetylcholine receptors

    PubMed Central

    Barrantes, Francisco J.

    2014-01-01

    Synapse efficacy heavily relies on the number of neurotransmitter receptors available at a given time. In addition to the equilibrium between the biosynthetic production, exocytic delivery and recycling of receptors on the one hand, and the endocytic internalization on the other, lateral diffusion and clustering of receptors at the cell membrane play key roles in determining the amount of active receptors at the synapse. Mobile receptors traffic between reservoir compartments and the synapse by thermally driven Brownian motion, and become immobilized at the peri-synaptic region or the synapse by: (a) clustering mediated by homotropic inter-molecular receptor–receptor associations; (b) heterotropic associations with non-receptor scaffolding proteins or the subjacent cytoskeletal meshwork, leading to diffusional “trapping,” and (c) protein-lipid interactions, particularly with the neutral lipid cholesterol. This review assesses the contribution of some of these mechanisms to the supramolecular organization and dynamics of the paradigm neurotransmitter receptor of muscle and neuronal cells -the nicotinic acetylcholine receptor (nAChR). Currently available information stemming from various complementary biophysical techniques commonly used to interrogate the dynamics of cell-surface components is critically discussed. The translational mobility of nAChRs at the cell surface differs between muscle and neuronal receptors in terms of diffusion coefficients and residence intervals at the synapse, which cover an ample range of time regimes. A peculiar feature of brain α7 nAChR is its ability to spend much of its time confined peri-synaptically, vicinal to glutamatergic (excitatory) and GABAergic (inhibitory) synapses. An important function of the α7 nAChR may thus be visiting the territories of other neurotransmitter receptors, differentially regulating the dynamic equilibrium between excitation and inhibition, depending on its residence time in each domain. PMID

  7. TGM2 A Cell Surface Marker in Esophageal Adenocarcinomas

    PubMed Central

    Leicht, Deborah T.; Kausar, Tasneem; Wang, Zhuwen; Ferrer-Torres, Daysha; Wang, Thomas D.; Thomas, Dafydd G.; Lin, Jules; Chang, Andrew C.; Lin, Lin; Beer, David G.

    2014-01-01

    Introduction Esophageal adenocarcinomas (EAC) are aggressive cancers that are increasing in incidence and associated with a poor prognosis. The identification of highly expressed genes in EAC relative to metaplastic Barrett’s esophagus (BE) may provide new targets for novel early cancer detection strategies using endoscopically administered, fluorescently labeled peptides. Methods Gene expression analysis of BE and EACs were used to identify the cell surface marker transglutaminase 2 (TGM2) as overexpressed in cancer. The expression of two major isoforms of TGM2 was determined by qRT-polymerase chain reaction in an independent cohort of 128 EACs. Protein expression was confirmed by tissue microarrays and immunoblot analysis of EAC cell lines. TGM2 DNA copy number was assessed using single nucleotide polymorphism microarrays and confirmed by qPCR. TGM2 expression in neoadjuvantly treated EACs and following small interfering RNA-mediated knockdown in cisplatin-treated EAC cells was used to determine its possible role in chemoresistance. Results TGM2 is overexpressed in 15 EACs relative to 26 BE samples. Overexpression of both TGM2 isoforms was confirmed in 128 EACs and associated with higher tumor stage, poor differentiation, and increased inflammatory and desmoplastic response. Tissue microarrays and immunohistochemistry confirmed elevated TGM2 protein expression in EAC. Single nucleotide polymorphism and qPCR analysis revealed increased TGM2 gene copy number as one mechanism underlying elevated TGM2 expression. TGM2 was highly expressed in resistant EAC after patient treatment with neoadjuvant chemotherapy/radiation suggesting a role for TGM2 in chemoresistance. Conclusion TGM2 may be a useful cell surface biomarker for early detection of EAC. PMID:24828664

  8. Schizosaccharomyces pombe sxa1+ and sxa2+ encode putative proteases involved in the mating response.

    PubMed

    Imai, Y; Yamamoto, M

    1992-04-01

    The Schizosaccharomyces pombe sxa1 and sxa2 mutants showed an exaggerated response to mating pheromones, producing excessively long conjugation tubes and exhibiting mating deficiency. This phenotype was similar to phenotypes of cells bearing an activated allele of ras1, such as ras1Val-17 or ras1Leu-66, and phenotypes of cells defective in gap1. However, genetic evidence suggested that the sxa1 and sxa2 gene products are not directly involved in the Ras1 pathway. The gene products of sxa1 and sxa2, as deduced from their nucleotide sequences, were homologous to aspartyl proteases and serine carboxypeptidases, respectively. The sxa1 gene function was required for efficient mating only in h+ cells, although even disruption of sxa1 did not completely abolish the mating ability. Conversely, the sxa2 gene function was required only in h- cells. Wild-type cells produced a diffusible substance, which may be the sxa2 gene product itself, that could confer fertility to sxa2 mutant cells placed at a distance. These observations are consistent with the possibility that the sxa gene products are involved in degradation or processing of the mating pheromones and that their loss cause a persistent response to the pheromones. PMID:1549128

  9. A Putative PP2C-Encoding Gene Negatively Regulates ABA Signaling in Populus euphratica

    PubMed Central

    Chen, Jinhuan; Zhang, Dongzhi; Zhang, Chong; Xia, Xinli; Yin, Weilun; Tian, Qianqian

    2015-01-01

    A PP2C homolog gene was cloned from the drought-treated cDNA library of Populus euphratica. Multiple sequence alignment analysis suggested that the gene is a potential ortholog of HAB1. The expression of this HAB1 ortholog (PeHAB1) was markedly induced by drought and moderately induced by ABA. To characterize its function in ABA signaling, we generated transgenic Arabidopsis thaliana plants overexpressing this gene. Transgenic lines exhibited reduced responses to exogenous ABA and reduced tolerance to drought compared to wide-type lines. Yeast two-hybrid analyses indicated that PeHAB1 could interact with the ABA receptor PYL4 in an ABA-independent manner. Taken together; these results indicated that PeHAB1 is a new negative regulator of ABA responses in poplar. PMID:26431530

  10. The C. elegans sex-determining gene fem-2 encodes a putative protein phosphatase.

    PubMed Central

    Pilgrim, D; McGregor, A; Jäckle, P; Johnson, T; Hansen, D

    1995-01-01

    The genetic and molecular analysis of genes involved in the regulation of sex determination in Caenorhabditis elegans suggests that the gene fem-2 plays an important role in regulating a pathway transducing a non-cell-autonomous signal to a nuclear transcription factor. The wild-type fem-2 gene was cloned by identifying sequences from the C. elegans physical map that could restore normal Fem-2 function to homozygous mutant fem-2 transgenic animals. cDNA sequences mapping to the minimal rescuing region correspond to an open reading frame with a sequence similar to protein phosphatase 2C enzymes from systems as diverse as yeast, humans, and plants, but the alignments suggest that FEM-2 falls into a separate class of proteins than the canonical homologues. Several fem-2 mutant alleles were sequenced, and the mutations are predicted to cause protein changes consistent with their observed phenotypes, such as missense mutations in conditional alleles, and a nonsense mutation in a predicted null allele. This is the first evidence implicating phosphorylation and/or dephosphorylation as a control mechanism in C. elegans sex determination. Images PMID:8534913

  11. rmtA, encoding a putative anginine methyltransferase, regulates secondary metabolism and development in Aspergillus flavus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspergillus flavus is found colonizing numerous oil seed crops such as corn, peanuts, sorghum, treenuts and cotton worldwide, contaminating them with aflatoxin and other harmful potent toxins. In the phylogenetically related model fungus Aspergillus nidulans, the methyltransferase, RmtA, has been de...

  12. Identification and characterization of kraken, a gene encoding a putative hydrolytic enzyme in Drosophila melanogaster.

    PubMed

    Edwin Chan, H Y; Harris, S J; O'Kane, C J

    1998-11-19

    Kraken, a novel Drosophila gene isolated from a 4-8-h-old Drosophila embryo cDNA library, shows homology to a family of serine hydrolases whose common feature is that they all catalyse breakage of substrates with a carbonyl-containing group. It is a single-copy gene with at least two introns and maps to position 21D on polytene chromosomes. kraken is a member of a conserved gene family. Messenger RNA of kraken is expressed ubiquitously in early embryogenesis. Later, it is concentrated in the foregut and the posterior midgut primordium. Towards the end of embryogenesis, expression of kraken is confined to the gastric caeca. During the third-instar larval stage, kraken is expressed at low levels in the gastric caeca and parts of the gut, and at higher levels in the fat body. We suggest a role for Kraken in detoxification and digestion during embryogenesis and larval development. PMID:9831651

  13. A Putative PP2C-Encoding Gene Negatively Regulates ABA Signaling in Populus euphratica.

    PubMed

    Chen, Jinhuan; Zhang, Dongzhi; Zhang, Chong; Xia, Xinli; Yin, Weilun; Tian, Qianqian

    2015-01-01

    A PP2C homolog gene was cloned from the drought-treated cDNA library of Populus euphratica. Multiple sequence alignment analysis suggested that the gene is a potential ortholog of HAB1. The expression of this HAB1 ortholog (PeHAB1) was markedly induced by drought and moderately induced by ABA. To characterize its function in ABA signaling, we generated transgenic Arabidopsis thaliana plants overexpressing this gene. Transgenic lines exhibited reduced responses to exogenous ABA and reduced tolerance to drought compared to wide-type lines. Yeast two-hybrid analyses indicated that PeHAB1 could interact with the ABA receptor PYL4 in an ABA-independent manner. Taken together; these results indicated that PeHAB1 is a new negative regulator of ABA responses in poplar. PMID:26431530

  14. Toddlers' Duration of Attention toward Putative Threat

    ERIC Educational Resources Information Center

    Kiel, Elizabeth J.; Buss, Kristin A.

    2011-01-01

    Although individual differences in reactions to novelty in the toddler years have been consistently linked to risk of developing anxious behavior, toddlers' attention toward a novel, putatively threatening stimulus while in the presence of other enjoyable activities has rarely been examined as a precursor to such risk. The current study examined…

  15. Neural signals encoding shifts in beliefs.

    PubMed

    Schwartenbeck, Philipp; FitzGerald, Thomas H B; Dolan, Ray

    2016-01-15

    Dopamine is implicated in a diverse range of cognitive functions including cognitive flexibility, task switching, signalling novel or unexpected stimuli as well as advance information. There is also longstanding line of thought that links dopamine with belief formation and, crucially, aberrant belief formation in psychosis. Integrating these strands of evidence would suggest that dopamine plays a central role in belief updating and more specifically in encoding of meaningful information content in observations. The precise nature of this relationship has remained unclear. To directly address this question we developed a paradigm that allowed us to decompose two distinct types of information content, information-theoretic surprise that reflects the unexpectedness of an observation, and epistemic value that induces shifts in beliefs or, more formally, Bayesian surprise. Using functional magnetic-resonance imaging in humans we show that dopamine-rich midbrain regions encode shifts in beliefs whereas surprise is encoded in prefrontal regions, including the pre-supplementary motor area and dorsal cingulate cortex. By linking putative dopaminergic activity to belief updating these data provide a link to false belief formation that characterises hyperdopaminergic states associated with idiopathic and drug induced psychosis. PMID:26520774

  16. Neural signals encoding shifts in beliefs

    PubMed Central

    Schwartenbeck, Philipp; FitzGerald, Thomas H.B.; Dolan, Ray

    2016-01-01

    Dopamine is implicated in a diverse range of cognitive functions including cognitive flexibility, task switching, signalling novel or unexpected stimuli as well as advance information. There is also longstanding line of thought that links dopamine with belief formation and, crucially, aberrant belief formation in psychosis. Integrating these strands of evidence would suggest that dopamine plays a central role in belief updating and more specifically in encoding of meaningful information content in observations. The precise nature of this relationship has remained unclear. To directly address this question we developed a paradigm that allowed us to decompose two distinct types of information content, information-theoretic surprise that reflects the unexpectedness of an observation, and epistemic value that induces shifts in beliefs or, more formally, Bayesian surprise. Using functional magnetic-resonance imaging in humans we show that dopamine-rich midbrain regions encode shifts in beliefs whereas surprise is encoded in prefrontal regions, including the pre-supplementary motor area and dorsal cingulate cortex. By linking putative dopaminergic activity to belief updating these data provide a link to false belief formation that characterises hyperdopaminergic states associated with idiopathic and drug induced psychosis. PMID:26520774

  17. Alignment system for encoders

    NASA Technical Reports Server (NTRS)

    Villani, Daniel D. (Inventor)

    1988-01-01

    An improved encoder alignment system is disclosed which provides an indication of the extent of misalignment and a measure of the rate at which the misalignment may be changing. The invention is adapted for use with a conventional encoder which provides a digital coarse word having at least significant bit and a digital fine word having a least significant bit and a most significant bit. The invention generates the exclusive or of the least significant bit of the coarse digital signal and the least significant bit of the fine digital signal to provide a first signal. The invention then generates the exclusive or of the first signal and the complement of the most significant bit of the fine digital signal to provide an output signal which represents the misalignment of the encoder.

  18. Cloning, protein expression and display of synthetic multi-epitope mycobacterial antigens on Salmonella typhi Ty21a cell surface.

    PubMed

    Sarhan, Mohammed A A; Musa, Mustaffa; Zainuddin, Zainul F

    2011-09-01

    Expressing proteins of interest as fusion to proteins of bacterial envelope is a powerful technique for biotechnological and medical applications. The synthetic gene (VacII) encoding for T-cell epitopes of selected genes of Mycobacterium tuberculosis namely, ESAT6, MTP40, 38 kDa, and MPT64 was fused with N- terminus of Pseudomonas syringae ice nucleation protein (INP) outer membrane protein. The fused genes were cloned into a bacterial expression vector pKK223-3. The recombinant protein was purified by Ni-NAT column. VacII gene was displayed on the cell surface of Salmonella typhi Ty21a using N-terminal region of ice nucleation proteins (INP) as an anchoring motif. Glycine method confirmed that VacII was anchored on the cell surface. Western blot analysis further identified the synthesis of INP derivatives containing the N-terminal domain INP- VacII fusion protein of the expected size (52 kDa). PMID:21941936

  19. Developmental expression of a cell surface protein involved in sea urchin skeleton formation. [Strongylocentrotus purpuratus; Lytechinus pictus

    SciTech Connect

    Farach, M.C.; Valdizan, M.; Park, H.R.; Decker, G.L.; Lennarz, W.J.

    1986-05-01

    The authors have previously used a monoclonal antibody (1223) to identify a 130 Kd cell surface protein involved in skeleton formation is sea urchin embryos. In the current study the authors have examined the expression of the 1223 antigen over the course of development of embryos of two species, Strongylocentrotus purpuratus and Lytechinus pictus. The 130 Kd protein is detected in S. purp eggs on immunoblots. Labeling with (/sup 3/H) leucine and immunoaffinity chromatography show that it also is synthesized shortly after fertilization. Immunofluroescence reveals that at this early stage the 1223 antigen is uniformly distributed on all of the cells. Synthesis decreases to a minimum by the time of hatching (18 h), as does the total amount of antigen present in the embryo. A second period of synthesis commences at the mesenchyme blastula stage, when the spicule-forming primary mesenchyme cells (PMCs) have appeared. During this later stage, synthesis and cell surface expression are restricted to the PMCs. In contrast to S. purp., in L. pictus the 130 Kd protein does not appear until the PMCs are formed. Hybrid embryos demonstrate a pattern of expression of the maternal species. These results suggest that early expression of 1223 antigen in S. purp. is due to utilization of maternal transcripts present in the egg. In both species later expression in PMCs appears to be the result of cell-type specific synthesis, perhaps encoded by embryonic transcripts.

  20. Polarization encoded color camera.

    PubMed

    Schonbrun, Ethan; Möller, Guðfríður; Di Caprio, Giuseppe

    2014-03-15

    Digital cameras would be colorblind if they did not have pixelated color filters integrated into their image sensors. Integration of conventional fixed filters, however, comes at the expense of an inability to modify the camera's spectral properties. Instead, we demonstrate a micropolarizer-based camera that can reconfigure its spectral response. Color is encoded into a linear polarization state by a chiral dispersive element and then read out in a single exposure. The polarization encoded color camera is capable of capturing three-color images at wavelengths spanning the visible to the near infrared. PMID:24690806

  1. AMP-Activated Protein Kinase Regulates the Cell Surface Proteome and Integrin Membrane Traffic

    PubMed Central

    Thavarajah, Thanusi; Medvedev, Sergei; Bowden, Peter; Marshall, John G.; Antonescu, Costin N.

    2015-01-01

    The cell surface proteome controls numerous cellular functions including cell migration and adhesion, intercellular communication and nutrient uptake. Cell surface proteins are controlled by acute changes in protein abundance at the plasma membrane through regulation of endocytosis and recycling (endomembrane traffic). Many cellular signals regulate endomembrane traffic, including metabolic signaling; however, the extent to which the cell surface proteome is controlled by acute regulation of endomembrane traffic under various conditions remains incompletely understood. AMP-activated protein kinase (AMPK) is a key metabolic sensor that is activated upon reduced cellular energy availability. AMPK activation alters the endomembrane traffic of a few specific proteins, as part of an adaptive response to increase energy intake and reduce energy expenditure. How increased AMPK activity during energy stress may globally regulate the cell surface proteome is not well understood. To study how AMPK may regulate the cell surface proteome, we used cell-impermeable biotinylation to selectively purify cell surface proteins under various conditions. Using ESI-MS/MS, we found that acute (90 min) treatment with the AMPK activator A-769662 elicits broad control of the cell surface abundance of diverse proteins. In particular, A-769662 treatment depleted from the cell surface proteins with functions in cell migration and adhesion. To complement our mass spectrometry results, we used other methods to show that A-769662 treatment results in impaired cell migration. Further, A-769662 treatment reduced the cell surface abundance of β1-integrin, a key cell migration protein, and AMPK gene silencing prevented this effect. While the control of the cell surface abundance of various proteins by A-769662 treatment was broad, it was also selective, as this treatment did not change the cell surface abundance of the transferrin receptor. Hence, the cell surface proteome is subject to acute

  2. Atomic Force Microscopy in Microbiology: New Structural and Functional Insights into the Microbial Cell Surface

    PubMed Central

    2014-01-01

    ABSTRACT Microbial cells sense and respond to their environment using their surface constituents. Therefore, understanding the assembly and biophysical properties of cell surface molecules is an important research topic. With its ability to observe living microbial cells at nanometer resolution and to manipulate single-cell surface molecules, atomic force microscopy (AFM) has emerged as a powerful tool in microbiology. Here, we survey major breakthroughs made in cell surface microbiology using AFM techniques, emphasizing the most recent structural and functional insights. PMID:25053785

  3. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

    PubMed

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte; Corbeil, Denis; Hoflack, Bernard

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  4. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry

    PubMed Central

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  5. Antifouling property of highly oleophobic substrates for solar cell surfaces

    NASA Astrophysics Data System (ADS)

    Fukada, Kenta; Nishizawa, Shingo; Shiratori, Seimei

    2014-03-01

    Reduction of solar cell conversion efficiency by bird spoor or oil smoke is a common issue. Maintaining the surface of solar cells clean to retain the incident light is of utmost importance. In this respect, there has been growing interest in the area of superhydrophobicity for developing water repelling and self-cleaning surfaces. This effect is inspired by lotus leaves that have micro papillae covered with hydrophobic wax nanostructures. Superhydrophobic surfaces on transparent substrates have been developed for removing contaminants from solar cell surfaces. However, oil cannot be removed by superhydrophobic effect. In contrast, to prevent bird spoor, a highly oleophobic surface is required. In a previous study, we reported transparent-type fabrics comprising nanoparticles with a nano/micro hierarchical structure that ensured both oleophobicity and transparency. In the current study, we developed new highly oleophobic stripes that were constructed into semi-transparent oleophobic surfaces for solar cells. Solar cell performance was successfully maintained; the total transmittance was a key factor for determining conversion efficiency.

  6. Cell Surface Access Is Modulated by Tethered Bottlebrush Proteoglycans.

    PubMed

    Chang, Patrick S; McLane, Louis T; Fogg, Ruth; Scrimgeour, Jan; Temenoff, Johnna S; Granqvist, Anna; Curtis, Jennifer E

    2016-06-21

    The hyaluronan-rich pericellular matrix (PCM) plays physical and chemical roles in biological processes ranging from brain plasticity, to adhesion-dependent phenomena such as cell migration, to the onset of cancer. This study investigates how the spatial distribution of the large negatively charged bottlebrush proteoglycan, aggrecan, impacts PCM morphology and cell surface access. The highly localized pericellular milieu limits transport of nanoparticles in a size-dependent fashion and sequesters positively charged molecules on the highly sulfated side chains of aggrecan. Both rat chondrocyte and human mesenchymal stem cell PCMs possess many unused binding sites for aggrecan, showing a 2.5x increase in PCM thickness from ∼7 to ∼18 μm when provided exogenous aggrecan. Yet, full extension of the PCM occurs well below aggrecan saturation. Hence, cells equipped with hyaluronan-rich PCM can in principle manipulate surface accessibility or sequestration of molecules by tuning the bottlebrush proteoglycan content to alter PCM porosity and the number of electrostatic binding sites. PMID:27332132

  7. Extracellular Protease Digestion to Evaluate Membrane Protein Cell Surface Localization

    PubMed Central

    Besingi, Richard N.; Clark, Patricia L.

    2016-01-01

    Membrane proteins play crucial roles in signaling and as anchors for cell surface display. Proper secretion of a membrane protein can be evaluated by its susceptibility to digestion by an extracellular protease, but this requires a crucial control to confirm membrane integrity during digestion. This protocol describes how to use this approach to determine how efficiently a protein is secreted to the outer surface of Gram-negative bacteria. Its success relies upon careful selection of an appropriate intracellular reporter protein that will remain undigested if the membrane barrier remains intact, but is rapidly digested when cells are lysed prior to evaluation. Reporter proteins that are resistant to proteases (e.g. maltose-binding protein) do not return accurate results; in contrast, proteins that are more readily digested (e.g. SurA) serve as more sensitive reporters of membrane integrity, yielding more accurate measurements of membrane protein localization. Similar considerations apply when evaluating membrane protein localization in other contexts, including eukaryotic cells and organelle membranes. Evaluating membrane protein localization using this approach requires only standard biochemistry laboratory equipment for cell lysis, gel electrophoresis and western blotting. After expression of the protein of interest, this procedure can be completed in 4 h. PMID:26584447

  8. EXAFS Study of Uranyl Complexation at Pseudomonas fluorescens Cell Surfaces

    NASA Astrophysics Data System (ADS)

    Bencheikh, R.; Bargar, J. R.; Tebo, B. M.

    2002-12-01

    Little is known about the roles of microbial biomass as a sink and source for uranium in contaminated aquifers, nor of the impact of bacterial biochemistry on uranium speciation in the subsurface. A significant role is implied by the high affinities of both Gram positive and Gram negative cells for binding uranyl (UO2{ 2+}). In the present study, Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy was used to identify membrane functional groups involved in uranyl binding to the Gram negative bacterium Pseudomonas fluorescens from pH 3 to pH 8. Throughout this pH-range, EXAFS spectra can be described primarily in terms of coordination of carboxylic groups to uranyl. U-C distances characteristic of 4-, 5- and 8- membered rings were observed, as well as the possibility of phosphato groups. Both shell-by-shell fits and principle component analyses indicate that the functional groups involved in binding of uranyl to the cell surface do not vary systematically across the pH range investigated. This result contrasts with EXAFS results of uranyl sorbed to Gram positive bacteria, and suggests an important role for long-chain carboxylate-terminated membrane functional groups in binding uranyl.

  9. Sorption of heavy metals by prepared bacterial cell surfaces

    SciTech Connect

    Churchill, S.A.; Walters, J.V.; Churchill, P.F.

    1995-10-01

    Prepared biomass from two Gram-negative and one Gram-positive bacterial strains was examined for single, binary, and quaternary mixtures of polyvalent metal cation binding to cell surfaces. The biosorption of {sub 24}Cr{sup 3+}, {sub 27}Co{sup 2+}, {sub 28}Ni{sup 2+}, and {sub 29}Cu{sup 2+} for each bacterial cell type was evaluated using a batch equilibrium method. The binding of each metal by all three bacterial cells could be described by the Freundlich sorption model. The isotherm binding constants suggest that E. coli cells are the most efficient at binding copper, chromium, and nickel; and M. luteus adsorbs cobalt most efficiently. The K-values for copper bound to P. aeruginosa and E. coli are > 2-fold and > 8-fold greater, respectively, than previous reported for intact cells. The general metal-affinity series observed was Cr{sup 3+} > Cu{sup 2+} > Ni{sup 2+} > Co{sup 2+}. There was a marked lower affinity of all biosorbents for Co{sup 2+} and Ni{sup 2+}. M. luteus and E. coli had a strong preference for Co{sup 2+} over Ni{sup 2+}. Metal-binding enhancement could be ascribed to increased cell barrier surface porosity to metal-bearing solutions.

  10. Specific cell surface labeling of GPCRs using split GFP.

    PubMed

    Jiang, Wen-Xue; Dong, Xu; Jiang, Jing; Yang, Yu-Hong; Yang, Ju; Lu, Yun-Bi; Fang, San-Hua; Wei, Er-Qing; Tang, Chun; Zhang, Wei-Ping

    2016-01-01

    Specific cell surface labeling is essential for visualizing the internalization processes of G-protein coupled receptors (GPCRs) and for gaining mechanistic insight of GPCR functions. Here we present a rapid, specific, and versatile labeling scheme for GPCRs at living-cell membrane with the use of a split green fluorescent protein (GFP). Demonstrated with two GPCRs, GPR17 and CysLT2R, we show that two β-stands (β-stands 10 and 11) derived from a superfolder GFP (sfGFP) can be engineered to one of the three extracellular loop of a GPCR. The complementary fragment of sfGFP has nine β-strands (β-stands 1-9) that carries the mature fluorophore, and can be proteolytically derived from the full-length sfGFP. Separately the GFP fragments are non-fluorescent, but become fluorescent upon assembly, thus allowing specific labeling of the target proteins. The two GFP fragments rapidly assemble and the resulting complex is extremely tight under non-denaturing conditions, which allows real-time and quantitative assessment of the internalized GPCRs. We envision that this labeling scheme will be of great use for labeling other membrane proteins in various biological and pharmacological applications. PMID:26857153

  11. RPE cell surface proteins in normal and dystrophic rats

    SciTech Connect

    Clark, V.M.; Hall, M.O.

    1986-02-01

    Membrane-bound proteins in plasma membrane enriched fractions from cultured rat RPE were analyzed by two-dimensional gel electrophoresis. Membrane proteins were characterized on three increasingly specific levels. Total protein was visualized by silver staining. A maximum of 102 separate proteins were counted in silver-stained gels. Glycoproteins were labeled with 3H-glucosamine or 3H-fucose and detected by autoradiography. Thirty-eight fucose-labeled and 61-71 glucosamine-labeled proteins were identified. All of the fucose-labeled proteins were labeled with glucosamine-derived radioactivity. Proteins exposed at the cell surface were labeled by lactoperoxidase-catalyzed radioiodination prior to preparation of membranes for two-dimensional analysis. Forty separate 125I-labeled surface proteins were resolved by two-dimensional electrophoresis/autoradiography. Comparison with the glycoprotein map showed that a number of these surface labeled proteins were glycoproteins. Two-dimensional maps of total protein, fucose-labeled, and glucosamine-labeled glycoproteins, and 125I-labeled surface proteins of membranes from dystrophic (RCS rdy-p+) and normal (Long Evans or RCS rdy+p+) RPE were compared. No differences in the total protein or surface-labeled proteins were observed. However, the results suggest that a 183K glycoprotein is more heavily glycosylated with glucosamine and fucose in normal RPE membranes as compared to membranes from dystrophic RPE.

  12. Cell surface lectin array: parameters affecting cell glycan signature.

    PubMed

    Landemarre, Ludovic; Cancellieri, Perrine; Duverger, Eric

    2013-04-01

    Among the "omics", glycomics is one of the most complex fields and needs complementary strategies of analysis to decipher the "glycan dictionary". As an alternative method, which has developed since the beginning of the 21st century, lectin array technology could generate relevant information related to glycan motifs, accessibility and a number of other valuable insights from molecules (purified and non-purified) or cells. Based on a cell line model, this study deals with the key parameters that influence the whole cell surface glycan interaction with lectin arrays and the consequences on the interpretation and reliability of the results. The comparison between the adherent and suspension forms of Chinese Hamster Ovary (CHO) cells, showed respective glycan signatures, which could be inhibited specifically by neoglycoproteins. The modifications of the respective glycan signatures were also revealed according to the detachment modes and cell growth conditions. Finally the power of lectin array technology was highlighted by the possibility of selecting and characterizing a specific clone from the mother cell line, based on the slight difference determination in the respective glycan signatures. PMID:22899543

  13. TARM1 is a novel LRC-encoded ITAM receptor that co-stimulates pro-inflammatory cytokine secretion by macrophages and neutrophils

    PubMed Central

    Radjabova, Valeria; Mastroeni, Piero; Skjødt, Karsten; Zaccone, Paola; de Bono, Bernard; Goodall, Jane C; Chilvers, Edwin R; Juss, Jatinder K; Jones, Des C; Trowsdale, John; Barrow, Alexander David

    2015-01-01

    We identified a novel, evolutionarily conserved receptor encoded within the human Leukocyte Receptor Complex (LRC) and syntenic region of mouse chromosome 7, named T cell-interacting, activating receptor on myeloid cells-1 (TARM1). The transmembrane region of TARM1 contained a conserved arginine residue, consistent with association with a signaling adaptor. TARM1 associated with the ITAM adaptor Fc receptor common γ chain but not with DAP10 or DAP12. In healthy mice, TARM1 is constitutively expressed on the cell-surface of mature and immature CD11b+ Gr-1+ neutrophils within the bone marrow. Following intraperitoneal lipopolysaccharide (LPS) treatment or systemic bacterial challenge TARM1 expression was upregulated by neutrophils and inflammatory monocytes and TARM1+ cells were rapidly recruited to sites of inflammation. TARM1 expression was also upregulated by bone marrow-derived macrophages and dendritic cells following stimulation with TLR agonists in vitro. Ligation of TARM1 receptor in the presence of TLR ligands, such as LPS, enhanced the secretion of pro-inflammatory cytokines by macrophages and primary mouse neutrophils, whereas TARM1 stimulation alone had no effect. Finally, an immobilized TARM1-Fc fusion protein suppressed CD4+ T cell activation and proliferation in vitro. These results suggest that a putative T cell ligand can interact with TARM1 receptor resulting in bi-directional signaling, raising the T cell activation threshold whilst co-stimulating the release of pro-inflammatory cytokines by macrophages and neutrophils. PMID:26311901

  14. Time-Encoded Imagers.

    SciTech Connect

    Marleau, Peter; Brubaker, Erik

    2014-11-01

    This report provides a short overview of the DNN R&D funded project, Time-Encoded Imagers. The project began in FY11 and concluded in FY14. The Project Description below provides the overall motivation and objectives for the project as well as a summary of programmatic direction. It is followed by a short description of each task and the resulting deliverables.

  15. Video Time Encoding Machines

    PubMed Central

    Lazar, Aurel A.; Pnevmatikakis, Eftychios A.

    2013-01-01

    We investigate architectures for time encoding and time decoding of visual stimuli such as natural and synthetic video streams (movies, animation). The architecture for time encoding is akin to models of the early visual system. It consists of a bank of filters in cascade with single-input multi-output neural circuits. Neuron firing is based on either a threshold-and-fire or an integrate-and-fire spiking mechanism with feedback. We show that analog information is represented by the neural circuits as projections on a set of band-limited functions determined by the spike sequence. Under Nyquist-type and frame conditions, the encoded signal can be recovered from these projections with arbitrary precision. For the video time encoding machine architecture, we demonstrate that band-limited video streams of finite energy can be faithfully recovered from the spike trains and provide a stable algorithm for perfect recovery. The key condition for recovery calls for the number of neurons in the population to be above a threshold value. PMID:21296708

  16. Plasmids encoding therapeutic agents

    DOEpatents

    Keener, William K.

    2007-08-07

    Plasmids encoding anti-HIV and anti-anthrax therapeutic agents are disclosed. Plasmid pWKK-500 encodes a fusion protein containing DP178 as a targeting moiety, the ricin A chain, an HIV protease cleavable linker, and a truncated ricin B chain. N-terminal extensions of the fusion protein include the maltose binding protein and a Factor Xa protease site. C-terminal extensions include a hydrophobic linker, an L domain motif peptide, a KDEL ER retention signal, another Factor Xa protease site, an out-of-frame buforin II coding sequence, the lacZ.alpha. peptide, and a polyhistidine tag. More than twenty derivatives of plasmid pWKK-500 are described. Plasmids pWKK-700 and pWKK-800 are similar to pWKK-500 wherein the DP178-encoding sequence is substituted by RANTES- and SDF-1-encoding sequences, respectively. Plasmid pWKK-900 is similar to pWKK-500 wherein the HIV protease cleavable linker is substituted by a lethal factor (LF) peptide-cleavable linker.

  17. Reed-Solomon Encoder

    NASA Technical Reports Server (NTRS)

    Troung, T. K.; Reed, I. S.; Deutsch, L. J.; Hsu, I. S.; Wang, K.; Yeh, C. S.

    1985-01-01

    Report presents mathematical principles of Berlekamp bit serial multiplier algorithm and its application to design of very-large-scale integrated (VLSI) encoders for Reed-Solomon error-correcting codes. Structure made readily on single chip of negatively doped channel metal oxide semiconductor.

  18. Magnetism and the putative early Martian life

    NASA Astrophysics Data System (ADS)

    Rochette, P.

    2001-08-01

    A short critical review is provided on three questions linking magnetism and the putative early Mars life. Was there a large internal Martian magnetic field, during which period, and is it a requisite for life? What is the origin of the paleomagnetic signal of Martian meteorites, including ALH84001? What is the present credibility of the case for fossil bacterial magnetite grains in ALH84001?

  19. An efficient delivery of DAMPs on the cell surface by the unconventional secretion pathway

    SciTech Connect

    Zhu, Haiyan; Wang, Lan; Ruan, Yuanyuan; Zhou, Lei; Zhang, Dongmei; Min, Zhihui; Xie, Jianhui; Yu, Min; Gu, Jianxin

    2011-01-21

    Research highlights: {yields} Hsp60 transported to cell surface through the classical secretory pathway was modified with N-glycosylation. {yields} HSAPB-N18 could efficiently deliver Hsp60 to the cell surface via the unconventional secretory pathway. {yields} Cell surface Hsp60 delivered by HASPB-N18 has a proper conformation. {yields} HASPB-N18 is an efficient delivery signal for other DAMP molecules such as Hsp70 and HMGB1. -- Abstract: Damage-associated molecular patterns (DAMPs) are signals released from dying cells evoking the immune system response in several inflammatory disorders. In normal situations, many of DAMPs are nuclear or cytosolic proteins with defined intracellular function, but they could be found on the cell surface following tissue injury. The biological function of the translocated DAMPs is still not well known and an efficient delivery of these molecules on the cell surface is required to clarify their biological effects. In this study, we demonstrated that an unclassical secretory signal peptide, N-terminal 18 amino acids of HASPB (HASPB-N18), could efficiently deliver Hsp60, Hsp70, and HMGB1 on the cell surface. Furthermore, the delivery of these molecules on the cell surface by HASPB-N18 is not limited to a special cell line because several cell lines could use this delivery signal to deliver these molecules on the cell surface. Moreover, we demonstrated that Hsp60 on the cell surface delivered by HASPB-N18 could be recognized by a soluble form of LOX-1, which implies that DAMPs on the cell surface delivered by HASPB-N18 have a proper conformation during transport. Therefore, delivery of DAMPs by HASPB-N18 is a reliable model to further understand the biological significance of DAMPs on the cell surface.

  20. Hydrophobic and Electrostatic Cell Surface Properties of Thermophilic Dairy Streptococci

    PubMed Central

    van der Mei, H. C.; de Vries, J.; Busscher, H. J.

    1993-01-01

    Microbial adhesion to hydrocarbons (MATH) and microelectrophoresis were done in 10 mM potassium phosphate solutions to characterize the surfaces of thermophilic dairy streptococci, isolated from pasteurizers. Regardless of whether they were grown (in M17 broth) with lactose, sucrose, or glucose added, strains were relatively hydrophilic (showing low initial removal rates by hexadecane) and slightly negatively charged. A tendency exists for cells grown with sucrose added to be more hydrophilic than cells grown with glucose or lactose added. Also, the lowest isoelectric points, i.e., the pH values for which the zeta potentials are zero, were measured for strains with glucose added to the growth medium. The isoelectric points for the strains were all rather high, between pH 3 and 5, indicative of protein-rich surfaces, although X-ray photoelectron spectroscopy did not measure excessively large amounts of nitrogen on the cell surfaces. Both MATH and microelectrophoresis were done as a function of pH. Maxima in hydrophobicity were observed at certain pH values. Usually these pH values were in the range of the isoelectric points of the cells. Thus it appears that MATH measures an interplay of hydrophobicity and electrostatic interactions. MATH measures solely hydrophobicity only when electrostatic interactions are absent, i.e., close to the isoelectric points of the cells. Considering that these thermophilic streptococci are all rather hydrophilic, a possible pathway to prevent fouling in the pasteurization process might be to render the heat exchanger plates of the pasteurizer more hydrophobic. PMID:16349127

  1. The β1-subunit of Na+/K+-ATPase interacts with BKCa channels and affects their steady-state expression on the cell surface

    PubMed Central

    Jha, Smita; Dryer, Stuart E.

    2009-01-01

    Large conductance Ca2+- activated K+ channels (BKCa) encoded by the Slo1 gene play a role in the physiological regulation of many cell types. Here, we show that the β1 subunit of Na+/K+-ATPase (NKβ1) interacts with the cytoplasmic COOH-terminal region of Slo1 proteins. Reduced expression of endogenous NKβ1 markedly inhibits evoked BKCa currents with no apparent effect on their gating. In addition, NKβ1 down-regulated cells show decreased density of Slo1 subunits on the cell surface. PMID:19729011

  2. A novel protein, ubiquitous in marine phytoplankton, concentrates iron at the cell surface and facilitates uptake.

    PubMed

    Morrissey, Joe; Sutak, Robert; Paz-Yepes, Javier; Tanaka, Atsuko; Moustafa, Ahmed; Veluchamy, Alaguraj; Thomas, Yann; Botebol, Hugo; Bouget, François-Yves; McQuaid, Jeffrey B; Tirichine, Leila; Allen, Andrew E; Lesuisse, Emmanuel; Bowler, Chris

    2015-02-01

    Numerous cellular functions including respiration require iron. Plants and phytoplankton must also maintain the iron-rich photosynthetic electron transport chain, which most likely evolved in the iron-replete reducing environments of the Proterozoic ocean [1]. Iron bioavailability has drastically decreased in the contemporary ocean [1], most likely selecting for the evolution of efficient iron acquisition mechanisms among modern phytoplankton. Mesoscale iron fertilization experiments often result in blooms dominated by diatoms [2], indicating that diatoms have adaptations that allow survival in iron-limited waters and rapid multiplication when iron becomes available. Yet the genetic and molecular bases are unclear, as very few iron uptake genes have been functionally characterized from marine eukaryotic phytoplankton, and large portions of diatom iron starvation transcriptomes are genes encoding unknown functions [3-5]. Here we show that the marine diatom Phaeodactylum tricornutum utilizes ISIP2a to concentrate Fe(III) at the cell surface as part of a novel, copper-independent and thermodynamically controlled iron uptake system. ISIP2a is expressed in response to iron limitation several days prior to the induction of ferrireductase activity, and it facilitates significant Fe(III) uptake during the initial response to Fe limitation. ISIP2a is able to directly bind Fe(III) and increase iron uptake when heterologously expressed, whereas knockdown of ISIP2a in P. tricornutum decreases iron uptake, resulting in impaired growth and chlorosis during iron limitation. ISIP2a is expressed by diverse marine phytoplankton, indicating that it is an ecologically significant adaptation to the unique nutrient composition of marine environments. PMID:25557662

  3. Variation of expression defects in cell surface 190-kDa protein antigen of Streptococcus mutans.

    PubMed

    Lapirattanakul, Jinthana; Nomura, Ryota; Matsumoto-Nakano, Michiyo; Srisatjaluk, Ratchapin; Ooshima, Takashi; Nakano, Kazuhiko

    2015-05-01

    Streptococcus mutans, which consists of four serotypes, c, e, f, and k, possesses a 190-kDa cell surface protein antigen (PA) for initial tooth adhesion. We used Western blot analysis to determine PA expression in 750 S. mutans isolates from 150 subjects and found a significantly higher prevalence of the isolates with PA expression defects in serotypes f and k compared to serotypes c and e. Moreover, the defect patterns could be classified into three types; no PA expression on whole bacterial cells and in their supernatant samples (Type N1), PA expression mainly seen in supernatant samples (Type N2), and only low expression of PA in the samples of whole bacterial cells (Type W). The underlying reasons for the defects were mutations in the gene encoding PA as well as in the transcriptional processing of this gene for Type N1, defects in the sortase gene for Type N2, and low mRNA expression of PA for Type W. Since cellular hydrophobicity and phagocytosis susceptibility of the PA-defective isolates were significantly lower than those of the normal expression isolates, the potential implication of such defective isolates in systemic diseases involving bacteremia other than dental caries was suggested. Additionally, multilocus sequence typing was utilized to characterize S. mutans clones that represented a proportion of isolates with PA defects of 65-100%. Therefore, we described the molecular basis for variation defects in PA expression of S. mutans. Furthermore, we also emphasized the strong association between PA expression defects and serotypes f and k as well as the clonal relationships among these isolates. PMID:25792295

  4. Oxidation of cell surface thiol groups by contact sensitizers triggers the maturation of dendritic cells.

    PubMed

    Kagatani, Saori; Sasaki, Yoshinori; Hirota, Morihiko; Mizuashi, Masato; Suzuki, Mie; Ohtani, Tomoyuki; Itagaki, Hiroshi; Aiba, Setsuya

    2010-01-01

    p38 mitogen-activated protein kinase (MAPK) has a crucial role in the maturation of dendritic cells (DCs) by sensitizers. Recently, it has been reported that the oxidation of cell surface thiols by an exogenous impermeant thiol oxidizer can phosphorylate p38 MAPK. In this study, we examined whether sensitizers oxidize cell surface thiols of monocyte-derived DCs (MoDCs). When cell surface thiols were quantified by flow cytometry using Alexa fluor maleimide, all the sensitizers that we examined decreased cell surface thiols on MoDCs. To examine the effects of decreased cell surface thiols by sensitizers on DC maturation, we analyzed the effects of an impermeant thiol oxidizer, o-phenanthroline copper complex (CuPhen). The treatment of MoDCs with CuPhen decreased cell surface thiols, phosphorylated p38 MAPK, and induced MoDC maturation, that is, the augmentation of CD83, CD86, HLA-DR, and IL-8 mRNA, as well as the downregulation of aquaporin-3 mRNA. The augmentation of CD86 was significantly suppressed when MoDCs were pretreated with N-acetyl-L-cystein or treated with SB203580. Finally, we showed that epicutaneous application of 2,4-dinitrochlorobenzene on mouse skin significantly decreased cell surface thiols of Langerhans cells in vivo. These data suggest that the oxidation of cell surface thiols has some role in triggering DC maturation by sensitizers. PMID:19641517

  5. The cell surface GRP78 facilitates the invasion of hepatocellular carcinoma cells.

    PubMed

    Zhang, Xiu-Xiu; Li, Hong-Dan; Zhao, Song; Zhao, Liang; Song, Hui-Juan; Wang, Guan; Guo, Qing-Jun; Luan, Zhi-Dong; Su, Rong-Jian

    2013-01-01

    Invasion is a major characteristic of hepatocellular carcinoma and one of the main causes of refractory to treatment. We have previously reported that GRP78 promotes the invasion of hepatocellular carcinoma although the mechanism underlying this change remains uncertain. In this paper, we explored the role of the cell surface GRP78 in the regulation of cancer cell invasion in hepatocellular carcinoma cells. We found that neutralization of the endogenous cell surface GRP78 with the anti-GRP78 antibody inhibited the adhesion and invasion in hepatocellular carcinoma cell lines Mahlavu and SMMC7721. However, forced expression of the cell surface GRP78 facilitated the adhesion and invasion in SMMC7721. We further demonstrated that inhibition of the endogenous cell surface GRP78 specifically inhibited the secretion and activity of MMP-2 but did not affect the secretion and activity of MMP-9. We also found that inhibition of the cell surface GRP78 increased E-Cadherin expression and decreased N-Cadherin level. On the contrary, forced expression of the cell surface GRP78 increased N-Cadherin expression and decreased E-Cadherin level, suggesting that the cell surface GRP78 plays critical role in the regulation of EMT process. These findings suggest that the cell surface GRP78 plays a stimulatory role in the invasion process and may be a potential anti-invasion target for the treatment of hepatocellular carcinoma. PMID:24383061

  6. An efficient delivery of DAMPs on the cell surface by the unconventional secretion pathway.

    PubMed

    Zhu, Haiyan; Wang, Lan; Ruan, Yuanyuan; Zhou, Lei; Zhang, Dongmei; Min, Zhihui; Xie, Jianhui; Yu, Min; Gu, Jianxin

    2011-01-21

    Damage-associated molecular patterns (DAMPs) are signals released from dying cells evoking the immune system response in several inflammatory disorders. In normal situations, many of DAMPs are nuclear or cytosolic proteins with defined intracellular function, but they could be found on the cell surface following tissue injury. The biological function of the translocated DAMPs is still not well known and an efficient delivery of these molecules on the cell surface is required to clarify their biological effects. In this study, we demonstrated that an unclassical secretory signal peptide, N-terminal 18 amino acids of HASPB (HASPB-N18), could efficiently deliver Hsp60, Hsp70, and HMGB1 on the cell surface. Furthermore, the delivery of these molecules on the cell surface by HASPB-N18 is not limited to a special cell line because several cell lines could use this delivery signal to deliver these molecules on the cell surface. Moreover, we demonstrated that Hsp60 on the cell surface delivered by HASPB-N18 could be recognized by a soluble form of LOX-1, which implies that DAMPs on the cell surface delivered by HASPB-N18 have a proper conformation during transport. Therefore, delivery of DAMPs by HASPB-N18 is a reliable model to further understand the biological significance of DAMPs on the cell surface. PMID:21168385

  7. Functional analysis of the putative integrin recognition motif on adeno-associated virus 9.

    PubMed

    Shen, Shen; Berry, Garrett E; Castellanos Rivera, Ruth M; Cheung, Roland Y; Troupes, Andrew N; Brown, Sarah M; Kafri, Tal; Asokan, Aravind

    2015-01-16

    Adeno-associated viruses (AAVs) display a highly conserved NGR motif on the capsid surface. Earlier studies have established this tripeptide motif as being essential for integrin-mediated uptake of recombinant AAV serotype 2 (AAV2) in cultured cells. However, functional attributes of this putative integrin recognition motif in other recombinant AAV serotypes displaying systemic transduction in vivo remain unknown. In this study, we dissect the biology of an integrin domain capsid mutant derived from the human isolate AAV9 in mice. The AAV9/NGA mutant shows decreased systemic transduction in mice. This defective phenotype was accompanied by rapid clearance of mutant virions from the blood circulation and nonspecific sequestration by the spleen. Transient vascular hyperpermeability, induced by histamine coinjection, exacerbated AAV9/NGA uptake by the spleen but not the liver. However, such treatment did not affect AAV9 virions, suggesting a potential entry/post-entry defect for the mutant in different tissues. Further characterization revealed modestly decreased cell surface binding but a more pronounced defect in the cellular entry of mutant virions. These findings were corroborated by the observation that blocking multiple integrins adversely affected recombinant AAV9 transduction in different cell types, albeit with variable efficiencies. From a structural perspective, we observed that the integrin recognition motif is located in close proximity to the galactose binding footprint on AAV9 capsids and postulate that this feature could influence cell surface attachment, cellular uptake at the tissue level, and systemic clearance by the reticuloendothelial system. PMID:25404742

  8. Functional analysis of putative phosphoenolpyruvate transporters localized to the Golgi apparatus in Schizosaccharomyces pombe.

    PubMed

    Yoritsune, Ken-ichi; Higuchi, Yujiro; Matsuzawa, Tomohiko; Takegawa, Kaoru

    2014-11-01

    The cell surface of Schizosaccharomyces pombe is negatively charged due to the presence of pyruvylated oligosaccharides, which is important for cell-cell recognition. However, the mechanism of pyruvate supply to oligosaccharides is not clearly understood. Here, we analyzed three putative phosphoenolpyruvate (PEP) transporter genes (pet1(+) , pet2(+) , and pet3(+) ) in S. pombe, identified by sequence homology search against the Arabidopsis thaliana PEP transporter AtPPT1. Schizosaccharomyces pombe strain carrying a disruption in pet1(+) (pet1Δ) or in pet2(+) (pet2Δ), but not the strain carrying a disruption in pet3(+) (pet3Δ), showed reduced pyruvate level on the cell surface. This reduction in pyruvate level was restored to the control level by expressing green fluorescent protein (GFP)-tagged Pet1p and Pet2p in respective disruptants. Fluorescence microscope studies revealed that GFP-tagged Pet1p and Pet2p were localized to the Golgi apparatus. Although expression of neither AtPPT1 nor AtPPT2 suppressed the pet1Δ phenotype, that of chimeric constructs, where the N-terminal regions of AtPPT1 and AtPPT2 were replaced by the N-terminal region of Pet1p, partially suppressed the pet1Δ phenotype. Furthermore, the reduction in cell surface negative charge in pet1Δ cells was restored by incubating these cells with recombinant Pvg1p and PEP. Thus, Pet1p and Pet2p are likely involved in transporting PEP from the cytoplasm into the Golgi. PMID:25195688

  9. The Text Encoding Initiative: Flexible and Extensible Document Encoding.

    ERIC Educational Resources Information Center

    Barnard, David T.; Ide, Nancy M.

    1997-01-01

    The Text Encoding Initiative (TEI), an international collaboration aimed at producing a common encoding scheme for complex texts, examines the requirement for generality versus the requirement to handle specialized text types. Discusses how documents and users tax the limits of fixed schemes requiring flexible extensible encoding to support…

  10. Insulin stimulates movement of sorting nexin 9 between cellular compartments: a putative role mediating cell surface receptor expression and insulin action.

    PubMed Central

    MaCaulay, S Lance; Stoichevska, Violet; Grusovin, Julian; Gough, Keith H; Castelli, Laura A; Ward, Colin W

    2003-01-01

    SNX9 (sorting nexin 9) is one member of a family of proteins implicated in protein trafficking. This family is characterized by a unique PX (Phox homology) domain that includes a proline-rich sequence and an upstream phospholipid binding domain. Many sorting nexins, including SNX9, also have a C-terminal coiled region. SNX9 additionally has an N-terminal SH3 (Src homology 3) domain. Here we have investigated the cellular localization of SNX9 and the potential role it plays in insulin action. SNX9 had a cytosolic and punctate distribution, consistent with endosomal and cytosolic localization, in 3T3L1 adipocytes. It was excluded from the nucleus. The SH3 domain was responsible, at least in part, for the membrane localization of SNX9, since expression of an SH3-domain-deleted GFP (green fluorescent protein)-SNX9 fusion protein in HEK293T cells rendered the protein cytosolic. Membrane localization may also be attributed in part to the PX domain, since in vitro phospholipid binding studies demonstrated SNX9 binding to polyphosphoinositides. Insulin induced movement of SNX9 to membrane fractions from the cytosol. A GST (glutathione S-transferase)-SNX9 fusion protein was associated with IGF1 (insulin-like growth factor 1) and insulin receptors in vitro. A GFP-SNX9 fusion protein, overexpressed in 3T3L1 adipocytes, co-immunoprecipitated with insulin receptors. Furthermore, overexpression of this GFP-SNX9 fusion protein in CHOT cells decreased insulin binding, consistent with a role for SNX9 in the trafficking of insulin receptors. Microinjection of 3T3L1 cells with an antibody against SNX9 inhibited stimulation by insulin of GLUT4 translocation. These results support the involvement of SNX9 in insulin action, via an influence on the processing/trafficking of insulin receptors. A secondary role in regulation of the cellular processing, transport and/or subcellular localization of GLUT4 is also suggested. PMID:12917015

  11. Wellcome Prize Lecture. Cell surface, ion-sensing receptors.

    PubMed

    Riccardi, Daniela

    2002-07-01

    Changes in extracellular calcium (Ca(2+)o) concentration ([Ca2+]o) affect kidney function both under basal and hormone-stimulated conditions. The molecular identification of an extracellular Ca(2+)-sensing receptor (CaR) has confirmed a direct role of Ca(2+)o on parathyroid and kidney function (i.e. independent of calciotropic hormones) as a modulator of Ca2+ homeostasis. In addition, evidence accumulated over the last 10 years has shown that CaR is also expressed in regions outside the calcium homeostatic system where its role is largely undefined but seems to be linked to regulation of local ionic homeostasis. The parathyroid and kidney CaRs are 1081 and 1079 amino acids long, respectively, and belong to the type III family of G protein-coupled receptors (GPCRs), which includes other CaRs, metabotropic glutamate receptors and putative vomeronasal organ receptors. For the CaR, its low (millimolar) affinity for Ca2+, its positive cooperativity and its large ion-sensing extracellular domain, indicate that the receptor is more sensitive to changes in net cationic charge rather than to a specific ligand. Mg2+, trivalent cations of the lanthanide series and polyvalent cations such as spermine and aminoglycoside antibiotics can all activate the receptor in vitro with EC50 values in the micromolar range for trivalent and polyvalent cations or in the millimolar range for Ca2+ and Mg2+. In addition to true CaR agonists, CaR sensitivity to Ca(2+)o is also susceptible to allosteric modulation by ionic strength, L-amino acids and by pharmacological agents. This review will address endogenous and exogenous CaR agonists, the role of the receptor in the calcium homeostatic system and some speculation on possible role(s) of the CaR in regions not involved in mineral ion homeostasis. PMID:12392104

  12. Putative Excitatory and Putative Inhibitory Inputs Localize to Different Dendritic Domains in a Drosophila Flight Motoneuron

    PubMed Central

    Kuehn, Claudia; Duch, Carsten

    2012-01-01

    Input-output computations of individual neurons may be affected by the three-dimensional structure of their dendrites and by the targeting of input synapses to specific parts of their dendrites. However, only few examples exist where dendritic architecture can be related to behaviorally relevant computations of a neuron. By combining genetic, immunohistochemical, and confocal laser scanning methods this study estimates the location of the spike initiating zone and the dendritic distribution patterns of putative synaptic inputs on an individually identified Drosophila flight motorneuron, MN5. MN5 is a monopolar neuron with more than 4000 dendritic branches. The site of spike initiation was estimated by mapping sodium channel immunolabel onto geometric reconstructions of MN5. Maps of putative excitatory cholinergic and of putative inhibitory GABAergic inputs on MN5 dendrites were created by charting tagged Dα7 nicotinic acetylcholine receptors and Rdl GABAA receptors onto MN5 dendritic surface reconstructions. Although these methods provided only an estimate of putative input synapse distributions, the data indicated that inhibitory and excitatory synapses were targeted preferentially to different dendritic domains of MN5, and thus, computed mostly separately. Most putative inhibitory inputs were close to spike initiation, which was consistent with sharp inhibition, as predicted previously based on recordings of motoneuron firing patterns during flight. By contrast, highest densities of putative excitatory inputs at more distant dendritic regions were consistent with the prediction that in response to different power demands during flight, tonic excitatory drive to flight motoneuron dendrites must be smoothly translated into different tonic firing frequencies. PMID:23279094

  13. VLSI Reed-Solomon Encoder

    NASA Technical Reports Server (NTRS)

    Liu, K. Y.

    1983-01-01

    Modular Reed-solomon encoder uses identical custom VLSI chips called "symbol slices." By cascading and properly interconnecting group of these chips, encoder is made for any desired error-correcting capability and interleaving level. VLSI encoder requires only one-tenth the number of chips required by conventional Reed-Solomon Circuit implemented with discrete IC's.

  14. Ketone isosteres of 2-N-acetamidosugars as substrates for metabolic cell surface engineering

    SciTech Connect

    Hang, Howard C.; Bertozzi, Carolyn R.

    2000-08-22

    Novel chemical reactivity can be engendered on cell surfaces by the metabolic incorporation of unnatural sugars into cell surface glycoconjuagtes. 2-N-Acetamido sugars such as GalNAc and GlcNAc are abundant components of cell surface glycoconjugates, and hence attractive targets for metabolic cell surface engineering. Here we report (1) the synthesis of isosteric analogs bearing a ketone group in place of the N-acetamido group, and (2) evaluation of their metabolic incorporation into mammalian cell surface glycans. A ketone isostere of GalNAc was metabolized by CHO cells through the salvage pathway and delivered to O-linked glycoproteins on the cell surface. Its residence at the core position of O-linked glycans is suggested by studies with a-benzyl GalNAc, an inhibitor of O-linked oligosaccharide extension. A mutant CHO cell line lacking endogenous UDP-GalNAc demonstrated enhanced metabolism of the GalNAc analog, suggesting that competition with native intermediates might limits enzymatic transformation in mammalian cells. A ketone isostere of GlcNAc could not be detected on CHO or human cell surfaces after incubation. Thus, the enzymes in the GlcNAc salvage pathway might be less permissive of unnatural substrates than those comprising the GalNAc salvage pathway. Alternatively, high levels of endogenous GlcNAc derivatives might compete with the ketone isostere and prevent its incorporation into oligosaccharides.

  15. Multiple transcripts encode glucose 6-phosphate dehydrogenase in the southern cattle tick, Rhipicephalus (Boophilus) microplus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glucose 6-phosphate dehydrogenase (G6PDH) is an enzyme that plays a critical role in the production of NADPH. Here we describe the identification of four transcripts (G6PDH-A, -B, -C, and -D) that putatively encode the enzyme in the southern cattle tick, Rhipicephalus (Boophilus) microplus. The geno...

  16. Structure of PA4019, a putative aromatic acid decarboxylase from Pseudomonas aeruginosa

    PubMed Central

    Kopec, Jolanta; Schnell, Robert; Schneider, Gunter

    2011-01-01

    The ubiX gene (PA4019) of Pseudomonas aeruginosa has been annotated as encoding a putative 3-octaprenyl-4-hydroxybenzoate decarboxylase from the ubiquinone-biosynthesis pathway. Based on a transposon mutagenesis screen, this gene was also implicated as being essential for the survival of this organism. The crystal structure of recombinant UbiX determined to 1.5 Å resolution showed that the protein belongs to the superfamily of homo-oligomeric flavine-containing cysteine decarboxylases. The enzyme assembles into a dodecamer with 23 point symmetry. The subunit displays a typical Rossmann fold and contains one FMN molecule bound at the interface between two subunits. PMID:22102023

  17. Ten Putative Contributors to the Obesity Epidemic

    PubMed Central

    McAllister, Emily J.; Dhurandhar, Nikhil V.; Keith, Scott W.; Aronne, Louis J.; Barger, Jamie; Baskin, Monica; Benca, Ruth M.; Biggio, Joseph; Boggiano, Mary M.; Eisenmann, Joe C.; Elobeid, Mai; Fontaine, Kevin R.; Gluckman, Peter; Hanlon, Erin C.; Katzmarzyk, Peter; Pietrobelli, Angelo; Redden, David T.; Ruden, Douglas M.; Wang, Chenxi; Waterland, Robert A.; Wright, Suzanne M.; Allison, David B.

    2010-01-01

    The obesity epidemic is a global issue and shows no signs of abating, while the cause of this epidemic remains unclear. Marketing practices of energy-dense foods and institutionally-driven declines in physical activity are the alleged perpetrators for the epidemic, despite a lack of solid evidence to demonstrate their causal role. While both may contribute to obesity, we call attention to their unquestioned dominance in program funding and public efforts to reduce obesity, and propose several alternative putative contributors that would benefit from equal consideration and attention. Evidence for microorganisms, epigenetics, increasing maternal age, greater fecundity among people with higher adiposity, assortative mating, sleep debt, endocrine disruptors, pharmaceutical iatrogenesis, reduction in variability of ambient temperatures, and intrauterine and intergenerational effects, as contributing factors to the obesity epidemic are reviewed herein. While the evidence is strong for some contributors such as pharmaceutical-induced weight gain, it is still emerging for other reviewed factors. Considering the role of such putative etiological factors of obesity may lead to comprehensive, cause specific, and effective strategies for prevention and treatment of this global epidemic. PMID:19960394

  18. Quantification of differential ErbB1 and ErbB2 cell surface expression and spatial nanoclustering through plasmon coupling.

    PubMed

    Wang, Jing; Yu, Xinwei; Boriskina, Svetlana V; Reinhard, Björn M

    2012-06-13

    Cell surface receptors play ubiquitous roles in cell signaling and communication and their expression levels are important biomarkers for many diseases. Expression levels are, however, only one factor that determines the physiological activity of a receptor. For some surface receptors, their distribution on the cell surface, especially their clustering, provides additional mechanisms for regulation. To access this spatial information robust assays are required that provide detailed insight into the organization of cell surface receptors on nanometer length scales. In this manuscript, we demonstrate through combination of scattering spectroscopy, electron microscopy, and generalized multiple particle Mie theory (GMT) simulations that the density- and morphology-dependent spectral response of Au nanoparticle (NP) immunolabels bound to the epidermal growth factor receptors ErbB1 and ErbB2 encodes quantitative information of both the cell surface expression and spatial clustering of the two receptors in different unliganded in vitro cancer cell lines (SKBR3, MCF7, A431). A systematic characterization of the collective spectral responses of NPs targeted at ErbB1 and ErbB2 at various NP concentrations indicates differences in the large-scale organization of ErbB1 and ErbB2 in cell lines that overexpress these receptors. Validation experiments in the scanning electron microscope (SEM) confirm that NPs targeted at ErbB1 on A431 are more strongly clustered than NPs bound to ErbB2 on SKBR3 or MCF7 at overall comparable NP surface densities. This finding is consistent with the existence of larger receptor clusters for ErbB1 than for ErbB2 in the plasma membranes of the respective cells. PMID:22587495

  19. Genetic Modification of Mesenchymal Stem Cells to Express a Single-Chain Antibody Against EGFRvIII on the Cell Surface

    PubMed Central

    Balyasnikova, Irina V.; Franco-Gou, Rosa; Mathis, J. Michael; Lesniak, Maciej S.

    2010-01-01

    Human adult mesenchymal stem cells (hMSC) are under active investigation as cellular carriers for gene therapy. hMSC possess natural tropism toward tumors, however, the targeting of hMSC to specific cell populations within tumors is unexplored. In the case of glioblastoma multiforme (GBM), at least half of the tumors express EGFRvIII on the cell surface, an ideal target for antibody-mediated gene/drug delivery. In this study, we investigated the feasibility of genetically modifying hMSC to express a single-chain antibody (scFv) to EGFRvIII on their surface. Nucleofection was used to transfect hMSC with cDNA encoding scFv EGFRvIII fused with PDGFR or human B7-1 transmembrane domains. The expression of scFv EGFRvIII on the cell surface was assessed by FACS. A stable population of scFv EGFRvIII-expressing hMSC was selected based on antibiotic resistance and enriched using FACS. We found that nucleofection allows the efficient expression of scFv EGFRvIII on the cell surface of hMSC. hMSCs transfected with the construct encoding scFv EGFRvIII as a fusion with PDGFRtm showed scFv EGFRvIII expression in up to 86% of cells. Most importantly, human MSC expressing scFv against EGFRvIII demonstrated enhanced binding to U87-EGFRvIII cells in vitro and at least 7-fold increased retention in human U87-EGFRvIII expressing tumors in vivo. In summary, we provide the first conclusive evidence of genetic modification of hMSC with a single-chain antibody against an antigen expressed on the surface of tumor cells, thereby opening up a new venue for enhanced delivery of gene therapy applications in the context of malignant brain cancer. PMID:19937911

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

    PubMed Central

    2014-01-01

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

  1. Influence of manufacturing processes on cell surface properties of probiotic strain Lactobacillus rhamnosus Lcr35®.

    PubMed

    Nivoliez, Adrien; Veisseire, Philippe; Alaterre, Elina; Dausset, Caroline; Baptiste, Fabrice; Camarès, Olivier; Paquet-Gachinat, Marylise; Bonnet, Muriel; Forestier, Christiane; Bornes, Stéphanie

    2015-01-01

    The influence of the industrial process on the properties of probiotics, administered as complex manufactured products, has been poorly investigated. In the present study, we comparatively assessed the cell wall characteristics of the probiotic strain Lactobacillus rhamnosus Lcr35® together with three of its commercial formulations with intestinal applications. Putative secreted and transmembrane-protein-encoding genes were initially searched in silico in the genome of L. rhamnosus Lcr35®. A total of 369 candidate genes were identified which expressions were followed using a custom Lactobacillus DNA chip. Among them, 60 or 67 genes had their expression either upregulated or downregulated in the Lcr Restituo® packet or capsule formulations, compared to the native Lcr35® strain. Moreover, our data showed that the probiotic formulations (Lcr Lenio®, Lcr restituo® capsule and packet) showed a better capacity to adhere to intestinal epithelial Caco-2 cells than the native Lcr35® strain. Microbial (MATS) tests showed that the probiotic was an electron donor and that they were more hydrophilic than the native strain. The enhanced adhesion capacity of the active pharmaceutical ingredients (APIs) to epithelial Caco-2 cells and their antipathogen effect could be due to this greater surface hydrophilic character. These findings suggest that the manufacturing process influences the protein composition and the chemical properties of the cell wall. It is therefore likely that the antipathogen effect of the formulation is modulated by the industrial process. Screening of the manufactured products' properties would therefore represent an essential step in evaluating the effects of probiotic strains. PMID:25280746

  2. Time encoded radiation imaging

    DOEpatents

    Marleau, Peter; Brubaker, Erik; Kiff, Scott

    2014-10-21

    The various technologies presented herein relate to detecting nuclear material at a large stand-off distance. An imaging system is presented which can detect nuclear material by utilizing time encoded imaging relating to maximum and minimum radiation particle counts rates. The imaging system is integrated with a data acquisition system that can utilize variations in photon pulse shape to discriminate between neutron and gamma-ray interactions. Modulation in the detected neutron count rates as a function of the angular orientation of the detector due to attenuation of neighboring detectors is utilized to reconstruct the neutron source distribution over 360 degrees around the imaging system. Neutrons (e.g., fast neutrons) and/or gamma-rays are incident upon scintillation material in the imager, the photons generated by the scintillation material are converted to electrical energy from which the respective neutrons/gamma rays can be determined and, accordingly, a direction to, and the location of, a radiation source identified.

  3. Rotary encoding device

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B. (Inventor)

    1993-01-01

    A device for position encoding of a rotating shaft in which a polygonal mirror having a number of facets is mounted to the shaft and a light beam is directed towards the facets is presented. The facets of the polygonal mirror reflect the light beam such that a light spot is created on a linear array detector. An analog-to-digital converter is connected to the linear array detector for reading the position of the spot on the linear array detector. A microprocessor with memory is connected to the analog-to-digital converter to hold and manipulate the data provided by the analog-to-digital converter on the position of the spot and to compute the position of the shaft based upon the data from the analog-to-digital converter.

  4. Linear encoding device

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B. (Inventor)

    1993-01-01

    A Linear Motion Encoding device for measuring the linear motion of a moving object is disclosed in which a light source is mounted on the moving object and a position sensitive detector such as an array photodetector is mounted on a nearby stationary object. The light source emits a light beam directed towards the array photodetector such that a light spot is created on the array. An analog-to-digital converter, connected to the array photodetector is used for reading the position of the spot on the array photodetector. A microprocessor and memory is connected to the analog-to-digital converter to hold and manipulate data provided by the analog-to-digital converter on the position of the spot and to compute the linear displacement of the moving object based upon the data from the analog-to-digital converter.

  5. Proteomic analysis and identification of cell surface-associated proteins of Clostridium chauvoei.

    PubMed

    Jayaramaiah, Usharani; Singh, Neetu; Thankappan, Sabarinath; Mohanty, Ashok Kumar; Chaudhuri, Pallab; Singh, Vijendra Pal; Nagaleekar, Viswas Konasagara

    2016-06-01

    Blackleg is a highly fatal disease of cattle and sheep, caused by Clostridium chauvoei, a Gram positive, anaerobic, spore forming bacteria. Cell surface-associated proteins play a major role in inducing the protective immunity. However, the identity of a majority of cell surface-associated proteins of C. chauvoei is not known. In the present investigation, we have used SDS-PAGE, 2D-gel electrophoresis and Western blotting followed by mass spectrometry to identify cell surface-associated proteins of C. chauvoei. Among the identified proteins, which have shown to offer protective antigencity in other bacteria, Enolase, Chaperonin, Ribosomal protein L10, Glycosyl Hydrolase and Flavoprotein were characterized by sequencing and their overexpression in Escherichia coli. In conclusion, cell surface-associated proteins were identified using proteomic approach and the genes for the immunoreactive proteins were expressed, which may prove to be potential diagnostic or vaccine candidates. PMID:26971466

  6. Microbial cell surface characteristics: Elucidating attachment/detachment using hydrophobicity and electrokinetic measurements

    EPA Science Inventory

    The surface properties of microorganisms play an important role in their behavior within the environment. Electrophoretic mobility and cell surface hydrophobicity of bacterial cells influence their initial interaction with surfaces and mediate their stability within an aqueous su...

  7. The expression of superoxide dismutase (SOD) and a putative ABC transporter permease is inversely correlated during biofilm formation in Listeria monocytogenes 4b G

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little is known about the molecular basis of biofilm formation in Listeria monocytogenes. The superoxide dismutase (SOD) of the deletion mutant of lm.G_1771 gene, which encodes for a putative ABC_transporter permease, is highly expressed in biofilm. In this study, the sod gene deletion mutant delta ...

  8. Effects of entrapment on nucleic acid content, cell morphology, cell surface property, and stress of pure cultures commonly found in biological wastewater treatment.

    PubMed

    Pramanik, Sudipta; Khanna, Rohit; Katti, Kalpana; McEvoy, John; Khan, Eakalak

    2011-10-01

    The effects of cell entrapment on nucleic acid content, cell morphology, cell surface property, and stress of major groups of bacteria (betaproteobacteria and gammaproteobacteria) in biological municipal wastewater treatment were investigated. Three different entrapment media (alginate, carrageenan, and polyvinyl alcohol) were examined. Results indicated that the entrapment and type of entrapment media affected nucleic acid content, cell morphology, cell surface property, and stress of the three representative species (Alcaligenes faecalis, Comamonas testosteroni, and Pseudomonas putida) studied. The highest deoxyribonucleic acid and ribonucleic acid increases were observed with the alginate and polyvinyl alcohol (PVA) entrapment, respectively. A cell morphological change from bacilli to coccoidal was observed in the case of alginate entrapment while the PVA-entrapped cells had a slim morphology when compared to non-entrapped cells and formed putative nanowires. The entrapment increased or decreased the surface roughness of cells depending on the type of entrapment media. Expression of a nitrosative stress gene, which is linked to oxygen deprivation, was observed more in the alginate-entrapped cells. These research findings advance the fundamental understanding of the entrapped cell physiology which can lead to more efficient entrapped cell-based wastewater treatment. PMID:21660542

  9. Cell surface nucleolin as a target for anti-cancer therapies.

    PubMed

    Koutsioumpa, Marina; Papadimitriou, Evangelia

    2014-05-01

    A large number of mostly recent reports show enhanced expression of the multi-functional protein nucleolin (NCL) on the surface of activated lymphocytes, angiogenic endothelial and many different types of cancer cells. Translocation of NCL at the external side of the plasma membrane occurs via a secretory pathway independent of the endoplasmic reticulum-Golgi complex, requires intracellular intact actin cytoskeleton, and seems to be mediated by a variety of factors. Cell surface NCL serves as a binding partner of several molecules implicated in cell differentiation, adhesion, and leukocyte trafficking, inflammation, angiogenesis and tumor development, mediating their biological activities and in some cases, leading to their internalization. Accumulating evidence validates cell surface NCL as a strategic target for treatment of cancer, while its property of tumor-specific uptake of targeted ligands seems to be useful for the development of non-invasive imaging tools for the diagnosis of cancer and for the targeted release of chemotherapeutic drugs. The observation that cell surface NCL exists in complexes with several other proteins implicated in tumorigenesis and angiogenesis suggests that targeting cell surface NCL might trigger multi-inhibitory effects, depending on the cell type. This review summarizes papers and patents related to the redistribution and the biological functions of cell surface NCL, with emphasis on the potential importance and advantages of developing efficient anti-cell surface NCL strategies. PMID:24251811

  10. Cell-surface nucleolin is involved in lipopolysaccharide internalization and signalling in alveolar macrophages.

    PubMed

    Wang, Yi; Mao, Mei; Xu, Jian-cheng

    2011-07-01

    C23 (nucleolin) shuttling between the nucleus, cytoplasm and cell surface has been implicated in controlling regulatory processes and may play a role in pathogen infection and autoimmune diseases. It has been reported that cell surface-expressed C23 on THP-1 monocytes is involved in the inflammatory response induced by LPS (lipopolysaccharide). This study investigates whether C23 is a membrane receptor for LPS during LPS-induced AMs (alveolar macrophages) activation. First, using immunofluorescence and microscopy, we detected the expression of C23 on the surface of AMs. Second, using LPS affinity columns, we demonstrated that C23 directly binds to LPS. Third, we found that LPS colocalized with C23 on both the cell surface and in the cytoplasm. Finally, knockdown of C23 expression on the cell surface using siRNA (small interfering RNA) led to significant reductions in the internalization of LPS, in LPS-induced NF-κB (nuclear factor κB)-DNA binding and in the protein expression of TNF (tumour necrosis factor)-α and IL-6 (interleukin-6). These findings provide evidence that cell-surface C23 on AMs may serve as a receptor for LPS and are essential for internalization and transport of LPS. Furthermore, C23 participates in the regulation of LPS-induced inflammation of AMs, which indicates that cell-surface C23 is a new and promising therapeutic target for the treatment of bacterial infections. PMID:21309751

  11. Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis

    PubMed Central

    Özlü, Nurhan; Qureshi, Mohammad H; Toyoda, Yusuke; Renard, Bernhard Y; Mollaoglu, Gürkan; Özkan, Nazlı E; Bulbul, Selda; Poser, Ina; Timm, Wiebke; Hyman, Anthony A; Mitchison, Timothy J; Steen, Judith A

    2015-01-01

    The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti-mitotic cancer chemotherapy. PMID:25476450

  12. Cell surface nucleolin serves as receptor for DNA nanoparticles composed of pegylated polylysine and DNA.

    PubMed

    Chen, Xuguang; Kube, Dianne M; Cooper, Mark J; Davis, Pamela B

    2008-02-01

    Compacted DNA nanoparticles deliver transgenes efficiently to the lung following intrapulmonary dosing. Here we show that nucleolin, a protein known to shuttle between the nucleus, cytoplasm, and cell surface, is a receptor for DNA nanoparticles at the cell surface. By using surface plasmon resonance (SPR), we demonstrate that nucleolin binds to DNA nanoparticles directly. The presence of nucleolin on the surface of HeLa and 16HBEo- cells was confirmed by surface biotinylation assay and immunofluorescence. Rhodamine-labeled DNA nanoparticles colocalize with nucleolin on the cell surface, as well as in the cytoplasm and nucleus, but not with transferrin or markers of early endosome or lysosome following cellular uptake. Reducing nucleolin on the cell surface by serum-free medium or siRNA against nucleolin treatment leads to significant reduction in luciferase reporter gene activity, while overexpressing nucleolin has the opposite effect. Competition for binding to DNA nanoparticles with exogenous purified nucleolin decreases the transfection efficiency by 60-90% in a dose-dependent manner. Therefore, the data strongly suggest that cell surface nucleolin serves as a receptor for DNA nanoparticles, and that nucleolin is essential for internalization and/or transport of the nanoparticles from cell surface to the nucleus. PMID:18059369

  13. Space vehicle onboard command encoder

    NASA Technical Reports Server (NTRS)

    1975-01-01

    A flexible onboard encoder system was designed for the space shuttle. The following areas were covered: (1) implementation of the encoder design into hardware to demonstrate the various encoding algorithms/code formats, (2) modulation techniques in a single hardware package to maintain comparable reliability and link integrity of the existing link systems and to integrate the various techniques into a single design using current technology. The primary function of the command encoder is to accept input commands, generated either locally onboard the space shuttle or remotely from the ground, format and encode the commands in accordance with the payload input requirements and appropriately modulate a subcarrier for transmission by the baseband RF modulator. The following information was provided: command encoder system design, brassboard hardware design, test set hardware and system packaging, and software.

  14. N-Consecutive-Phase Encoder

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush; Lee, Ho-Kyoung; Weber, Charles

    1995-01-01

    N-consecutive-phase encoder (NCPE) is conceptual encoder for generating alphabet of N consecutive full-response continuous-phase-modulation (CPM) signals. Enables use of binary preencoder of higher rate than used with simple continuous-phase encoder (CPE). NCPE makes possible to achieve power efficiencies and bandwidth efficiencies greater than conventional trellis coders with continuous-phase frequency-shift keying (CPFSK).

  15. CopA:GFP localizes to putative Golgi equivalents in Aspergillus nidulans.

    PubMed

    Breakspear, Andrew; Langford, Kelly J; Momany, Michelle; Assinder, Susan J

    2007-12-01

    The Golgi complex is a main component of the eukaryotic secretory system and functions to modify nascent proteins sent from the endoplasmic reticulum. Ultrastructural studies of filamentous fungi have shown Golgi to be individual smooth membrane cisternae that are referred to as Golgi equivalents or dictyosomes. The Aspergillus nidulans copA gene encodes a homolog of mammalian coat protein (alpha-COP), a constituent of the Golgi-localized COPI vesicle coat. Here, the localization of A. nidulansalpha-COP was examined in live cells using the reporter green fluorescent protein (GFP). CopA:GFP localized to putative Golgi equivalents that were concentrated at hyphal tips. The localization was disrupted by the fungal metabolite brefeldin A. To investigate the significance of the microtubule cytoskeleton in the localization of putative Golgi equivalents, the copA:gfp fusion was expressed in a temperature-sensitive dynein mutant. In addition, a wild-type strain expressing copA:gfp was treated with the microtubule-disrupting drug nocodazole. The results suggest that the microtubule cytoskeleton is not the primary mechanism of localizing putative Golgi equivalents in A. nidulans. PMID:17986089

  16. GIP2, a Putative Transcription Factor That Regulates the Aurofusarin Biosynthetic Gene Cluster in Gibberella zeae

    PubMed Central

    Kim, Jung-Eun; Jin, Jianming; Kim, Hun; Kim, Jin-Cheol; Yun, Sung-Hwan; Lee, Yin-Won

    2006-01-01

    Gibberella zeae (anamorph: Fusarium graminearum) is an important pathogen of maize, wheat, and rice. Colonies of G. zeae produce yellow-to-tan mycelia with the white-to-carmine red margins. In this study, we focused on nine putative open reading frames (ORFs) closely linked to PKS12 and GIP1, which are required for aurofusarin biosynthesis in G. zeae. Among them is an ORF designated GIP2 (for Gibberella zeae pigment gene 2), which encodes a putative protein of 398 amino acids that carries a Zn(II)2Cys6 binuclear cluster DNA-binding domain commonly found in transcription factors of yeasts and filamentous fungi. Targeted gene deletion and complementation analyses confirmed that GIP2 is required for aurofusarin biosynthesis. Expression of GIP2 in carrot medium correlated with aurofusarin production by G. zeae and was restricted to vegetative mycelia. Inactivation of the 10 contiguous genes in the ΔGIP2 strain delineates an aurofusarin biosynthetic gene cluster. Overexpression of GIP2 in both the ΔGIP2 and the wild-type strains increases aurofusarin production and reduces mycelial growth. Thus, GIP2 is a putative positive regulator of the aurofusarin biosynthetic gene cluster, and aurofusarin production is negatively correlated with vegetative growth by G. zeae. PMID:16461721

  17. Cloning and molecular characterization of a putative voltage-gated sodium channel gene in the crayfish.

    PubMed

    Coskun, Cagil; Purali, Nuhan

    2016-06-01

    Voltage-gated sodium channel genes and associated proteins have been cloned and studied in many mammalian and invertebrate species. However, there is no data available about the sodium channel gene(s) in the crayfish, although the animal has frequently been used as a model to investigate various aspects of neural cellular and circuit function. In the present work, by using RNA extracts from crayfish abdominal ganglia samples, the complete open reading frame of a putative sodium channel gene has firstly been cloned and molecular properties of the associated peptide have been analyzed. The open reading frame of the gene has a length of 5793 bp that encodes for the synthesis of a peptide, with 1930 amino acids, that is 82% similar to the α-peptide of a sodium channel in a neighboring species, Cancer borealis. The transmembrane topology analysis of the crayfish peptide indicated a pattern of four folding domains with several transmembrane segments, as observed in other known voltage-gated sodium channels. Upon analysis of the obtained sequence, functional regions of the putative sodium channel responsible for the selectivity filter, inactivation gate, voltage sensor, and phosphorylation have been predicted. The expression level of the putative sodium channel gene, as defined by a qPCR method, was measured and found to be the highest in nervous tissue. PMID:27032955

  18. A multistep bioinformatic approach detects putative regulatory elements in gene promoters

    PubMed Central

    Bortoluzzi, Stefania; Coppe, Alessandro; Bisognin, Andrea; Pizzi, Cinzia; Danieli, Gian Antonio

    2005-01-01

    Background Searching for approximate patterns in large promoter sequences frequently produces an exceedingly high numbers of results. Our aim was to exploit biological knowledge for definition of a sheltered search space and of appropriate search parameters, in order to develop a method for identification of a tractable number of sequence motifs. Results Novel software (COOP) was developed for extraction of sequence motifs, based on clustering of exact or approximate patterns according to the frequency of their overlapping occurrences. Genomic sequences of 1 Kb upstream of 91 genes differentially expressed and/or encoding proteins with relevant function in adult human retina were analyzed. Methodology and results were tested by analysing 1,000 groups of putatively unrelated sequences, randomly selected among 17,156 human gene promoters. When applied to a sample of human promoters, the method identified 279 putative motifs frequently occurring in retina promoters sequences. Most of them are localized in the proximal portion of promoters, less variable in central region than in lateral regions and similar to known regulatory sequences. COOP software and reference manual are freely available upon request to the Authors. Conclusion The approach described in this paper seems effective for identifying a tractable number of sequence motifs with putative regulatory role. PMID:15904489

  19. Molecular cloning and functional characterization of a putative sulfite oxidase (SO) ortholog from Nicotiana benthamiana.

    PubMed

    Xia, Zongliang; Su, Xinhong; Wu, Jianyu; Wu, Ke; Zhang, Hua

    2012-03-01

    Sulfite oxidase (SO) catalyzes the oxidation of sulfite to sulfate and thus has important roles in diverse metabolic processes. However, systematic molecular and functional investigations on the putative SO from tobacco (Nicotiana benthamiana) have hitherto not been reported. In this work, a full-length cDNA encoding putative sulfite oxidase from N. benthamiana (NbSO) was isolated. The deduced NbSO protein shares high homology and typical structural features with other species SOs. Phylogenetic analysis indicates that NbSO cDNA clone encodes a tobacco SO isoform. Southern blot analysis suggests that NbSO is a single-copy gene in the N. benthamiana genome. The NbSO transcript levels were higher in aerial tissues and were up-regulated in N. benthamiana during sulfite stress. Reducing the SO expression levels through virus-induced gene silencing caused a substantial accumulation in sulfite content and less sulfate accumulation in N. benthamiana leaves when exposed to sulfite stress, and thus resulted in decreased tolerance to sulfite stress. Taken together, this study improves our understanding on the molecular and functional properties of plant SO and provides genetic evidence on the involvement of SO in sulfite detoxification in a sulfite-oxidizing manner in N. benthamiana plants. PMID:21667106

  20. Identification of putative rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of bioinformatics approaches.

    PubMed

    Voxeur, Aline; André, Aurélie; Breton, Christelle; Lerouge, Patrice

    2012-01-01

    Rhamnogalacturonan-II (RG-II) is a complex plant cell wall polysaccharide that is composed of an α(1,4)-linked homogalacturonan backbone substituted with four side chains. It exists in the cell wall in the form of a dimer that is cross-linked by a borate di-ester. Despite its highly complex structure, RG-II is evolutionarily conserved in the plant kingdom suggesting that this polymer has fundamental functions in the primary wall organisation. In this study, we have set up a bioinformatics strategy aimed at identifying putative glycosyltransferases (GTs) involved in RG-II biosynthesis. This strategy is based on the selection of candidate genes encoding type II membrane proteins that are tightly coexpressed in both rice and Arabidopsis with previously characterised genes encoding enzymes involved in the synthesis of RG-II and exhibiting an up-regulation upon isoxaben treatment. This study results in the final selection of 26 putative Arabidopsis GTs, including 10 sequences already classified in the CAZy database. Among these CAZy sequences, the screening protocol allowed the selection of α-galacturonosyltransferases involved in the synthesis of α4-GalA oligogalacturonides present in both homogalacturonans and RG-II, and two sialyltransferase-like sequences previously proposed to be involved in the transfer of Kdo and/or Dha on the pectic backbone of RG-II. In addition, 16 non-CAZy GT sequences were retrieved in the present study. Four of them exhibited a GT-A fold. The remaining sequences harbored a GT-B like fold and a fucosyltransferase signature. Based on homologies with glycosyltransferases of known functions, putative roles in the RG-II biosynthesis are proposed for some GT candidates. PMID:23272088

  1. Megaplasmids encode differing combinations of lantibiotics in Streptococcus salivarius.

    PubMed

    Wescombe, Philip A; Burton, Jeremy P; Cadieux, Peter A; Klesse, Nikolai A; Hyink, Otto; Heng, Nicholas C K; Chilcott, Chris N; Reid, Gregor; Tagg, John R

    2006-10-01

    Streptococcus salivarius strains commonly produce bacteriocins as putative anti-competitor or signalling molecules. Here we report that bacteriocin production by the oral probiotic strain S. salivarius K12 is encoded by a large (ca. 190 kb) plasmid. Oral cavity transmission of the plasmid from strain K12 to a plasmid-negative variant of this bacterium was demonstrated in two subjects. Tests of additional S. salivarius strains showed large (up to ca. 220 kb) plasmids present in bacteriocin-producing isolates. Various combinations (up to 3 per plasmid) of loci encoding the known streptococcal lantibiotics salivaricin A, salivaricin B, streptin and SA-FF22 were localised to these plasmids. Since all bacteriocin-producing strains of S. salivarius tested to date appear to harbour plasmids, it appears that they may function as mobile repositories for bacteriocin loci, especially those of the lantibiotic class. PMID:16871420

  2. The Biogeography of Putative Microbial Antibiotic Production.

    PubMed

    Morlon, Hélène; O'Connor, Timothy K; Bryant, Jessica A; Charkoudian, Louise K; Docherty, Kathryn M; Jones, Evan; Kembel, Steven W; Green, Jessica L; Bohannan, Brendan J M

    2015-01-01

    Understanding patterns in the distribution and abundance of functional traits across a landscape is of fundamental importance to ecology. Mapping these distributions is particularly challenging for species-rich groups with sparse trait measurement coverage, such as flowering plants, insects, and microorganisms. Here, we use likelihood-based character reconstruction to infer and analyze the spatial distribution of unmeasured traits. We apply this framework to a microbial dataset comprised of 11,732 ketosynthase alpha gene sequences extracted from 144 soil samples from three continents to document the spatial distribution of putative microbial polyketide antibiotic production. Antibiotic production is a key competitive strategy for soil microbial survival and performance. Additionally, novel antibiotic discovery is highly relevant to human health, making natural antibiotic production by soil microorganisms a major target for bioprospecting. Our comparison of trait-based biogeographical patterns to patterns based on taxonomy and phylogeny is relevant to our basic understanding of microbial biogeography as well as the pressing need for new antibiotics. PMID:26102275

  3. The Biogeography of Putative Microbial Antibiotic Production

    PubMed Central

    Bryant, Jessica A.; Charkoudian, Louise K.; Docherty, Kathryn M.; Jones, Evan; Kembel, Steven W.; Green, Jessica L.; Bohannan, Brendan J. M.

    2015-01-01

    Understanding patterns in the distribution and abundance of functional traits across a landscape is of fundamental importance to ecology. Mapping these distributions is particularly challenging for species-rich groups with sparse trait measurement coverage, such as flowering plants, insects, and microorganisms. Here, we use likelihood-based character reconstruction to infer and analyze the spatial distribution of unmeasured traits. We apply this framework to a microbial dataset comprised of 11,732 ketosynthase alpha gene sequences extracted from 144 soil samples from three continents to document the spatial distribution of putative microbial polyketide antibiotic production. Antibiotic production is a key competitive strategy for soil microbial survival and performance. Additionally, novel antibiotic discovery is highly relevant to human health, making natural antibiotic production by soil microorganisms a major target for bioprospecting. Our comparison of trait-based biogeographical patterns to patterns based on taxonomy and phylogeny is relevant to our basic understanding of microbial biogeography as well as the pressing need for new antibiotics. PMID:26102275

  4. Magnetic Pulse Affects a Putative Magnetoreceptor Mechanism

    PubMed Central

    Davila, Alfonso F.; Winklhofer, Michael; Shcherbakov, Valera P.; Petersen, Nikolai

    2005-01-01

    Clusters of superparamagnetic (SP) magnetite crystals have recently been identified in free nerve endings in the upper-beak skin of homing pigeons and are interpreted as being part of a putative magnetoreceptor system. Motivated by these findings, we developed a physical model that accurately predicts the dynamics of interacting SP clusters in a magnetic field. The main predictions are: 1), under a magnetic field, a group of SP clusters self-assembles into a chain-like structure that behaves like a compass needle under slowly rotating fields; 2), in a frequently changing field as encountered by a moving bird, a stacked chain is a structurally more stable configuration than a single chain; 3), chain-like structures of SP clusters disrupt under strong fields applied at oblique angles; and 4), reassemble on a timescale of hours to days (assuming a viscosity of the cell plasma η ∼ 1 P). Our results offer a novel mechanism for magnetic field perception and are in agreement with the response of birds observed after magnetic-pulse treatments, which have been conducted in the past to specifically test if ferrimagnetic material is involved in magnetoreception, but which have defied explanation so far. Our theoretical results are supported by experiments on a technical SP model system using a high-speed camera. We also offer new predictions that can be tested experimentally. PMID:15863473

  5. Toddlers’ Duration of Attention towards Putative Threat

    PubMed Central

    Kiel, Elizabeth J.; Buss, Kristin A.

    2010-01-01

    Although individual differences in reactions to novelty in the toddler years have been consistently linked to risk for developing anxious behavior, toddlers’ attention towards a novel, putatively threatening stimulus while in the presence of other enjoyable activities has rarely been examined as a precursor to such risk. The current study examined how attention towards an angry-looking gorilla mask in a room with alternative opportunities for play in 24-month-old toddlers predicted social inhibition when children entered kindergarten. Analyses examined attention to threat above and beyond and in interaction with both proximity to the mask and fear of novelty observed in other situations. Attention to threat interacted with proximity to the mask to predict social inhibition, such that attention to threat most strongly predicted social inhibition when toddlers stayed furthest from the mask. This relation occurred above and beyond the predictive relation between fear of novelty and social inhibition. Results are discussed within the broader literature of anxiety development and attentional processes in young children. PMID:21373365

  6. Biogenic Origin for Earth's Oldest Putative Microfossils

    SciTech Connect

    De Gregorio, B.; Sharp, T; Flynn, G; Wirick, S; Hervig, R

    2009-01-01

    Carbonaceous microbe-like features preserved within a local chert unit of the 3.5 Ga old Apex Basalt in Western Australia may represent some of the oldest evidence of life on Earth. However, the biogenicity of these putative microfossils has been called into question, primarily because the sample collection locality is a black, carbon-rich, brecciated chert dike representing an Archean submarine hydrothermal spring, suggesting a formation via an abiotic organic synthesis mechanism. Here we describe the macromolecular hydrocarbon structure, carbon bonding, functional group chemistry, and biotic element abundance of carbonaceous matter associated with these filamentous features. These characteristics are similar to those of biogenic kerogen from the ca. 1.9 Ga old Gunflint Formation. Although an abiotic origin cannot be entirely ruled out, it is unlikely that known abiotic synthesis mechanisms could recreate both the structural and compositional complexity of this ancient carbonaceous matter. Thus, we find that a biogenic origin for this material is more likely, implying that the Apex microbe-like features represent authentic biogenic organic matter.

  7. Prosodic Encoding in Silent Reading.

    ERIC Educational Resources Information Center

    Wilkenfeld, Deborah

    In silent reading, short-memory tasks, such as semantic and syntactic processing, require a stage of phonetic encoding between visual representation and the actual extraction of meaning, and this encoding includes prosodic as well as segmental features. To test for this suprasegmental coding, an experiment was conducted in which subjects were…

  8. Anomalous cell surface structure of sickle cell anemia erythrocytes as demonstrated by cell surface labeling and endo-beta-galactosidase treatment

    SciTech Connect

    Fukuda, M.; Fukuda, M.N.; Hakomori, S.; Papayannopoulou, T.

    1981-01-01

    Erythrocyte surface glycoproteins from patients with various types of sickle cell anemia have been analyzed and compared with those from normal individuals. By hemagglutination with various anti-carbohydrate antibodies, sickle cells showed profound increase of i antigens and moderate increase of GlcNAc beta 1 leads to 3Gal beta 1 leads to 3 Glc structure, whereas antigenicity toward globosidic structure was unchanged. In parallel to these findings, erythrocytes of sickle cell patients have additional sialylated lactosaminoglycan in Band 3. Thus, it can be concluded that erythrocytes of sickle cell patients are characterized by an altered cell surface structure which does not appear to be due to topographical changes of cell surface membrane. It is possible that the anemia or the ''stress'' hematopoiesis in these patients is responsible for these changes.

  9. Peri-encoding predictors of memory encoding and consolidation.

    PubMed

    Cohen, Noga; Pell, Liat; Edelson, Micah G; Ben-Yakov, Aya; Pine, Alex; Dudai, Yadin

    2015-03-01

    We review reports of brain activations that occur immediately prior to the onset or following the offset of to-be-remembered information and can predict subsequent mnemonic success. Memory-predictive pre-encoding processes, occurring from fractions of a second to minutes prior to event onset, are mainly associated with activations in the medial temporal lobe (MTL), amygdala and midbrain, and with enhanced theta oscillations. These activations may be considered as the neural correlates of one or more cognitive operations, including contextual processing, attention, and the engagement of distinct computational modes associated with prior encoding or retrieval. Post-encoding activations that correlate with subsequent memory performance are mainly observed in the MTL, sensory cortices and frontal regions. These activations may reflect binding of elements of the encoded information and initiation of memory consolidation. In all, the findings reviewed here illustrate the importance of brain states in the immediate peri-encoding time windows in determining encoding success. Understanding these brain states and their specific effects on memory may lead to optimization of the encoding of desired memories and mitigation of undesired ones. PMID:25446944

  10. Information encoder/decoder using chaotic systems

    DOEpatents

    Miller, Samuel Lee; Miller, William Michael; McWhorter, Paul Jackson

    1997-01-01

    The present invention discloses a chaotic system-based information encoder and decoder that operates according to a relationship defining a chaotic system. Encoder input signals modify the dynamics of the chaotic system comprising the encoder. The modifications result in chaotic, encoder output signals that contain the encoder input signals encoded within them. The encoder output signals are then capable of secure transmissions using conventional transmission techniques. A decoder receives the encoder output signals (i.e., decoder input signals) and inverts the dynamics of the encoding system to directly reconstruct the original encoder input signals.

  11. Information encoder/decoder using chaotic systems

    DOEpatents

    Miller, S.L.; Miller, W.M.; McWhorter, P.J.

    1997-10-21

    The present invention discloses a chaotic system-based information encoder and decoder that operates according to a relationship defining a chaotic system. Encoder input signals modify the dynamics of the chaotic system comprising the encoder. The modifications result in chaotic, encoder output signals that contain the encoder input signals encoded within them. The encoder output signals are then capable of secure transmissions using conventional transmission techniques. A decoder receives the encoder output signals (i.e., decoder input signals) and inverts the dynamics of the encoding system to directly reconstruct the original encoder input signals. 32 figs.

  12. A protein fragment of streptococcal cell surface antigen I/II which prevents adhesion of Streptococcus mutans.

    PubMed Central

    Munro, G H; Evans, P; Todryk, S; Buckett, P; Kelly, C G; Lehner, T

    1993-01-01

    Attachment of Streptococcus mutans to the tooth surface involves a cell surface protein with an M(r) of 185,000, termed streptococcal antigen (SA) I/II. Four overlapping fragments of the gene encoding SA I/II were amplified by polymerase chain reaction, cloned, and expressed in Escherichia coli. The recombinant polypeptides were assayed for adhesion-binding activity to salivary receptors and for recognition by a panel of monoclonal antibodies (MAbs) raised against SA I/II. Two of the MAbs which are known to prevent colonization of S. mutans in vivo bound the recombinant polypeptide comprising residues 816 to 1161. In vitro adhesion of S. mutans to saliva-coated hydroxyapatite beads was also inhibited specifically by a polypeptide (residues 816 to 1213) encompassing the same region. The evidence from the MAbs preventing colonization of S. mutans and the adherence inhibition assay suggests that an adhesion-binding activity resides within the portion of SA I/II comprising residues 816 to 1213, which is highly conserved among oral streptococcal species. Images PMID:7691754

  13. Improving the performance of industrial ethanol-producing yeast by expressing the aspartyl protease on the cell surface.

    PubMed

    Guo, Zhong-peng; Zhang, Liang; Ding, Zhong-yang; Wang, Zheng-Xiang; Shi, Gui-Yang

    2010-12-01

    The yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. The PEP4 gene, encoding a vacuolar aspartyl protease in Saccharomyces cerevisiae, was either secretively or cell-surface anchored expressed in industrial ethanol-producing S. cerevisiae. The obtained recombinant strains APA (expressing the protease secretively) and APB (expressing the protease on the cell wall) were studied under ethanol fermentation conditions in feed barley cultures. The effects of expression of the protease on product formation, growth and cell protein content were measured. The biomass yield of the wild-type was clearly lower than that of the recombinant strains (0.578 ± 0.12 g biomass/g glucose for APA and 0.582 ± 0.08 g biomass/g glucose for APB). In addition, nearly 98-99% of the theoretical maximum level of ethanol yield was achieved (relative to the amount of substrate consumed) for the recombinant strains, while limiting the nitrogen source resulted in dissatisfactory fermentation for the wild-type and more than 30 g/l residual sugar was detected at the end of fermentation. In addition, higher growth rate, viability and lower yields of byproducts such as glycerol and pyruvic acid for recombinant strains were observed. Expressing acid protease can be expected to lead to a significant increase in ethanol productivity. PMID:20737427

  14. Analysis of human hyaluronan synthase gene transcriptional regulation and downstream hyaluronan cell surface receptor mobility in myofibroblast differentiation.

    PubMed

    Midgley, Adam C; Bowen, Timothy

    2015-01-01

    The ubiquitous extracellular glycosaminoglycan hyaluronan (HA) is a polymer composed of repeated disaccharide units of alternating D-glucuronic acid and D-N-acetylglucosamine residues linked via alternating β-1,4 and β-1,3 glycosidic bonds. Emerging data continue to reveal functions attributable to HA in a variety of physiological and pathological contexts. Defining the mechanisms regulating expression of the human hyaluronan synthase (HAS) genes that encode the corresponding HA-synthesizing HAS enzymes is therefore important in the context of HA biology in health and disease. We describe here methods to analyze transcriptional regulation of the HAS and HAS2-antisense RNA 1 genes. Elucidation of mechanisms of HA interaction with receptors such as the cell surface molecule CD44 is also key to understanding HA function. To this end, we provide protocols for fluorescent recovery after photobleaching analysis of CD44 membrane dynamics in the process of fibroblast to myofibroblast differentiation, a phenotypic transition that is common to the pathology of fibrosis of large organs such as the liver and kidney. PMID:25325984

  15. Remote Control of Tissue Interactions via Engineered Photo-switchable Cell Surfaces

    NASA Astrophysics Data System (ADS)

    Luo, Wei; Pulsipher, Abigail; Dutta, Debjit; Lamb, Brian M.; Yousaf, Muhammad N.

    2014-09-01

    We report a general cell surface molecular engineering strategy via liposome fusion delivery to create a dual photo-active and bio-orthogonal cell surface for remote controlled spatial and temporal manipulation of microtissue assembly and disassembly. Cell surface tailoring of chemoselective functional groups was achieved by a liposome fusion delivery method and quantified by flow cytometry and characterized by a new cell surface lipid pull down mass spectrometry strategy. Dynamic co-culture spheroid tissue assembly in solution and co-culture tissue multilayer assembly on materials was demonstrated by an intercellular photo-oxime ligation that could be remotely cleaved and disassembled on demand. Spatial and temporal control of microtissue structures containing multiple cell types was demonstrated by the generation of patterned multilayers for controlling stem cell differentiation. Remote control of cell interactions via cell surface engineering that allows for real-time manipulation of tissue dynamics may provide tools with the scope to answer fundamental questions of cell communication and initiate new biotechnologies ranging from imaging probes to drug delivery vehicles to regenerative medicine, inexpensive bioreactor technology and tissue engineering therapies.

  16. Remote control of tissue interactions via engineered photo-switchable cell surfaces.

    PubMed

    Luo, Wei; Pulsipher, Abigail; Dutta, Debjit; Lamb, Brian M; Yousaf, Muhammad N

    2014-01-01

    We report a general cell surface molecular engineering strategy via liposome fusion delivery to create a dual photo-active and bio-orthogonal cell surface for remote controlled spatial and temporal manipulation of microtissue assembly and disassembly. Cell surface tailoring of chemoselective functional groups was achieved by a liposome fusion delivery method and quantified by flow cytometry and characterized by a new cell surface lipid pull down mass spectrometry strategy. Dynamic co-culture spheroid tissue assembly in solution and co-culture tissue multilayer assembly on materials was demonstrated by an intercellular photo-oxime ligation that could be remotely cleaved and disassembled on demand. Spatial and temporal control of microtissue structures containing multiple cell types was demonstrated by the generation of patterned multilayers for controlling stem cell differentiation. Remote control of cell interactions via cell surface engineering that allows for real-time manipulation of tissue dynamics may provide tools with the scope to answer fundamental questions of cell communication and initiate new biotechnologies ranging from imaging probes to drug delivery vehicles to regenerative medicine, inexpensive bioreactor technology and tissue engineering therapies. PMID:25204325

  17. Rapidly rendering cells phagocytic through a cell surface display technique and concurrent Rac activation.

    PubMed

    Onuma, Hiroki; Komatsu, Toru; Arita, Makoto; Hanaoka, Kenjiro; Ueno, Tasuku; Terai, Takuya; Nagano, Tetsuo; Inoue, Takanari

    2014-07-15

    Cell surfaces represent a platform through which extracellular signals that determine diverse cellular processes, including migration, division, adhesion, and phagocytosis, are transduced. Techniques to rapidly reconfigure the surface properties of living cells should thus offer the ability to harness these cellular functions. Although the molecular mechanism of phagocytosis is well characterized, the minimal molecular players that are sufficient to activate this elaborate process remain elusive. We developed and implemented a technique to present a molecule of interest at the cell surface in an inducible manner on a time scale of minutes. We simultaneously induced the cell surface display of the C2 domain of milk fat globule epidermal growth factor factor 8 (MFG-E8) and activated the intracellular small guanosine triphosphatase Rac, which stimulates actin polymerization at the cell periphery. The C2 domain binds to phosphatidylserine, a lipid exposed on the surface of apoptotic cells. By integrating the stimulation of these two processes, we converted HeLa cells into a phagocytic cell line that bound to and engulfed apoptotic human Jurkat cells. Inducing either the cell surface display of the C2 domain or activating Rac alone was not sufficient to stimulate phagocytosis, which suggests that attachment to the target cell and actin reorganization together constitute the minimal molecular events that are needed to induce phagocytosis. This cell surface display technique might be useful as part of a targeted, cell-based therapy in which unwanted cells with characteristic surface molecules could be rapidly consumed by engineered cells. PMID:25028719

  18. Rapidly rendering cells phagocytic through a cell-surface display technique and concurrent Rac activation

    PubMed Central

    Onuma, Hiroki; Arita, Makoto; Hanaoka, Kenjiro; Ueno, Tasuku; Terai, Takuya; Nagano, Tetsuo

    2014-01-01

    Cell surfaces represent a platform through which extracellular signals that determine diverse cellular processes, including migration, division, adhesion, and phagocytosis, are transduced. Techniques to rapidly reconfigure the surface properties of living cells should thus offer the ability to harness these cellular functions. Although the molecular mechanism of phagocytosis is well-characterized, the minimal molecular players that are sufficient to activate this elaborate process remain elusive. We developed and implemented a technique to present a molecule of interest at the cell surface in an inducible manner on a timescale of minutes. We simultaneously induced the cell-surface display of the C2 domain of milk fat globule-EGF factor 8 (MFG-E8) and activated the intracellular small guanosine triphosphatase Rac, which stimulates actin polymerization at the cell periphery. The C2 domain binds to phosphatidylserine, a lipid exposed on the surface of apoptotic cells. By integrating the stimulation of these two processes, we converted HeLa cells into a phagocytic cell line that bound to and engulfed apoptotic human Jurkat cells. Inducing either the cell-surface display of the C2 domain or activating Rac alone was not sufficient to stimulate phagocytosis, which suggests that attachment to the target cell and actin reorganization together constitute the minimal molecular events that are needed to induce phagocytosis. This cell-surface display technique might be useful as part of a targeted, cell-based therapy in which unwanted cells with characteristic surface molecules could be rapidly consumed by engineered cells. PMID:25028719

  19. A sensitive electrochemiluminescence cytosensor for quantitative evaluation of epidermal growth factor receptor expressed on cell surfaces.

    PubMed

    Tang, Yanjuan; Zhang, Shaolian; Wen, Qingqing; Huang, Hongxing; Yang, Peihui

    2015-06-30

    A sensitive electrochemiluminescence (ECL) strategy for evaluating the epidermal growth factor receptor (EGFR) expression level on cell surfaces was designed by integrating the specific recognition of EGFR expressed on MCF-7 cell surfaces with an epidermal growth factor (EGF)-funtionalized CdS quantum dots (CdSQDs)-capped magnetic bead (MB) probe. The high sensitivity of ECL probe of EGF-funtionalized CdSQD-capped-MB was used for competitive recognition with EGFR expressed on cell surfaces with recombinant EGFR protein. The changes of ECL intensity depended on both the cell number and the expression level of EGFR receptor on cell surfaces. A wide linear response to cells ranging from 80 to 4×10(6)cellsmL(-1) with a detection limit of 40cellsmL(-1) was obtained. The EGF-cytosensor was used to evaluate EGFR expression levels on MCF-7 cells, and the average number of EGFR receptor on single MCF-7 cells was 1.35×10(5) with the relative standard deviation of 4.3%. This strategy was further used for in-situ and real-time evaluating EGFR receptor expressed on cell surfaces in response to drugs stimulation at different concentration and incubation time. The proposed method provided potential applications in the detection of receptors on cancer cells and anticancer drugs screening. PMID:26041531

  20. Cell surface of sea urchin micromeres and primary mesenchyme. [Arbacia punctulata; Strongylocentrotus drobachiensis; Strongylocentrotus purpuratus

    SciTech Connect

    DeSimone, D.W.

    1985-01-01

    The cell surface and extracellular matrix (ECM) of the sea urchin embryo were studied during the early morphogenetic events involved in the differentiation of the micromere cell lineage. Sixteen-cell and early cleavage stage blastomeres were isolated and the protein composition of their cell surfaces examined by /sup 125/I-labelling followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Micromere-specific cell surface proteins are reported for Arbacia punctulata, Strongylocentrotus droebachiensis, and Strongylocentrotus purpuratus. Cell surface glycoproteins were characterized on the basis of lectin binding specificity with a novel lectin affinity transfer technique. Using this procedure, cell-type specific surface proteins, which are also lectin-binding specific, can be detected. In addition, fluorescein conjugated lectins were microinjected into the blastocoels of living S. drobachiensis and Lytechinus pictus embryos and the patterns of lectin bindings observed by fluorescence microscopy. The evidence presented in this thesis suggests that the differentiation of the primary mesenchyme cells is correlated with changes in the molecular composition of the cell-surface and the ECM.

  1. Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells.

    PubMed

    Xiong, Jimin; Menicanin, Danijela; Zilm, Peter S; Marino, Victor; Bartold, P Mark; Gronthos, Stan

    2016-01-01

    The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein "spots" were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population. PMID:27579043

  2. Characterization of fucosyltransferase activity during mouse spermatogenesis: Evidence for a cell surface fucosyltransferase

    SciTech Connect

    Cardullo, R.A.; Armant, D.R.; Millette, C.F. )

    1989-02-21

    Fucosyltransferase activity was quantified in mouse germ cells at different stages of spermatogenesis. Specifically, fucosyltransferase activities of pachytene spermatocytes, round spermatids, and cauda epididymal sperm were compared. Fucosyltranferase activity of mixed germ cells displayed an apparent V{sub max} of 17 pmol (mg of protein){sup {minus}1} min{sup {minus}1} and an apparent K{sub m} of approximately 13 {mu}M for GDP-L-({sup 14}C)fucose in the presence of saturating amounts of asialofetuin at 33{degree}C. Under these conditions, cellular fucosyltransferase activity was found to increase during spermatogenesis. In agreement with assays of intact cells, examination of subcellular fractions indicated that a large fraction of fucosyltransferase activity was associated with the cell surface. The fraction of fucosyltransferase activity that was associated with the cell surface progressively increased throughout spermatogenesis and epididymal maturation so that nearly all of the fucosyltransferase in epididymal sperm was on the cell surface. Specifically, by comparison of activities in the presence and absence of the detergent NP-40, the fraction of fucosyltransferase activity that was associated with the cell surface in pachytene spermatocytes, round spermatids, and epididymal sperm was 0.36, 0.5, and 0.85, respectively. These results suggest that a cell surface fucosyltransferase may be important during differentiation of spermatogenic cells in the testis as well as during epididymal maturation and fertilization.

  3. Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells

    PubMed Central

    Xiong, Jimin; Menicanin, Danijela; Marino, Victor

    2016-01-01

    The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein “spots” were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population. PMID:27579043

  4. Molecular Cloning and Analysis of a Putative Siderophore ABC Transporter from Staphylococcus aureus

    PubMed Central

    Morrissey, Julie A.; Cockayne, Alan; Hill, Philip J.; Williams, Paul

    2000-01-01

    From a mass-excised Staphylococcus aureus λZapII expression library, we cloned an operon encoding a novel ABC transporter with significant homology to bacterial siderophore transporter systems. The operon encodes four genes designated sstA, -B, -C, and -D encoding two putative cytoplasmic membrane proteins (sstA and sstB), an ATPase (sstC), and a membrane-bound 38-kDa lipoprotein (sstD). The sst operon is preceded by two putative Fur boxes, which indicated that expression of the sst operon was likely to be iron dependent. SstD was overexpressed in Escherichia coli, purified by Triton X-114 phase partitioning, and used to generate monospecific antisera in rats. Immunoblotting studies located SstD in the membrane fraction of S. aureus and showed that expression of the lipoprotein was reduced under iron-rich growth conditions. Triton X-114 partitioning studies on isolated membranes provided additional biochemical evidence that SstD in S. aureus is a lipoprotein. Immunoreactive polypeptides of approximately 38 kDa were detected in a wide range of staphylococcal species, but no antigenic homolog was detected in Bacillus subtilis. Expression of SstD in vivo was confirmed by immunoblotting studies with S. aureus recovered from a rat intraperitoneal chamber implant model. To further define the contribution of SstD in promoting growth of S. aureus in vitro and in vivo, we used antisense RNA technology to modulate expression of SstD. Expression of antisense sstD RNA in S. aureus resulted in a decrease in SstD expression under both iron-rich and iron-restricted growth conditions. However, this reduction in SstD levels did not affect the growth of S. aureus in vitro in an iron-limited growth medium or when grown in an intraperitoneal rat chamber implant model in vivo. PMID:11035736

  5. Putative Lineage of Novel African Usutu Virus, Central Europe

    PubMed Central

    Cadar, Daniel; Bosch, Stefan; Jöst, Hanna; Börstler, Jessica; Garigliany, Mutien-Marie; Becker, Norbert

    2015-01-01

    We characterized the complete genome of a putative novel Usutu virus (USUV) strain (Usutu-BONN) detected in a dead blackbird from Germany. Genomic analysis revealed several unique amino acid substitutions among the polyprotein gene. Phylogenetic analyses demonstrated that Usutu-BONN constitutes a putative novel African USUV lineage, which was probably recently introduced to central Europe. PMID:26291923

  6. Identification of Putative Fallopian Tube Stem Cells

    PubMed Central

    Snegovskikh, Victoria; Mutlu, Levent; Massasa, Effi

    2014-01-01

    Stem cells are used to repair and regenerate multiple tissues in the adult. We have previously shown that stem cells play a significant role in mediating endometrial repair and tissue regeneration. We hypothesized that the oviduct may possess a similar population of stem cells that contribute to the maintenance of this tissue. Here we identify label-retaining cells (LRCs) in the murine oviduct which indicate the presence of a stem/progenitor cell population in this tissue as well. Two-day-old CD-1 mice were injected intraperitoneally with 5-bromo-2-deoxyuridine (BrdU) or vehicle control. Female animals (n = 36 for each group) were killed at 6 weeks post injection. Reproductive tracts were removed, specimens were embedded in paraffin, and 5-µ sections were prepared. Oviduct was identified by hematoxylin and eosin staining and morphology. Immunofluorescence studies were performed on serial sections tissues (n = 12 per animal) using antibodies against BrdU. Confocal microscopy was used to identify 4′,6-diamidino-2-phenylindole (DAPI)- and BrdU-stained nuclei. In the group of mice exposed to BrdU, we identified a population of LRCs in all specimens and not in controls. The putative stem cells are located at the base of each villi, suggesting the location of the stem cell niche. The number of DAPI-stained nuclei divided by the number of LRCs; LRCs constituted 0.5% of all nucleated cells. The oviduct contains a population of progenitor cells, likely used in the repair and regeneration of fallopian tube. Defective or insufficient stem cell reserve may underlie common tubal diseases, including hydrosalpinx and ectopic pregnancy. PMID:25305130

  7. TnphoA and TnphoA' elements for making and switching fusions for study of transcription, translation, and cell surface localization.

    PubMed Central

    Wilmes-Riesenberg, M R; Wanner, B L

    1992-01-01

    We describe a set of elements based on the transposon TnphoA for making transcriptional fusions to the lacZ gene and for making translational fusions to the phoA or lacZ structural gene. Each element can be switched, one for another, by homologous recombination, thereby allowing testing for transcription, translation, or cell surface localization determinants at the same site within a gene. We describe three kinds of elements for making each fusion type. Two kinds are transposition proficient (Tnp+): one encodes kanamycin resistance, and the other encodes tetracycline resistance. The third kind is transposition defective (Tnp-) and encodes kanamycin resistance. In addition, we describe one Tnp- element that has no reporter gene and encodes chloramphenicol resistance; this element is used primarily as a tool to aid in switching fusions. Switching is efficient because each element has in common 254 bp of DNA at the phoA end and 187 bp (or more) of DNA at the IS50R end of TnphoA, and switching is straightforward because individual elements encode different drug resistances. Thus, switched recombinants can be selected as drug-resistant transductants, and they can be recognized as ones that have lost the parental drug resistance and fusion phenotype. Further, switching Tnp+ elements to Tnp- elements reduces problems due to transposition that can arise in P1 crosses or cloning experiments. Some TnphoA and TnphoA' elements cause polar mutations, while others provide an outward promoter for downstream transcription. This feature is especially useful in the determination of operon structures. Strategies for the use of TnphoA and TnphoA' elements in gene analysis are also described. PMID:1378054

  8. Interaction of Biofunctionalized Nanoparticles with Receptors on Cell Surfaces: MC Simulations

    NASA Astrophysics Data System (ADS)

    Dormidontova, Elena; Wang, Shihu

    2015-03-01

    One of the areas of active development of modern nanomedicine is drug/gene delivery and imaging application of nanoparticles functionalized by ligands, aptamers or antibodies capable of specific interactions with cell surface receptors. Being a complex multifunctional system different structural aspects of nanoparticles affect their interactions with cell surfaces and the surface properties of cells can be different (e.g. density, distribution and mobility of receptors). Computer simulations allow a systematic investigation of the influence of multiple factors and provide a unified platform for the comparison. Using Monte Carlo simulations we investigate the influence of the nanoparticle properties (nanoparticle size, polymer tether length, polydispersity, density, ligand energy, valence and density) on nanoparticle-cell surface interactions and make predictions regarding favorable nanoparticle design for achieving multiple ligand-receptor binding. We will also discuss the implications of nanoparticle design on the selectivity of attachment to cells with high receptor density while ``ignoring'' cells with a low density of receptors.

  9. Significance of Nano- and Microtopography for Cell-Surface Interactions in Orthopaedic Implants

    PubMed Central

    Jäger, M.; Zilkens, C.; Zanger, K.; Krauspe, R.

    2007-01-01

    Cell-surface interactions play a crucial role for biomaterial application in orthopaedics. It is evident that not only the chemical composition of solid substances influence cellular adherence, migration, proliferation and differentiation but also the surface topography of a biomaterial. The progressive application of nanostructured surfaces in medicine has gained increasing interest to improve the cytocompatibility and osteointegration of orthopaedic implants. Therefore, the understanding of cell-surface interactions is of major interest for these substances. In this review, we elucidate the principle mechanisms of nano- and microscale cell-surface interactions in vitro for different cell types onto typical orthopaedic biomaterials such as titanium (Ti), cobalt-chrome-molybdenum (CoCrMo) alloys, stainless steel (SS), as well as synthetic polymers (UHMWPE, XLPE, PEEK, PLLA). In addition, effects of nano- and microscaled particles and their significance in orthopaedics were reviewed. The significance for the cytocompatibility of nanobiomaterials is discussed critically. PMID:18274618

  10. Pharmacological induction of cell surface GRP78 contributes to apoptosis in triple negative breast cancer cells.

    PubMed

    Raiter, Annat; Yerushalmi, Rinat; Hardy, Britta

    2014-11-30

    Breast cancer tumor with triple-negative receptors (estrogen, progesterone and Her 2, receptors) is the most aggressive and deadly subtype, with high rates of disease recurrence and poor survival. Here, we show that induction in cell surface GRP78 by doxorubicin and tunicamycin was associated with CHOP/GADD153 upregulation and increase in apoptosis in triple negative breast cancer tumor cells. GRP78 is a major regulator of the stress induced unfolded protein response pathway and CHOP/GADD153 is a pro-apoptotic transcription factor associated exclusively with stress induced apoptosis. The blocking of cell surface GRP78 by anti-GRP78 antibody prevented apoptosis, suggesting that induction of cell surface GRP78 by doxorubicin and tunicamycin is required for apoptosis. A better understanding of stress induction of apoptotic signaling in triple negative breast cancer cells may help to define new therapeutic strategies. PMID:25360516

  11. Recent advances in yeast cell-surface display technologies for waste biorefineries.

    PubMed

    Liu, Zhuo; Ho, Shih-Hsin; Hasunuma, Tomohisa; Chang, Jo-Shu; Ren, Nan-Qi; Kondo, Akihiko

    2016-09-01

    Waste biorefinery aims to maximize the output of value-added products from various artificial/agricultural wastes by using integrated bioprocesses. To make waste biorefinery economically feasible, it is thus necessary to develop a low-cost, environment-friendly technique to perform simultaneous biodegradation and bioconversion of waste materials. Cell-surface display engineering is a novel, cost-effective technique that can auto-immobilize proteins on the cell exterior of microorganisms, and has been applied for use with waste biofinery. Through tethering different enzymes (e.g., cellulase, lipase, and protease) or metal-binding peptides on cell surfaces, various yeast strains can effectively produce biofuels and biochemicals from sugar/protein-rich waste materials, catalyze waste oils into biodiesels, or retrieve heavy metals from wastewater. This review critically summarizes recent applications of yeast cell-surface display on various types of waste biorefineries, highlighting its potential and future challenges with regard to commercializing this technology. PMID:27039354

  12. Two digital video encoder circuits

    NASA Astrophysics Data System (ADS)

    Eldon, John A.

    1992-11-01

    Central to `multimedia' image processing is the desire to encode computer graphics data into a standard television signal, complete with line, field, and color subcarrier synchronizing information. The numerous incompatibilities between television and computer display standards render this operation far less trivial than it sounds to anyone who hasn't worked with both types of signals. To simplify the task of encoding computer graphics signals into standard NTSC (North America and Japan) or PAL (most of Europe) television format for display, broadcast, or recording, TRW LSI Products Inc. has introduced the two newest members of it multimedia integrated circuit family, the TMC22090 and TMC22190 digital video encoders.

  13. Whole-Genome Survey of the Putative ATP-Binding Cassette Transporter Family Genes in Vitis vinifera

    PubMed Central

    Çakır, Birsen; Kılıçkaya, Ozan

    2013-01-01

    The ATP-binding cassette (ABC) protein superfamily constitutes one of the largest protein families known in plants. In this report, we performed a complete inventory of ABC protein genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with ABC protein members of Arabidopsis thaliana, we identified 135 putative ABC proteins with 1 or 2 NBDs in V. vinifera. Of these, 120 encode intrinsic membrane proteins, and 15 encode proteins missing TMDs. V. vinifera ABC proteins can be divided into 13 subfamilies with 79 “full-size,” 41 “half-size,” and 15 “soluble” putative ABC proteins. The main feature of the Vitis ABC superfamily is the presence of 2 large subfamilies, ABCG (pleiotropic drug resistance and white-brown complex homolog) and ABCC (multidrug resistance-associated protein). We identified orthologs of V. vinifera putative ABC transporters in different species. This work represents the first complete inventory of ABC transporters in V. vinifera. The identification of Vitis ABC transporters and their comparative analysis with the Arabidopsis counterparts revealed a strong conservation between the 2 species. This inventory could help elucidate the biological and physiological functions of these transporters in V. vinifera. PMID:24244377

  14. Analysis of the cell surface expression of cytokine receptors using the surface protein biotinylation method.

    PubMed

    Pavel, Mahmud Arif; Lam, Clarissa; Kashyap, Parul; Salehi-Najafabadi, Zahra; Singh, Gurpreet; Yu, Yong

    2014-01-01

    Cytokines are pleiotropic, low-molecular-weight proteins that regulate the immune responses to infection and inflammation. They stimulate the immune responses by binding to cytokine receptors on the cell plasma membrane. Thus, knowledge of the expression level of particular cytokine receptors on cell surface is crucial for understanding the cytokine function and regulation. One of the techniques to explore the membrane embedded cytokine receptors is cell surface biotinylation. Biotinylated surface proteins can be rapidly purified through the strong interaction between biotin and streptavidin. Here, we describe the procedure for surface biotinylation and purification of biotinylated cytokine receptors for further downstream analysis. PMID:24908305

  15. Hydroxy decenoic acid down regulates gtfB and gtfC expression and prevents Streptococcus mutans adherence to the cell surfaces

    PubMed Central

    2012-01-01

    Background 10-Hydroxy-2-decenoic acid, an unsaturated fatty acid is the most active and unique component to the royal jelly that has antimicrobial properties. Streptococcus mutans is associated with pathogenesis of oral cavity, gingivoperiodontal diseases and bacteremia following dental manipulations. In the oral cavity, S. mutans colonize the soft tissues including tongue, palate, and buccal mucosa. When considering the role of supragingival dental plaque in caries, the proportion of acid producing bacteria (particularly S. mutans), has direct relevance to the pathogenicity of the plaque. The genes that encode glucosyltransferases (gtfs) especially gtfB and gtfC are important in S. mutans colonization and pathogenesis. This study investigated the hydroxy-decenoic acid (HDA) effects on gtfB and gtfC expression and S. mutans adherence to cells surfaces. Methods Streptococcus mutans was treated by different concentrations of HPLC purified HDA supplied by Iran Beekeeping and Veterinary Association. Real time RT-PCR and western blot assays were conducted to evaluate gtfB and gtfC genes transcription and translation before and after HDA treatment. The bacterial attachment to the cell surfaces was evaluated microscopically. Results 500 μg ml-1 of HDA inhibited gtfB and gtfC mRNA transcription and its expression. The same concentration of HDA decreased 60% the adherence of S. mutans to the surface of P19 cells. Conclusion Hydroxy-decenoic acid prevents gtfB and gtfC expression efficiently in the bactericide sub-concentrations and it could effectively reduce S. mutans adherence to the cell surfaces. In the future, therapeutic approaches to affecting S. mutans could be selective and it’s not necessary to put down the oral flora completely. PMID:22839724

  16. Cloning and characterization of ADAM28: evidence for autocatalytic pro-domain removal and for cell surface localization of mature ADAM28.

    PubMed Central

    Howard, L; Maciewicz, R A; Blobel, C P

    2000-01-01

    The metalloprotease disintegrins are a family of membrane-anchored glycoproteins with diverse functions in fertilization, myoblast fusion, neurogenesis and protein ectodomain shedding. Here we report a cDNA sequence, encoding a metalloprotease disintegrin, termed ADAM28 ('a disintegrin and metalloprotease 28'), which was cloned from mouse lung. From protein sequence comparisons, ADAM28 is more closely related to snake venom metalloproteases (SVMPs) than to other ADAMs, and hence may cleave similar substrates to SVMPs, perhaps including components of the extracellular matrix. Northern blot analysis of selected mouse tissues revealed that ADAM28 is expressed highly and in alternatively spliced forms in the epididymis, suggesting a possible role in sperm maturation, and at lower levels in lung. The intracellular maturation of ADAM28 expressed in COS-7 cells resembles that of other ADAMs, in that ADAM28 is made as a precursor and processed to a mature form in a late Golgi compartment of the secretory pathway. Most or all of the mature, and thus presumably catalytically active, form of ADAM28 in COS-7 cells is accessible to cell surface trypsinization, suggesting that ADAM28 functions mainly on the cell surface. A mutation converting the catalytic-site glutamate residue into alanine abolishes pro-domain removal, even though this mutant form of ADAM28 can be transported to the cell surface in a manner similar to the wild-type protein. This suggests that pro-domain removal and maturation of ADAM28 may be, at least in part, autocatalytic. This is in contrast with several other ADAMs, for which furin-like proprotein convertases are involved in pro-domain removal, and in which a glutamate-to-alanine mutation in the catalytic site does not alter pro-domain removal. PMID:10794709

  17. Staphylococcus caprae Strains Carry Determinants Known To Be Involved in Pathogenicity: a Gene Encoding an Autolysin-Binding Fibronectin and the ica Operon Involved in Biofilm Formation

    PubMed Central

    Allignet, Jeanine; Aubert, Sylvie; Dyke, Keith G. H.; El Solh, Nevine

    2001-01-01

    The atlC gene (1,485 bp), encoding an autolysin which binds fibronectin, and the ica operon, involved in biofilm formation, were isolated from the chromosome of an infectious isolate of Staphylococcus caprae and sequenced. AtlC (155 kDa) is similar to the staphylococcal autolysins Atl, AtlE, Aas (48 to 72% amino acid identity) and contains a putative signal peptide of 29 amino acids and two enzymatic centers (N-acetylmuramoyl-l-alanine amidase and endo-β-N-acetylglucosaminidase) interconnected by three imperfect fibronectin-binding repeats. The glycine-tryptophan (GW) motif found in the central and end part of each repeat may serve for cell surface anchoring of AtlC as they do in Listeria monocytogenes. The S. caprae ica operon contains four genes closely related to S. epidermidis and S. aureus icaA, icaB, icaC, and icaD genes (≥ 68% similarity) and is preceded by a gene similar to icaR (≥70% similarity). The polypeptides deduced from the S. caprae ica genes exhibit 67 to 88% amino acid identity to those of S. epidermidis and S. aureus ica genes. The ica operon and icaR gene were analyzed in 14 S. caprae strains from human specimens or goats' milk. Some of the strains produced biofilm, and others did not. All strains carry the ica operon and icaR of the same sizes and in the same relative positions, suggesting that the absence of biofilm formation is not related to the insertion of a mobile element such as an insertion sequence or a transposon. PMID:11159959

  18. A High-Resolution Shape Fitting and Simulation Demonstrated Equatorial Cell Surface Softening during Cytokinesis and Its Promotive Role in Cytokinesis

    PubMed Central

    Koyama, Hiroshi; Umeda, Tamiki; Nakamura, Kazuyuki; Higuchi, Tomoyuki; Kimura, Akatsuki

    2012-01-01

    Different models for animal cell cytokinesis posit that the stiffness of the equatorial cortex is either increased or decreased relative to the stiffness of the polar cortex. A recent work has suggested that the critical cytokinesis signaling complex centralspindlin may reduce the stiffness of the equatorial cortex by inactivating the small GTPase Rac. To determine if such a reduction occurs and if it depends on centralspindlin, we devised a method to estimate cortical bending stiffness with high spatio-temporal resolution from in vivo cell shapes. Using the early Caenorhabditis elegans embryo as a model, we show that the stiffness of the equatorial cell surface is reduced during cytokinesis, whereas the stiffness of the polar cell surface remains stiff. The equatorial reduction of stiffness was compromised in cells with a mutation in the gene encoding the ZEN-4/kinesin-6 subunit of centralspindlin. Theoretical modeling showed that the absence of the equatorial reduction of stiffness could explain the arrest of furrow ingression in the mutant. By contrast, the equatorial reduction of stiffness was sufficient to generate a cleavage furrow even without the constriction force of the contractile ring. In this regime, the contractile ring had a supportive contribution to furrow ingression. We conclude that stiffness is reduced around the equator in a centralspindlin-dependent manner. In addition, computational modeling suggests that proper regulation of stiffness could be sufficient for cleavage furrow ingression. PMID:22359606

  19. Human Cytomegalovirus UL40 Signal Peptide Regulates Cell Surface Expression of the Natural Killer Cell Ligands HLA-E and gpUL18

    PubMed Central

    Prod’homme, Virginie; Tomasec, Peter; Cunningham, Charles; Lemberg, Marius K.; Stanton, Richard J.; McSharry, Brian P.; Wang, Eddie C.Y.; Cuff, Simone; Martoglio, Bruno; Davison, Andrew J.; Braud, Véronique M.; Wilkinson, Gavin W. G.

    2012-01-01

    Human cytomegalovirus (HCMV)-encoded NK cell evasion functions include an MHC-I homologue (UL18) with high affinity for the leukocyte inhibitory receptor LIR-1 (CD85j, ILT2 or LILRB1) and a signal peptide (SPUL40) that acts by upregulating cell surface expression of HLA-E. Detailed characterization of SPUL40 revealed that the N-terminal 14 amino acid residues bestowed TAP-independent upregulation of HLA-E, while c-region sequences delayed processing of SPUL40 by a signal peptide peptidase-type intramembrane protease. Most significantly, the consensus HLA-E-binding epitope within SPUL40 was shown to promote cell surface expression of both HLA-E and gpUL18. UL40 was found to possess two transcription start sites, with utilization of the downstream site resulting in translational being initiated within the HLA-E-binding epitope (P2). Remarkably, this truncated SPUL40 was functional and retained the capacity to upregulate gpUL18, but not HLA-E. Our findings thus identify an elegant mechanism by which an HCMV signal peptide differentially regulates two distinct NK cell evasion pathways. Moreover, we describe a natural SPUL40 mutant that provides the first example of an HCMV clinical virus with a defect in an NK cell evasion function and exemplifies issues that confront the virus when adapting to immunogenetic diversity in the host. PMID:22345649

  20. Human cytomegalovirus UL40 signal peptide regulates cell surface expression of the NK cell ligands HLA-E and gpUL18.

    PubMed

    Prod'homme, Virginie; Tomasec, Peter; Cunningham, Charles; Lemberg, Marius K; Stanton, Richard J; McSharry, Brian P; Wang, Eddie C Y; Cuff, Simone; Martoglio, Bruno; Davison, Andrew J; Braud, Véronique M; Wilkinson, Gavin W G

    2012-03-15

    Human CMV (HCMV)-encoded NK cell-evasion functions include an MHC class I homolog (UL18) with high affinity for the leukocyte inhibitory receptor-1 (CD85j, ILT2, or LILRB1) and a signal peptide (SP(UL40)) that acts by upregulating cell surface expression of HLA-E. Detailed characterization of SP(UL40) revealed that the N-terminal 14 aa residues bestowed TAP-independent upregulation of HLA-E, whereas C region sequences delayed processing of SP(UL40) by a signal peptide peptidase-type intramembrane protease. Most significantly, the consensus HLA-E-binding epitope within SP(UL40) was shown to promote cell surface expression of both HLA-E and gpUL18. UL40 was found to possess two transcription start sites, with utilization of the downstream site resulting in translation being initiated within the HLA-E-binding epitope (P2). Remarkably, this truncated SP(UL40) was functional and retained the capacity to upregulate gpUL18 but not HLA-E. Thus, our findings identify an elegant mechanism by which an HCMV signal peptide differentially regulates two distinct NK cell-evasion pathways. Moreover, we describe a natural SP(UL40) mutant that provides a clear example of an HCMV clinical virus with a defect in an NK cell-evasion function and exemplifies issues that confront the virus when adapting to immunogenetic diversity in the host. PMID:22345649

  1. A selective defect in IgM antigen receptor synthesis and transport causes loss of cell surface IgM expression on tolerant B lymphocytes.

    PubMed Central

    Bell, S E; Goodnow, C C

    1994-01-01

    To explore the biochemical basis for maintaining immunological tolerance by functional inactivation of self-reactive B lymphocytes, transgenic mice carrying rearranged anti-lysozyme immunoglobulin transgenes and a lysozyme transgene were used as a source of large numbers of tolerant self-reactive B cells. Antigen receptors of the IgD isotype were expressed at normal levels on tolerant B cells, contained the heterodimeric MB1/B29 signalling component of the receptor complex and were structurally indistinguishable from IgD on nontolerant B cells. In contrast, cell surface expression of IgM receptor complexes on tolerant B cells was greatly reduced, despite normal expression of mRNA encoding the receptor components. Three-fold fewer immunoreactive mu heavy chains were detectable after a short period of biosynthetic labelling and the immunoreactive mu chains produced were paired with kappa light chains and assembled normally into intact receptor complexes containing the MB1/B29 heterodimer. Nascent IgM receptor complexes nevertheless failed to be processed into an endoglycosidase H-resistant form in the tolerant B cells and thus appeared to be selectively blocked in their transport from the endoplasmic reticulum to the medial Golgi. These findings demonstrate that intracellular trafficking of antigen receptor complexes is regulated by exposure to receptor stimuli at the cell surface causing a long-lasting decrease in surface receptor expression on tolerant B cells. Images PMID:8112296

  2. Expression at the cell surface of biologically active fusion and hemagglutinin/neuraminidase proteins of the paramyxovirus simian virus 5 from cloned cDNA.

    PubMed Central

    Paterson, R G; Hiebert, S W; Lamb, R A

    1985-01-01

    cDNAs encoding the mRNAs for the fusion protein (F) and the hemagglutinin/neuraminidase protein (HN) of the paramyxovirus simian virus 5 have been inserted into a eukaryotic expression vector under the control of the simian virus 40 late promoter. The F and HN proteins synthesized in recombinant infected cells are indistinguishable in terms of electrophoretic mobility and glycosylation from the proteins synthesized in simian virus 5-infected cells. In addition, the expressed F and HN proteins have been shown to be anchored in the plasma membrane in a biologically active form by indirect live cell immunofluorescence, the F-mediated formation of syncytia, and the ability of HN to cause the hemadsorption of erythrocytes to the infected cell surface. Images PMID:3865176

  3. Serial position encoding of signs.

    PubMed

    Miozzo, Michele; Petrova, Anna; Fischer-Baum, Simon; Peressotti, Francesca

    2016-09-01

    Reduced short-term memory (STM) capacity has been reported for sign as compared to speech when items have to be recalled in a specific order. This difference has been attributed to a more precise and efficient serial position encoding in verbal STM (used for speech) than visuo-spatial STM (used for sign). We tested in the present investigation whether the reduced STM capacity with signs stems from a lack of positional encoding available in verbal STM. Error analyses reported in prior studies have revealed that positions are defined in verbal STM by distance from both the start and the end of the sequence (both-edges positional encoding scheme). Our analyses of the errors made by deaf participants with finger-spelled letters revealed that the both-edges positional encoding scheme underlies the STM representation of signs. These results indicate that the cause of the STM disadvantage is not the type of positional encoding but rather the difficulties in binding an item in visuo-spatial STM to its specific position in the sequence. Both-edges positional encoding scheme could be specific of sign, since it has not been found in visuo-spatial STM tasks conducted with hearing participants. PMID:27244095

  4. Edwardsiella ictaluri Encodes an Acid Activated Urease that is Required for Intracellular Replication in Channel Catfish Ictalurus punctatus Macrophages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genomic analysis indicated that Edwardsiella ictaluri encodes a putative ureasepathogenicity island containing 9 open reading frames, including urea and ammonium transporters. In vitro studies with the wild-type E. ictaluri and a ureG::kan urease mutant strain indicated that E. ictaluri is significa...

  5. Characterization of a highly conserved human homolog to the chicken neural cell surface protein Bravo/Nr-CAM that maps to chromosome band 7q31

    SciTech Connect

    Lane, R.P.; Vielmetter, J.; Dreyer, W.J.

    1996-08-01

    The neuronal cell adhesion molecule Bravo/Nr-CAM is a cell surface protein of the immunoglobulin (Ig) superfamily and is closely related to the L1/NgCAM and neurofascin molecules, all of which contain six immunoglobulin domains, five fibronectin repeats, a transmembrane region, and an intracellular domain. Chicken Bravo/Nr-CAM has been shown to interact with other cell surface molecules of the Ig superfamily and has been implicated in specific pathfinding roles of axonal growth cones in the developing nervous system. We now report the characterization of cDNA clones encoding the human Bravo/Nr-CAM protein, which, like its chicken homolog, is composed of six V-like Ig domains and five fibronectin type III repeats. The human Bravo/Nr-CAM homolog also contains a transmembrane and intracellular domain, both of which are 100% conserved at the amino acid level compared to its chicken homolog. Overall, the human Bravo/Nr-CAM homolog is 82% identical to the chicken Bravo/Nr-CAM amino acid sequence. Independent cDNAs encoding four different isoforms were also identified, all of which contain alternatively spliced variants around the fifth fibronectin type III repeat, including one isoform that had been previously identified for chicken Bravo/Nr-CAM. Northern blot analysis reveals one mRNA species of approximately 7.0 kb in adult human brain tissue. Fluorescence in situ hybridization maps the gene for human Bravo/Nr-CAM to human chromosome 7q31.1-q31.2. This chromosomal locus has been previously identified as containing a tumore suppressor candidate gene commonly deleted in certain human cancer tissues. 38 refs., 5 figs.

  6. Cloning, Expression, and Sequencing of a Cell Surface Antigen Containing a Leucine-Rich Repeat Motif from Bacteroides forsythus ATCC 43037

    PubMed Central

    Sharma, Ashu; Sojar, Hakimuddin T.; Glurich, Ingrid; Honma, Kiyonobu; Kuramitsu, Howard K.; Genco, Robert J.

    1998-01-01

    Bacteroides forsythus is a recently recognized human periodontopathogen associated with advanced, as well as recurrent, periodontitis. However, very little is known about the mechanism of pathogenesis of this organism. The present study was undertaken to identify the surface molecules of this bacterium that may play roles in its adherence to oral tissues or triggering of a host immune response(s). The gene (bspA) encoding a cell surface-associated protein of B. forsythus with an apparent molecular mass of 98 kDa was isolated by immunoscreening of a B. forsythus gene library constructed in a lambda ZAP II vector. The encoded 98-kDa protein (BspA) contains 14 complete repeats of 23 amino acid residues that show partial homology to leucine-rich repeat motifs. A recombinant protein containing the repeat region was expressed in Escherichia coli, purified, and utilized for antibody production, as well as in vitro binding studies. The purified recombinant protein bound strongly to fibronectin and fibrinogen in a dose-dependent manner and further inhibited the binding of B. forsythus cells to these extracellular matrix (ECM) components. In addition, adult patients with B. forsythus-associated periodontitis expressed specific antibodies against the BspA protein. We report here the cloning and expression of an immunogenic cell surface-associated protein (BspA) of B. forsythus and speculate that it mediates the binding of bacteria to ECM components and clotting factors (fibronectin and fibrinogen, respectively), which may be important in the colonization of the oral cavity by this bacterium and is also a target for the host immune response. PMID:9826345

  7. Immunochemical identification of the major cell surface agglutinogen of Acinetobacter calcoaceticus RAG-92.

    PubMed

    Bayer, E A; Skutelsky, E; Goldman, S; Rosenberg, E; Gutnick, D L

    1983-04-01

    The immunochemical and immunocytochemical characteristics of three Acinetobacter calcoaceticus RAG strains were compared in order to clarify the relationship between antibody-induced agglutination and the production of polyanionic extracellular emulsifier (termed emulsan). In addition to the parent, RAG-92, two mutant strains were examined: (1) a non-agglutinating emulsan-producer (AB15), and (2) an agglutinating mutant (16TLU) defective in the production of emulsan. A combined genetic-immunochemical approach was employed. This included the comparison of crossed immunoelectrophoresis patterns of parent and mutant supernates and the effect of absorption of anti-whole cell antiserum with mutant cells. In addition, agglutinability and competition studies were performed as well as electron microscopic cytochemistry. The results demonstrated that three major antigenic components were associated with the cell surface and the supernate. Mutant cells were altered both in their cell surface properties and in their extracellular products. One antigenic component, termed component C3, was the major cell surface agglutinogen; this component was absent in non-agglutinating mutants. Component C3 may be identical with or attached to the 300 nm projections on the parent cell surface, but it is not directly related to the presence of emulsan. It appears that emulsan plays little or no role in the phenomenon of antibody-induced agglutination of this organism. PMID:6688443

  8. Erratum: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing.

    PubMed

    2015-01-01

    The author's email has been corrected in the publication of Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing. There was an error with the author, Jerry Zhou's, email. The author's email has been updated to: j.zhou@uws.edu.au from: jzho7551@mail.usyd.edu.au. PMID:26167960

  9. Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells

    PubMed Central

    Zhuang, Hanyi; Matsunami, Hiroaki

    2009-01-01

    A fundamental question in olfaction is which odorant receptors (ORs) are activated by a given odorant. A major roadblock to investigate odorant-OR relationship in mammals has been an inability to express ORs in heterologous cells suitable for screening active ligands for ORs. The discovery of the receptor-transporting protein (RTP) family has facilitated the effective cell-surface expression of ORs in heterologous cells. The establishment of a robust heterologous expression system for mammalian ORs facilitates the high-throughput “deorphanization” of these receptors by matching them to their cognate ligands. This protocol details the method used for evaluating the cell-surface expression and measuring the functional activation of ORs of transiently-expressed mammalian odorant receptors in HEK293T cells. The stages of odorant receptor cell-surface expression include cell culture preparation, transfer of cells, transfection, and immunocytochemistry/flow cytometry, odorant stimulation, and luciferase assay. This protocol can be completed in a period of 3 days from transfer of cells to cell-surface expression detection and/or measurement of functional activation. PMID:18772867

  10. Cell surface properties of HLA antigens on Epstein-Barr virus-transformed cell lines.

    PubMed Central

    Smith, L M; Petty, H R; Parham, P; McConnell, H M

    1982-01-01

    A number of monoclonal antibodies have been used to investigate the distributions and rates of lateral motion of the HLA-A,B, and-DR antigens on several Epstein--Barr virus-transformed B-cell lines. The lateral diffusion coefficients (D) of fluorescein conjugates of the monoclonal antibodies bound to the cell surface were determined by fluorescence recovery after pattern photobleaching. Ds of HLA-A and-B were found to be comparable and of the order of 10(-9) to 10(-10) cm2/sec for each of the seven monoclonal antibodies and four cell lines examined. The HLA antigens appear to be monomeric on the cell surface based on experiments using mixtures of arsanilic acid-conjugated and fluorescein-conjugated antibodies. Four monoclonal antibodies against DR antigens were examined. Two of these, Genox 3.53 and L243, labeled the cell surface uniformly and gave Ds comparable to those obtained for the HLA-A and -B antigens. The other two, DA2 and 2.06, rapidly patched on the cell surface and were immobile. The DA2, L243, and Genox 3.53 antibodies bound outside of the caps formed with the arsanilic acid-conjugated 2.06 antibody and a second-step rhodamine-conjugated rabbit anti-arsanilate antibody. This is consistent with recent biochemical evidence that there are multiple distinct antigens coded for by the HLA-DR region. Images PMID:6281776

  11. Effects of Streptococcus sanguinis Bacteriocin on Cell Surface Hydrophobicity, Membrane Permeability, and Ultrastructure of Candida Thallus

    PubMed Central

    Ma, Shengli; Zhao, Yingnan; Xia, Xue; Dong, Xue; Ge, Wenyu; Li, Hui

    2015-01-01

    Candida albicans (C.a) and Candida tropicalis (C.t) were treated with Streptococcus sanguinis bacteriocin (S.s bacteriocin), respectively; the bacteriostatic dynamics of S.s bacteriocin, their effects on cell surface hydrophobicity, leakage of inorganic phosphorus and macromolecular substance, cytosolic calcium concentration, and ultrastructure changes of Candida thallus were detected and analyzed. The results showed that inhibitory effect of S.s bacteriocin on C.a and C.t reached peak level at 24 h, the cell-surface hydrophobicity decreased significantly (P < 0.05) after S.s bacteriocin treatment, and there was leakage of cytoplasmic inorganic phosphorus and macromolecular substance from C.a and C.t; cytosolic calcium concentration decreased greatly. After 24 h treatment by S.s bacteriocin, depressive deformity and defect could be found in the cell surface of C.a and C.t; the thallus displayed irregular forms: C.a was shrunken, there was unclear margins abutting upon cell wall and cell membrane, nucleus disappeared, and cytoplasm was inhomogeneous; likewise, C.t was first plasmolysis, and then the cytoplasm was shrunk, the ultrastructure of cell wall and cell membrane was continuously damaged, and the nucleus was karyolysis. It was illustrated that S.s bacteriocin had similar antifungal effect on C.a and C.t; their cell surface hydrophobicity, membrane permeability, and ultrastructure were changed significantly on exposure to S.s bacteriocin. PMID:26064919

  12. A cell-surface-anchored ratiometric i-motif sensor for extracellular pH detection.

    PubMed

    Ying, Le; Xie, Nuli; Yang, Yanjing; Yang, Xiaohai; Zhou, Qifeng; Yin, Bincheng; Huang, Jin; Wang, Kemin

    2016-06-14

    A FRET-based sensor is anchored on the cell surface through streptavidin-biotin interactions. Due to the excellent properties of the pH-sensitive i-motif structure, the sensor can detect extracellular pH with high sensitivity and excellent reversibility. PMID:27241716

  13. Cell surface nucleolin is crucial in the activation of the CXCL12/CXCR4 signaling pathway.

    PubMed

    Yang, Xiangshan; Xu, Zhongfa; Li, Daotang; Cheng, Shaomei; Fan, Kaixi; Li, Chengjun; Li, Aiping; Zhang, Jing; Feng, Man

    2014-01-01

    Recently, CXCL12-CXCR4 has been focused on therapeutic strategies for papillary thyroid carcinoma (PTC) and other cancers. At the same time, cell surface nucleolin is also over-expressed in PTC and others. Interestingly, a few reports suggest that either CXCR4 or cell surface nucleolin is a co-receptor for HIV-1 entry into CD4+ T cells, which indicates that there is a relationship between CXCR4 and nucleolin. In this study, antibody and siRNA were used to identify effects of cell surface nucleolin and CXCR4 on cell signaling; soft-agar colony formation assay and Transwell assay were used to determine roles of nucleolin and CXCR4 in cell proliferation and migration. Importantly, co-immunoprecipitation was used to demonstrate the relationship between CXCR4 and nucleolin. Results showed CXCR4 and nucleolin were co-expressed in PTC cell line K1, B-CPAP, and TPC-1. Either cell surface nucleolin or CXCR4 was necessary to prompt extracellular signal-regulated kinase phosphorylation. When blocked, CXCR4 or nucleolin can significantly affect TPC-1 proliferation and migration (p < 0.01). Co-immunoprecipitation analysis identified that nucleolin can bind and interact with CXCR4 to activate CXCR4 signaling. This study suggests that nucleolin is crucial in the activation of CXCR4 signaling, which affects cell growth, migration, and invasiveness. Further, nucleolin may interact with other receptors. Our study also offers new ideas for cancer therapy. PMID:23918302

  14. A Dual Receptor and Reporter for Multi-Modal Cell Surface Engineering.

    PubMed

    Luo, Wei; Westcott, Nathan; Dutta, Debjit; Pulsipher, Abigail; Rogozhnikov, Dmitry; Chen, Jean; Yousaf, Muhammad N

    2015-10-16

    The rapid development of new small molecule drugs, nanomaterials, and genetic tools to modulate cellular function through cell surface manipulation has revolutionized the diagnosis, study, and treatment of disorders in human health. Since the cell membrane is a selective gateway barrier that serves as the first line of defense/offense and communication to its environment, new approaches that molecularly engineer or tailor cell membrane surfaces would allow for a new era in therapeutic design, therapeutic delivery, complex coculture tissue construction, and in situ imaging probe tracking technologies. In order to develop the next generation of multimodal therapies, cell behavior studies, and biotechnologies that focus on cell membrane biology, new tools that intersect the fields of chemistry, biology, and engineering are required. Herein, we develop a liposome fusion and delivery strategy to present a novel dual receptor and reporter system at cell surfaces without the use of molecular biology or metabolic biosynthesis. The cell surface receptor is based on bio-orthogonal functional groups that can conjugate a range of ligands while simultaneously reporting the conjugation through the emission of fluorescence. We demonstrate this dual receptor and reporter system by conjugating and tracking various cell surface ligands for temporal control of cell fluorescent signaling, cell-cell interaction, and tissue assembly construction. PMID:26204094

  15. Cell surface energy, contact angles and phase partition. II. Bacterial cells in biphasic aqueous mixtures.

    PubMed

    Gerson, D F; Akit, J

    1980-11-01

    Partition coefficients in biphasic mixtures of poly(ethylene glycol) and Dextran are compared to cell surface energies obtained from contact angles of each liquid phase on cell layers. Linear relationships are observed between these two independent measurements for a variety of bacterial cells. The results demonstrate the importance of interfacial phenomena and contact angles in the phase-partition process. PMID:6159003

  16. Noncovalent Cell Surface Engineering: Incorporation of Bioactive Synthetic Glycopolymers into Cellular Membranes

    PubMed Central

    Rabuka, David; Forstner, Martin B.; Groves, Jay T.; Bertozzi, Carolyn R.

    2009-01-01

    The controlled addition of structurally defined components to live cell membranes can facilitate the molecular level analysis of cell surface phenomena. Here we demonstrate that cell surfaces can be engineered to display synthetic bioactive polymers at defined densities by exogenous membrane insertion. The polymers were designed to mimic native cell-surface mucin glycoproteins, which are defined by their dense glycosylation patterns and rod-like structures. End-functionalization with a hydrophobic anchor permitted incorporation into the membranes of live cultured cells. We probed the dynamic behavior of cell-bound glycopolymers bearing various hydrophobic anchors and glycan structures using fluorescence correlation spectroscopy (FCS). Their diffusion properties mirrored those of many natural membrane-associated biomolecules. Furthermore, the membrane-bound glycopolymers were internalized into early endosomes similarly to endogenous membrane components and were capable of specific interactions with protein receptors. This system provides a platform to study cell-surface phenomena with a degree of chemical control that cannot be achieved using conventional biological tools. PMID:18402449

  17. Evidence of cell surface iron speciation of acidophilic iron-oxidizing microorganisms in indirect bioleaching process.

    PubMed

    Nie, Zhen-yuan; Liu, Hong-chang; Xia, Jin-lan; Yang, Yi; Zhen, Xiang-jun; Zhang, Li-Juan; Qiu, Guan-zhou

    2016-02-01

    While indirect model has been widely accepted in bioleaching, but the evidence of cell surface iron speciation has not been reported. In the present work the iron speciation on the cell surfaces of four typically acidophilic iron-oxidizing microorganism (mesophilic Acidithiobacillus ferrooxidans ATCC 23270, moderately thermophilic Leptospirillum ferriphilum YSK and Sulfobacillus thermosulfidooxidans St, and extremely thermophilic Acidianus manzaensis YN25) grown on different energy substrates (chalcopyrite, pyrite, ferrous sulfate and elemental sulfur (S(0))) were studied in situ firstly by using synchrotron-based micro- X-ray fluorescence analysis and X-ray absorption near-edge structure spectroscopy. Results showed that the cells grown on iron-containing substrates had apparently higher surface iron content than the cells grown on S(0). Both ferrous iron and ferric iron were detected on the cell surface of all tested AIOMs, and the Fe(II)/Fe(III) ratios of the same microorganism were affected by different energy substrates. The iron distribution and bonding state of single cell of A. manzaensis were then studied in situ by scanning transmission soft X-ray microscopy based on dual-energy contrast analysis and stack analysis. Results showed that the iron species distributed evenly on the cell surface and bonded with amino, carboxyl and hydroxyl groups. PMID:26645388

  18. Cell surface annexins regulate ADAM-mediated ectodomain shedding of proamphiregulin

    PubMed Central

    Nakayama, Hironao; Fukuda, Shinji; Inoue, Hirofumi; Nishida-Fukuda, Hisayo; Shirakata, Yuji; Hashimoto, Koji; Higashiyama, Shigeki

    2012-01-01

    A disintegrin and metalloproteinase (ADAM) is a family of enzymes involved in ectodomain shedding of various membrane proteins. However, the molecular mechanism underlying substrate recognition by ADAMs remains unknown. In this study, we successfully captured and analyzed cell surface transient assemblies between the transmembrane amphiregulin precursor (proAREG) and ADAM17 during an early shedding phase, which enabled the identification of cell surface annexins as components of their shedding complex. Annexin family members annexin A2 (ANXA2), A8, and A9 interacted with proAREG and ADAM17 on the cell surface. Shedding of proAREG was increased when ANXA2 was knocked down but decreased with ANXA8 and A9 knockdown, because of enhanced and impaired association with ADAM17, respectively. Knockdown of ANXA2 and A8 in primary keratinocytes altered wound-induced cell migration and ultraviolet B–induced phosphorylation of epidermal growth factor receptor (EGFR), suggesting that annexins play an essential role in the ADAM-mediated ectodomain shedding of EGFR ligands. On the basis of these data, we propose that annexins on the cell surface function as “shedding platform” proteins to determine the substrate selectivity of ADAM17, with possible therapeutic potential in ADAM-related diseases. PMID:22438584

  19. Effects of Streptococcus sanguinis Bacteriocin on Cell Surface Hydrophobicity, Membrane Permeability, and Ultrastructure of Candida Thallus.

    PubMed

    Ma, Shengli; Zhao, Yingnan; Xia, Xue; Dong, Xue; Ge, Wenyu; Li, Hui

    2015-01-01

    Candida albicans (C.a) and Candida tropicalis (C.t) were treated with Streptococcus sanguinis bacteriocin (S.s bacteriocin), respectively; the bacteriostatic dynamics of S.s bacteriocin, their effects on cell surface hydrophobicity, leakage of inorganic phosphorus and macromolecular substance, cytosolic calcium concentration, and ultrastructure changes of Candida thallus were detected and analyzed. The results showed that inhibitory effect of S.s bacteriocin on C.a and C.t reached peak level at 24 h, the cell-surface hydrophobicity decreased significantly (P < 0.05) after S.s bacteriocin treatment, and there was leakage of cytoplasmic inorganic phosphorus and macromolecular substance from C.a and C.t; cytosolic calcium concentration decreased greatly. After 24 h treatment by S.s bacteriocin, depressive deformity and defect could be found in the cell surface of C.a and C.t; the thallus displayed irregular forms: C.a was shrunken, there was unclear margins abutting upon cell wall and cell membrane, nucleus disappeared, and cytoplasm was inhomogeneous; likewise, C.t was first plasmolysis, and then the cytoplasm was shrunk, the ultrastructure of cell wall and cell membrane was continuously damaged, and the nucleus was karyolysis. It was illustrated that S.s bacteriocin had similar antifungal effect on C.a and C.t; their cell surface hydrophobicity, membrane permeability, and ultrastructure were changed significantly on exposure to S.s bacteriocin. PMID:26064919

  20. A rapid and selective assay for measuring cell surface hydrophobicity of brewer's yeast cells.

    PubMed

    Straver, M H; Kijne, J W

    1996-03-15

    A rapid and selective assay was developed to measure cell surface hydrophobicity of brewer's yeast cells. During this so-called magnobead assay, bottom-fermenting yeast cells adhere to paramagnetic, polystyrene-coated latex beads which can easily be removed from the cell suspension by using a (samarium-cobalt) magnet. At pH 4 center dot 5, electrostatic repulsion between yeast cells and latex beads was found to be minimal and yeast cell adhesion was predominantly based on hydrophobic interactions. The percentage of cells adhering to the beads could be calculated and provided a measure for cell surface hydrophobicity. Cell surface hydrophobicity measured by the magnobead assay was found to yield similar results, as did determination of contact angles of water droplets on a layer of yeast cells, a standard method for measuring surface hydrophobicity. However, the magnobead assay has the following advantages: (i) it is a quick and simple method, and, more significantly, (ii) hydrophobicity can be measured under physiological conditions. Use of the magnobead assay confirmed that a higher level of cell surface hydrophobicity is correlated with stronger flocculence of brewer's lager yeast cells. PMID:8904332

  1. Amyloid precursor protein enhances Nav1.6 sodium channel cell surface expression.

    PubMed

    Liu, Chao; Tan, Francis Chee Kuan; Xiao, Zhi-Cheng; Dawe, Gavin S

    2015-05-01

    Amyloid precursor protein (APP) is commonly associated with Alzheimer disease, but its physiological function remains unknown. Nav1.6 is a key determinant of neuronal excitability in vivo. Because mouse models of gain of function and loss of function of APP and Nav1.6 share some similar phenotypes, we hypothesized that APP might be a candidate molecule for sodium channel modulation. Here we report that APP colocalized and interacted with Nav1.6 in mouse cortical neurons. Knocking down APP decreased Nav1.6 sodium channel currents and cell surface expression. APP-induced increases in Nav1.6 cell surface expression were Go protein-dependent, enhanced by a constitutively active Go protein mutant, and blocked by a dominant negative Go protein mutant. APP also regulated JNK activity in a Go protein-dependent manner. JNK inhibition attenuated increases in cell surface expression of Nav1.6 sodium channels induced by overexpression of APP. JNK, in turn, phosphorylated APP. Nav1.6 sodium channel surface expression was increased by T668E and decreased by T668A, mutations of APP695 mimicking and preventing Thr-668 phosphorylation, respectively. Phosphorylation of APP695 at Thr-668 enhanced its interaction with Nav1.6. Therefore, we show that APP enhances Nav1.6 sodium channel cell surface expression through a Go-coupled JNK pathway. PMID:25767117

  2. Incorporation of Nasutitermes takasagoensis endoglucanase into cell surface-displayed minicellulosomes in Pichia pastoris X33.

    PubMed

    Ou, Jingshen; Cao, Yicheng

    2014-09-01

    In this study, the yeast Pichia pastoris was genetically modified to assemble minicellulosomes on its cell surface by the heterologous expression of a truncated scaffoldin CipA from Clostridium acetobutylicum. Fluorescence microscopy and western blot analysis confirmed that CipA was targeted to the yeast cell surface and that NtEGD, the Nasutitermes takasagoensis endoglucanase that was fused with dockerin, interacted with CipA on the yeast cell surface, suggesting that the cohesin and dockerin domains and cellulose-binding module of C. acetobutylicum were functional in the yeasts. The enzymatic activities of the cellulases in the minicellulosomes that were displayed on the yeast cell surfaces increased dramatically following interaction with the cohesin-dockerin domains. Additionally, the hydrolysis efficiencies of NtEGD for carboxymethyl cellulose, microcrystal cellulose, and filter paper increased up to 1.4-fold, 2.0-fold, and 3.2-fold, respectively. To the best of our knowledge, this is the first report describing the expression of C. acetobutylicum minicellulosomes in yeast and the incorporation of animal cellulases into cellulosomes. This strategy of heterologous cellulase incorporation lends novel insight into the process of cellulosome assembly. Potentially, the surface display of cellulosomes, such as that reported in this study, may be utilized in the engineering of S. cerevisiae for ethanol production from cellulose and additional future applications. PMID:24851815

  3. Requirement of aggregation propensity of Alzheimer amyloid peptides for neuronal cell surface binding

    PubMed Central

    Bateman, David A; McLaurin, JoAnne; Chakrabartty, Avijit

    2007-01-01

    Background Aggregation of the amyloid peptides, Aβ40 and Aβ42, is known to be involved in the pathology of Alzheimer's disease (AD). Here we investigate the relationship between peptide aggregation and cell surface binding of three forms of Aβ (Aβ40, Aβ42, and an Aβ mutant). Results Using confocal microscopy and flow cytometry with fluorescently labelled Aβ, we demonstrate a correlation between the aggregation propensity of the Alzheimer amyloid peptides and their neuronal cell surface association. We find that the highly aggregation prone Aβ42 associates with the surface of neuronal cells within one hour, while the less aggregation prone Aβ40 associates over 24 hours. We show that a double mutation in Aβ42 that reduces its aggregation propensity also reduces its association with the cell surface. Furthermore, we find that a cell line that is resistant to Aβ cytotoxicity, the non-neuronal human lymphoma cell line U937, does not bind either Aβ40 or Aβ42. Conclusion Taken together, our findings reveal that amyloid peptide aggregation propensity is an essential determinant of neuronal cell surface association. We anticipate that our approach, involving Aβ imaging in live cells, will be highly useful for evaluating the efficacy of therapeutic drugs that prevent toxic Aβ association with neuronal cells. PMID:17475015

  4. Quantitatively Resolving Ligand–Receptor Bonds on Cell Surfaces Using Force-Induced Remnant Magnetization Spectroscopy

    PubMed Central

    2016-01-01

    Molecule-specific noncovalent bonding on cell surfaces is the foundation for cellular recognition and functioning. A major challenge in probing these bonds is to resolve the specific bonds quantitatively and efficiently from the nonspecific interactions in a complex environment. Using force-induced remnant magnetization spectroscopy (FIRMS), we were able to resolve quantitatively three different interactions for magnetic beads bearing anti-CD4 antibodies with CD4+ T cell surfaces based upon their binding forces. The binding force of the CD4 antibody–antigen bonds was determined to be 75 ± 3 pN. For comparison, the same bonds were also studied on a functionalized substrate surface, and the binding force was determined to be 90 ± 6 pN. The 15 pN difference revealed by high-resolution FIRMS illustrates the significant impact of the bonding environment. Because the force difference was unaffected by the cell number or the receptor density on the substrate, we attributed it to the possible conformational or local environmental differences of the CD4 antigens between the cell surface and substrate surface. Our results show that the high force resolution and detection efficiency afforded by FIRMS are valuable for studying protein–protein interactions on cell surfaces. PMID:27163031

  5. Ultrastructural comparison of porcine putative embryonic stem cells derived by in vitro fertilization and somatic cell nuclear transfer.

    PubMed

    Yoo, Hyunju; Kim, Eunhye; Hwang, Seon-Ung; Yoon, Junchul David; Jeon, Yubyeol; Park, Kyu-Mi; Kim, Kyu-Jun; Jin, Minghui; Lee, Chang-Kyu; Lee, Eunsong; Kim, Hyunggee; Kim, Gonhyung; Hyun, Sang-Hwan

    2016-04-22

    The ultrastructure of porcine putative embryonic stem cells and porcine fetal fibroblasts (PFFs) was analyzed by transmission electron microscopy. The aim of this study was to compare the features of organelles in in vitro fertilization (IVF) derived porcine embryonic stem cells (IVF-pESCs) and somatic cell nuclear transfer (SCNT) derived pESCs (SCNT-pESCs). Also, the features of organelles in high-passage IVF-pESCs were compared with those in low-passage cells. The ultrastructure of PFFs showed rare microvilli on the cell surfaces, polygonal or irregular nuclei with one to two reticular-shaped nucleoli and euchromatin, low cytoplasm-to-nucleus ratios, rare ribosomes, rare rough endoplasmic reticulum, elongated mitochondria, rich lysosomes and rich phagocytic vacuoles. IVF-pESCs showed rare microvilli on the cell surfaces, round or irregular nuclei with one to two reticular-shaped nucleoli and euchromatin, low cytoplasm-to-nucleus ratios, rich ribosomes, long stacks of rough endoplasmic reticulum, elongated mitochondria, rare lysosomes and rare autophagic vacuoles. By contrast, SCNT-pESCs showed rich microvilli with various lengths and frequencies on the cell surfaces, polygonal nuclei with one reticular shaped nucleoli and heterochromatin, high cytoplasm-to-nucleus ratios, rare ribosomes, rare rough endoplasmic reticulum, round mitochondria, rich lysosomes and rich phagocytic vacuoles with clear intercellular junctions. Furthermore, high-passage IVF-pESCs showed irregularly shaped colonies, pyknosis and numerous lysosomes associated with autophagic vacuoles showing signs of apoptosis. In conclusion, this study confirms that the ultrastructural characteristics of pESCs differ depending on their origin. These ultrastructural characteristics might be useful in biomedical research using pESCs, leading to new insights regarding regenerative medicine and tissue repair. PMID:26821870

  6. Putative Nonribosomal Peptide Synthetase and Cytochrome P450 Genes Responsible for Tentoxin Biosynthesis in Alternaria alternata ZJ33

    PubMed Central

    Li, You-Hai; Han, Wen-Jin; Gui, Xi-Wu; Wei, Tao; Tang, Shuang-Yan; Jin, Jian-Ming

    2016-01-01

    Tentoxin, a cyclic tetrapeptide produced by several Alternaria species, inhibits the F1-ATPase activity of chloroplasts, resulting in chlorosis in sensitive plants. In this study, we report two clustered genes, encoding a putative non-ribosome peptide synthetase (NRPS) TES and a cytochrome P450 protein TES1, that are required for tentoxin biosynthesis in Alternaria alternata strain ZJ33, which was isolated from blighted leaves of Eupatorium adenophorum. Using a pair of primers designed according to the consensus sequences of the adenylation domain of NRPSs, two fragments containing putative adenylation domains were amplified from A. alternata ZJ33, and subsequent PCR analyses demonstrated that these fragments belonged to the same NRPS coding sequence. With no introns, TES consists of a single 15,486 base pair open reading frame encoding a predicted 5161 amino acid protein. Meanwhile, the TES1 gene is predicted to contain five introns and encode a 506 amino acid protein. The TES protein is predicted to be comprised of four peptide synthase modules with two additional N-methylation domains, and the number and arrangement of the modules in TES were consistent with the number and arrangement of the amino acid residues of tentoxin, respectively. Notably, both TES and TES1 null mutants generated via homologous recombination failed to produce tentoxin. This study provides the first evidence concerning the biosynthesis of tentoxin in A. alternata. PMID:27490569

  7. Putative Nonribosomal Peptide Synthetase and Cytochrome P450 Genes Responsible for Tentoxin Biosynthesis in Alternaria alternata ZJ33.

    PubMed

    Li, You-Hai; Han, Wen-Jin; Gui, Xi-Wu; Wei, Tao; Tang, Shuang-Yan; Jin, Jian-Ming

    2016-01-01

    Tentoxin, a cyclic tetrapeptide produced by several Alternaria species, inhibits the F₁-ATPase activity of chloroplasts, resulting in chlorosis in sensitive plants. In this study, we report two clustered genes, encoding a putative non-ribosome peptide synthetase (NRPS) TES and a cytochrome P450 protein TES1, that are required for tentoxin biosynthesis in Alternaria alternata strain ZJ33, which was isolated from blighted leaves of Eupatorium adenophorum. Using a pair of primers designed according to the consensus sequences of the adenylation domain of NRPSs, two fragments containing putative adenylation domains were amplified from A. alternata ZJ33, and subsequent PCR analyses demonstrated that these fragments belonged to the same NRPS coding sequence. With no introns, TES consists of a single 15,486 base pair open reading frame encoding a predicted 5161 amino acid protein. Meanwhile, the TES1 gene is predicted to contain five introns and encode a 506 amino acid protein. The TES protein is predicted to be comprised of four peptide synthase modules with two additional N-methylation domains, and the number and arrangement of the modules in TES were consistent with the number and arrangement of the amino acid residues of tentoxin, respectively. Notably, both TES and TES1 null mutants generated via homologous recombination failed to produce tentoxin. This study provides the first evidence concerning the biosynthesis of tentoxin in A. alternata. PMID:27490569

  8. Purification and crystallization of a putative transcriptional regulator of the benzoate oxidation pathway in Burkholderia xenovorans LB400

    PubMed Central

    Law, Adrienne M.; Bains, Jasleen; Boulanger, Martin J.

    2009-01-01

    Burkholderia xenovorans LB400 harbours two paralogous copies of the recently discovered benzoate oxidation (box) pathway. While both copies are functional, the paralogues are differentially regulated and flanked by putative transcriptional regulators from distinct families. The putative LysR-type transcriptional regulator (LTTR) adjacent to the megaplasmid-encoded box enzymes, Bxe_C0898, has been produced recombinantly in Escherichia coli and purified to homogeneity. Gel-filtration studies show that Bxe_C0898 is a tetramer in solution, consistent with previously characterized LTTRs. Bxe_C0898 crystallized with four molecules in the asymmetric unit of the P43212/P41212 unit cell with a solvent content of 61.19%, as indicated by processing of the X-ray diffraction data. DNA-protection assays are currently under way in order to identify potential operator regions for this LTTR and to define its role in regulation of the box pathway. PMID:19851006

  9. A Fruit-Specific Putative Dihydroflavonol 4-Reductase Gene Is Differentially Expressed in Strawberry during the Ripening Process1

    PubMed Central

    Moyano, Enriqueta; Portero-Robles, Ignacio; Medina-Escobar, Nieves; Valpuesta, Victoriano; Muñoz-Blanco, Juan; Luis Caballero, José

    1998-01-01

    A cDNA clone encoding a putative dihydroflavonol 4-reductase gene has been isolated from a strawberry (Fragaria × ananassa cv Chandler) DNA subtractive library. Northern analysis showed that the corresponding gene is predominantly expressed in fruit, where it is first detected during elongation (green stages) and then declines and sharply increases when the initial fruit ripening events occur, at the time of initiation of anthocyanin accumulation. The transcript can be induced in unripe green fruit by removing the achenes, and this induction can be partially inhibited by treatment of de-achened fruit with naphthylacetic acid, indicating that the expression of this gene is under hormonal control. We propose that the putative dihydroflavonol 4-reductase gene in strawberry plays a main role in the biosynthesis of anthocyanin during color development at the late stages of fruit ripening; during the first stages the expression of this gene could be related to the accumulation of condensed tannins. PMID:9625725

  10. Cloning and characterization of two Lactobacillus casei genes encoding a cystathionine lyase.

    PubMed

    Irmler, Stefan; Raboud, Sylvie; Beisert, Beata; Rauhut, Doris; Berthoud, Hélène

    2008-01-01

    Volatile sulfur compounds are key flavor compounds in several cheese types. To better understand the metabolism of sulfur-containing amino acids, which certainly plays a key role in the release of volatile sulfur compounds, we searched the genome database of Lactobacillus casei ATCC 334 for genes encoding putative homologs of enzymes known to degrade cysteine, cystathionine, and methionine. The search revealed that L. casei possesses two genes that putatively encode a cystathionine beta-lyase (CBL; EC 4.4.1.8). The enzyme has been implicated in the degradation of not only cystathionine but also cysteine and methionine. Recombinant CBL proteins catalyzed the degradation of L-cystathionine, O-succinyl-L-homoserine, L-cysteine, L-serine, and L-methionine to form alpha-keto acid, hydrogen sulfide, or methanethiol. The two enzymes showed notable differences in substrate specificity and pH optimum. PMID:17993563

  11. Characterization of the gene encoding a fibrinogen-related protein expressed in Crassostrea gigas hemocytes.

    PubMed

    Skazina, M A; Gorbushin, A M

    2016-07-01

    Four exons of the CgFrep1 gene (3333 bp long) encode a putative fibrinogen-related protein (324 aa) bearing a single C-terminal FBG domain. Transcripts of the gene obtained from hemocytes of different Pacific oysters show prominent individual variation based on SNP and indels of tandem repeats resulted in polymorphism of N-terminus of the putative CgFrep1 polypeptide. The polypeptide chain bears N-terminal coiled-coil region potentially acting as inter-subunit interface in the protein oligomerization. It is suggested that CgFrep1 gene encodes the oligomeric lectin composed of at least two subunits. PMID:27189918

  12. Identification of CBX3 and ABCA5 as Putative Biomarkers for Tumor Stem Cells in Osteosarcoma

    PubMed Central

    Saini, Vaibhav; Hose, Curtis D.; Monks, Anne; Nagashima, Kunio; Han, Bingnan; Newton, Dianne L.; Millione, Angelena; Shah, Jalpa; Hollingshead, Melinda G.; Hite, Karen M.; Burkett, Mark W.; Delosh, Rene M.; Silvers, Thomas E.; Scudiero, Dominic A.; Shoemaker, Robert H.

    2012-01-01

    Recently, there has been renewed interest in the role of tumor stem cells (TSCs) in tumorigenesis, chemoresistance, and relapse of malignant tumors including osteosarcoma. The potential exists to improve osteosarcoma treatment through characterization of TSCs and identification of therapeutic targets. Using transcriptome, proteome, immunophenotyping for cell-surface markers, and bioinformatic analyses, heterogeneous expression of previously reported TSC or osteosarcoma markers, such as CD133, nestin, POU5F1 (OCT3/4), NANOG, SOX2, and aldehyde dehydrogenase, among others, was observed in vitro. However, consistently significantly lower CD326, CD24, CD44, and higher ABCG2 expression in TSC-enriched as compared with un-enriched osteosarcoma cultures was observed. In addition, consistently higher CBX3 expression in TSC-enriched osteosarcoma cultures was identified. ABCA5 was identified as a putative biomarker of TSCs and/or osteosarcoma. Lastly, in a high-throughput screen we identified epigenetic (5-azacytidine), anti-microtubule (vincristine), and anti-telomerase (3,11-difluoro-6,8,13-trimethyl- 8H-quino [4,3,2-kl] acridinium methosulfate; RHPS4)-targeted therapeutic agents as candidates for TSC ablation in osteosarcoma. PMID:22870217

  13. Measurement of receptor cross-linking at the cell surface via multiparameter flow cytometry

    NASA Astrophysics Data System (ADS)

    Posner, Richard G.; Bold, Jennifer; Bernstein, Yael; Rasor, Joe; Braslow, Joshua; Hlavacek, William S.; Perelson, Alan S.

    1998-05-01

    Many cellular responses, particularly in the immune system, are triggered by ligand binding to a cell-surface receptor. However, as indicated by bell-shaped dose-response curves, ligand binding alone is sometimes insufficient to trigger a response. Often, ligand binding must also induce the aggregation of cell-surface receptors through crosslinking, which occurs when a ligand binds simultaneously to two or more receptors. Thus, an important goal in cell biology has been to establish quantitative relationships between the amount of ligand present on a cell surface and the number of crosslinked ligand-specific cell-surface receptors. To better understand ligand-induced receptor aggregation, we have been investigating the binding of a model multivalent antigen (DNP25PE) to cell-surface anti-DNP FITC-labeled IgE (FITC- IgE). To determine the kinetic and equilibrium parameters that characterize crosslinking in this system, we have developed a combined theoretical and experimental approach that is based on multiparameter flow cytometry. With this approach, we can measure both the average number of ligand molecules that are bound per cell and the average number of receptor binding sites that are bound per cell. The average number of DNP25PE per cell is determined by measuring the fluorescence of phycoerythrin. The average number of occupied IgE sites per cell is determined by measuring the fluorescence of FITC, which is quenched upon ligand binding. This novel approach, together with conventional methods for changes in intracellular calcium, allows us to correlate for the first time the dynamics of IgE crosslinking with cell activation.

  14. Pigment Epithelium-derived Factor (PEDF) Binds to Cell-surface F1-ATP Synthase

    PubMed Central

    Notari, Luigi; Arakaki, Naokatu; Mueller, David; Meier, Scott; Amaral, Juan; Becerra, S. Patricia

    2010-01-01

    Pigment epithelium-derived factor (PEDF), a potent blocker of angiogenesis in vivo, and of endothelial cell migration and tubule formation, binds with high affinity to a yet unknown protein on the surface of endothelial cells. Given that protein fingerprinting suggested a match of a ~60-kDa PEDF-binding protein in bovine retina to Bos taurus F1-ATP synthase β-subunit, and that F1F0-ATP synthase components have been identified recently as cell-surface receptors, we examined the direct binding of PEDF to F1. Size-exclusion ultrafiltration assays showed that recombinant human PEDF formed a complex with recombinant yeast F1. Real-time binding by surface plasmon resonance demonstrated that yeast F1 interacted specifically and reversibly with human PEDF. Kinetic evaluations revealed high binding affinity for PEDF, in agreement with PEDF affinities for endothelial cell-surfaces. PEDF blocked interactions between F1 and angiostatin, another antiangiogenic factor, suggesting overlapping PEDF- and angiostatin-binding sites on F1. Surfaces of endothelial cells exhibited affinity for PEDF-binding proteins of ~60-kDa. Antibodies to F1 β-subunit specifically captured PEDF-binding components in endothelial plasma membranes. Extracellular ATP synthesis activity of endothelial cells was examined in the presence of PEDF. PEDF significantly inhibited the extracellular ATP produced by endothelial cells, in agreement with direct interactions between cell-surface ATP synthase and PEDF. In addition to demonstrating that PEDF binds to cell-surface F1, these results show that PEDF is a ligand for endothelial cell-surface F1F0-ATP synthase. They suggest that PEDF-mediated inhibition of ATP synthase may be part of the biochemical mechanisms by which PEDF exerts its antiangiogenic activity. PMID:20412062

  15. The Mouse C2C12 Myoblast Cell Surface N-Linked Glycoproteome

    PubMed Central

    Gundry, Rebekah L.; Raginski, Kimberly; Tarasova, Yelena; Tchernyshyov, Irina; Bausch-Fluck, Damaris; Elliott, Steven T.; Boheler, Kenneth R.; Van Eyk, Jennifer E.; Wollscheid, Bernd

    2009-01-01

    Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing technology was used here to uncover the cell surface N-linked glycoprotein subproteome of myoblasts and to identify potential markers of myoblast differentiation. 128 bona fide cell surface-exposed N-linked glycoproteins, including 117 transmembrane, four glycosylphosphatidylinositol-anchored, five extracellular matrix, and two membrane-associated proteins were identified from mouse C2C12 myoblasts. The data set revealed 36 cluster of differentiation-annotated proteins and confirmed the occupancy for 235 N-linked glycosylation sites. The identification of the N-glycosylation sites on the extracellular domain of the proteins allowed for the determination of the orientation of the identified proteins within the plasma membrane. One glycoprotein transmembrane orientation was found to be inconsistent with Swiss-Prot annotations, whereas ambiguous annotations for 14 other proteins were resolved. Several of the identified N-linked glycoproteins, including aquaporin-1 and β-sarcoglycan, were found in validation experiments to change in overall abundance as the myoblasts differentiate toward myotubes. Therefore, the strategy and data presented shed new light on the complexity of the myoblast cell surface subproteome and reveal new targets for the clinically important characterization of cell intermediates during myoblast differentiation into myotubes. PMID:19656770

  16. Cell Wall Mannan and Cell Surface Hydrophobicity in Candida albicans Serotype A and B Strains

    PubMed Central

    Masuoka, James; Hazen, Kevin C.

    2004-01-01

    Cell surface hydrophobicity contributes to the pathogenesis of the opportunistic fungal pathogen Candida albicans. Previous work demonstrated a correlation between hydrophobicity status and changes in the acid-labile, phosphodiester-linked β-1,2-oligomannoside components of the N-linked glycans of cell wall mannoprotein. Glycan composition also defines the two major serotypes, A and B, of C. albicans strains. Here, we show that the cell surface hydrophobicity of the two serotypes is qualitatively different, suggesting that the serotypes may differ in how they modulate cell surface hydrophobicity status. The cell wall mannoproteins from hydrophilic and hydrophobic cells of both serotypes were compared to determine whether the glycan differences due to serotype affect the glycan differences due to hydrophobicity status. Composition analysis showed that the protein, hexose, and phosphate contents of the mannoprotein fraction did not differ significantly among the strains tested. Electrophoretic profiles of the acid-labile mannan differed only with hydrophobicity status, not serotype, though some strain-specific differences were observed. Furthermore, a newly available β-1,2-oligomannoside ladder allowed unambiguous identification of acid-labile mannan components. Finally, to assess whether the acid-stable mannan also affects cell surface hydrophobicity status, this fraction was fragmented into its component branches by acetolysis. The electrophoretic profiles of the acid-stable branches were very similar regardless of hydrophobicity status. However, differences were observed between serotypes. These results support and extend our current model that modification of the acid-labile β-1,2-oligomannoside chain length but not modification of the acid-stable region is one common mechanism by which switching of cell surface hydrophobicity status of C. albicans strains occurs. PMID:15501748

  17. Isthmin targets cell-surface GRP78 and triggers apoptosis via induction of mitochondrial dysfunction.

    PubMed

    Chen, M; Zhang, Y; Yu, V C; Chong, Y-S; Yoshioka, T; Ge, R

    2014-05-01

    Isthmin (ISM) is a secreted 60-kDa protein that potently induces endothelial cell (EC) apoptosis. It suppresses tumor growth and angiogenesis in mice when stably overexpressed in cancer cells. Although αvβ5 integrin serves as a low-affinity receptor for ISM, the mechanism by which ISM mediates antiangiogenesis and apoptosis in ECs remain to be fully resolved. In this work, we report the identification of cell-surface glucose-regulated protein 78 kDa (GRP78) as a high-affinity receptor for ISM (Kd=8.6 nM). We demonstrated that ISM-GRP78 interaction triggers apoptosis not only in activated ECs but also in cancer cells expressing high level of cell-surface GRP78. Normal cells and benign tumor cells tend to express low level of cell-surface GRP78 and are resistant to ISM-induced apoptosis. Upon binding to GRP78, ISM is internalized into ECs through clathrin-dependent endocytosis that is essential for its proapoptotic activity. Once inside the cell, ISM co-targets with GRP78 to mitochondria where it interacts with ADP/ATP carriers on the inner membrane and blocks ATP transport from mitochondria to cytosol, thereby causing apoptosis. Hence, ISM is a novel proapoptotic ligand that targets cell-surface GRP78 to trigger apoptosis by inducing mitochondrial dysfunction. The restricted and high-level expression of cell-surface GRP78 on cancer cells and cancer ECs make them uniquely susceptible to ISM-targeted apoptosis. Indeed, systemic delivery of recombinant ISM potently suppressed subcutaneous 4T1 breast carcinoma and B16 melanoma growth in mice by eliciting apoptosis selectively in the cancer cells and cancer ECs. Together, this work reveals a novel ISM-GRP78 apoptosis pathway and demonstrates the potential of ISM as a cancer-specific and dual-targeting anticancer agent. PMID:24464222

  18. Identification of Novel Tumor-Associated Cell Surface Sialoglycoproteins in Human Glioblastoma Tumors Using Quantitative Proteomics

    PubMed Central

    Autelitano, François; Loyaux, Denis; Roudières, Sébastien; Déon, Catherine; Guette, Frédérique; Fabre, Philippe; Ping, Qinggong; Wang, Su; Auvergne, Romane; Badarinarayana, Vasudeo; Smith, Michael; Guillemot, Jean-Claude; Goldman, Steven A.; Natesan, Sridaran; Ferrara, Pascual; August, Paul

    2014-01-01

    Glioblastoma multiform (GBM) remains clinical indication with significant “unmet medical need”. Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells. PMID:25360666

  19. 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

  20. Identification of the Repressor-Encoding Gene of the Lactobacillus Bacteriophage A2

    PubMed Central

    Ladero, Victor; García, Pilar; Bascarán, Victoria; Herrero, Mónica; Alvarez, Miguel A.; Suárez, Juan E.

    1998-01-01

    The repressor gene of the Lactobacillus phage A2 has the following properties: it (i) encodes a 224-residue polypeptide with DNA binding and RecA cleavage motifs, (ii) is expressed in lysogenic cultures, and (iii) confers superinfection immunity on the host. Adjacent, but divergently transcribed, lies another open reading frame whose product resembles the λ Cro protein. In the 161-bp intergenic segment, putative promoters and operators have been detected. PMID:9642205

  1. Discovering putative prion sequences in complete proteomes using probabilistic representations of Q/N-rich domains

    PubMed Central

    2013-01-01

    Background Prion proteins conform a special class among amyloids due to their ability to transmit aggregative folds. Prions are known to act as infectious agents in neurodegenerative diseases in animals, or as key elements in transcription and translation processes in yeast. It has been suggested that prions contain specific sequential domains with distinctive amino acid composition and physicochemical properties that allow them to control the switch between soluble and β-sheet aggregated states. Those prion-forming domains are low complexity segments enriched in glutamine/asparagine and depleted in charged residues and prolines. Different predictive methods have been developed to discover novel prions by either assessing the compositional bias of these stretches or estimating the propensity of protein sequences to form amyloid aggregates. However, the available algorithms hitherto lack a thorough statistical calibration against large sequence databases, which makes them unable to accurately predict prions without retrieving a large number of false positives. Results Here we present a computational strategy to predict putative prion-forming proteins in complete proteomes using probabilistic representations of prionogenic glutamine/asparagine rich regions. After benchmarking our predictive model against large sets of non-prionic sequences, we were able to filter out known prions with high precision and accuracy, generating prediction sets with few false positives. The algorithm was used to scan all the proteomes annotated in public databases for the presence of putative prion proteins. We analyzed the presence of putative prion proteins in all taxa, from viruses and archaea to plants and higher eukaryotes, and found that most organisms encode evolutionarily unrelated proteins with susceptibility to behave as prions. Conclusions To our knowledge, this is the first wide-ranging study aiming to predict prion domains in complete proteomes. Approaches of this kind could

  2. Crystal Structure of a Putative HTH-Type Transcriptional Regulator yxaF from Bacillus subtilis

    SciTech Connect

    Seetharaman,J.; Kumaran, D.; Bonanno, J.; Burley, S.; Swaminathan, S.

    2006-01-01

    The New York Structural GenomiX Research Consortium (NYSGXRC) has selected the protein coded by yxaF gene from Bacillus subtilis as a target for structure determination. The yxaF protein has 191 residues with a molecular mass of 21 kDa and had no sequence homology to any structure in the Protein Data Bank (PDB) at the time of target selection. We aimed to elucidate the three-dimensional structure for the putative protein yxaF to better understand the relationship between protein sequence, structure, and function. This protein is annotated as a putative helix-turn-helix (HTH) type transcriptional regulator. Many transcriptional regulators like TetR and QacR use a structurally well-defined DNA-binding HTH motif to recognize the target DNA sequences. DNA-HTH motif interactions have been extensively studied. As the HTH motif is structurally conserved in many regulatory proteins, these DNA-protein complexes show some similarity in DNA recognition patterns. Many such regulatory proteins have a ligand-binding domain in addition to the DNA-binding domain. Structural studies on ligand-binding regulatory proteins provide a wealth of information on ligand-, and possibly drug-, binding mechanisms. Understanding the ligand-binding mechanism may help overcome problems with drug resistance, which represent increasing challenges in medicine. The protein encoded by yxaF, hereafter called T1414, shows fold similar to QacR repressor and TetR/CamR repressor and possesses putative DNA and ligand-binding domains. Here, we report the crystal structure of T1414 and compare it with structurally similar drug and DNA-binding proteins.

  3. The post-translational modification of the Clostridium difficile flagellin affects motility, cell surface properties and virulence

    PubMed Central

    Faulds-Pain, Alexandra; Twine, Susan M; Vinogradov, Evgeny; Strong, Philippa C R; Dell, Anne; Buckley, Anthony M; Douce, Gillian R; Valiente, Esmeralda; Logan, Susan M; Wren, Brendan W

    2014-01-01

    Clostridium difficile is a prominent nosocomial pathogen, proliferating and causing enteric disease in individuals with a compromised gut microflora. We characterized the post-translational modification of flagellin in C. difficile 630. The structure of the modification was solved by nuclear magnetic resonance and shown to contain an N-acetylglucosamine substituted with a phosphorylated N-methyl-l-threonine. A reverse genetics approach investigated the function of the putative four-gene modification locus. All mutants were found to have truncated glycan structures by LC-MS/MS, taking into account bioinformatic analysis, we propose that the open reading frame CD0241 encodes a kinase involved in the transfer of the phosphate to the threonine, the CD0242 protein catalyses the addition of the phosphothreonine to the N-acetylglucosamine moiety and CD0243 transfers the methyl group to the threonine. Some mutations affected motility and caused cells to aggregate to each other and abiotic surfaces. Altering the structure of the flagellin modification impacted on colonization and disease recurrence in a murine model of infection, showing that alterations in the surface architecture of C. difficile vegetative cells can play a significant role in disease. We show that motility is not a requirement for colonization, but that colonization was compromised when the glycan structure was incomplete. PMID:25135277

  4. Face and object encoding under perceptual load: ERP evidence.

    PubMed

    Neumann, Markus F; Mohamed, Tarik N; Schweinberger, Stefan R

    2011-02-14

    According to the perceptual load theory, processing of a task-irrelevant distractor is abolished when attentional resources are fully consumed by task-relevant material. As an exception, however, famous faces have been shown to elicit repetition modulations in event-related potentials - an N250r - despite high load at initial presentation, suggesting preserved face-encoding. Here, we recorded N250r repetition modulations by unfamiliar faces, hands, and houses, and tested face specificity of preserved encoding under high load. In an immediate (S1-S2) repetition priming paradigm, participants performed a letter identification task on S1 by indicating whether an "X" vs. "N" was among 6 different (high load condition) or 6 identical (low load condition) letters. Letter strings were superimposed on distractor faces, hands, or houses. Subsequent S2 probes were either identical repetitions of S1 distractors, non-repeated exemplars from the same category, or infrequent butterflies, to which participants responded. Independent of attentional load at S1, an occipito-temporal N250r was found for unfamiliar faces. In contrast, no repetition-related neural modulation emerged for houses or hands. This strongly suggests that a putative face-selective attention module supports encoding under high load, and that similar mechanisms are unavailable for other natural or artificial objects. PMID:21044688

  5. Revealing the density of encoded functions in a viral RNA

    PubMed Central

    Patel, Nikesh; Dykeman, Eric C.; Coutts, Robert H. A.; Lomonossoff, George P.; Rowlands, David J.; Phillips, Simon E. V.; Ranson, Neil; Twarock, Reidun; Tuma, Roman; Stockley, Peter G.

    2015-01-01

    We present direct experimental evidence that assembly of a single-stranded RNA virus occurs via a packaging signal-mediated mechanism. We show that the sequences of coat protein recognition motifs within multiple, dispersed, putative RNA packaging signals, as well as their relative spacing within a genomic fragment, act collectively to influence the fidelity and yield of capsid self-assembly in vitro. These experiments confirm that the selective advantages for viral yield and encapsidation specificity, predicted from previous modeling of packaging signal-mediated assembly, are found in Nature. Regions of the genome that act as packaging signals also function in translational and transcriptional enhancement, as well as directly coding for the coat protein, highlighting the density of encoded functions within the viral RNA. Assembly and gene expression are therefore direct molecular competitors for different functional folds of the same RNA sequence. The strongest packaging signal in the test fragment, encodes a region of the coat protein that undergoes a conformational change upon contact with packaging signals. A similar phenomenon occurs in other RNA viruses for which packaging signals are known. These contacts hint at an even deeper density of encoded functions in viral RNA, which if confirmed, would have profound consequences for the evolution of this class of pathogens. PMID:25646435

  6. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120.

    PubMed

    Frébortová, Jitka; Greplová, Marta; Seidl, Michael F; Heyl, Alexander; Frébort, Ivo

    2015-01-01

    Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants. PMID:26376297

  7. Duplications and losses in gene families of rust pathogens highlight putative effectors

    PubMed Central

    Pendleton, Amanda L.; Smith, Katherine E.; Feau, Nicolas; Martin, Francis M.; Grigoriev, Igor V.; Hamelin, Richard; Nelson, C. Dana; Burleigh, J. Gordon; Davis, John M.

    2014-01-01

    Rust fungi are a group of fungal pathogens that cause some of the world's most destructive diseases of trees and crops. A shared characteristic among rust fungi is obligate biotrophy, the inability to complete a lifecycle without a host. This dependence on a host species likely affects patterns of gene expansion, contraction, and innovation within rust pathogen genomes. The establishment of disease by biotrophic pathogens is reliant upon effector proteins that are encoded in the fungal genome and secreted from the pathogen into the host's cell apoplast or within the cells. This study uses a comparative genomic approach to elucidate putative effectors and determine their evolutionary histories. We used OrthoMCL to identify nearly 20,000 gene families in proteomes of 16 diverse fungal species, which include 15 basidiomycetes and one ascomycete. We inferred patterns of duplication and loss for each gene family and identified families with distinctive patterns of expansion/contraction associated with the evolution of rust fungal genomes. To recognize potential contributors for the unique features of rust pathogens, we identified families harboring secreted proteins that: (i) arose or expanded in rust pathogens relative to other fungi, or (ii) contracted or were lost in rust fungal genomes. While the origin of rust fungi appears to be associated with considerable gene loss, there are many gene duplications associated with each sampled rust fungal genome. We also highlight two putative effector gene families that have expanded in Cqf that we hypothesize have roles in pathogenicity. PMID:25018762

  8. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120

    PubMed Central

    Frébortová, Jitka; Greplová, Marta; Seidl, Michael F.; Heyl, Alexander; Frébort, Ivo

    2015-01-01

    Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants. PMID:26376297

  9. Functional analysis of conserved residues in the putative "finger" domain of telomerase reverse transcriptase.

    PubMed

    Bosoy, D; Lue, N F

    2001-12-01

    Telomerase is a ribonucleoprotein reverse transcriptase (RT) responsible for the maintenance of one strand of telomere terminal repeats. The catalytic protein subunit of telomerase, known generically as telomerase reverse transcriptase (TERT), exhibits significant homology to RTs encoded by retroviruses and retroelements. The polymerization mechanisms of telomerase may therefore be similar to those of the "conventional" RTs. In this study, we explored the extent of mechanistic conservation by analyzing mutations of conserved residues within the putative "finger" domain of TERT. Previous analysis has implicated this domain of retroviral RTs in nucleotide and RNA binding and in processivity control. Our results demonstrate that residues conserved between TERT and human immunodeficiency virus-1 RT are more likely than TERT-specific residues to be required for enzyme activity. In addition, residues presumed to make direct contact with either the RNA or nucleotide substrate appear to be functionally more important. Furthermore, distinct biochemical defects can be observed for alterations in the putative RNA- and nucleotide-binding TERT residues in a manner that can be rationalized by their postulated mechanisms of action. This study thus supports a high degree of mechanistic conservation between telomerase and retroviral RTs and underscores the roles of distinct aspects of telomerase biochemistry in telomere length maintenance. PMID:11581271

  10. Comparative Transcriptome Analysis Identifies Putative Genes Involved in the Biosynthesis of Xanthanolides in Xanthium strumarium L.

    PubMed Central

    Li, Yuanjun; Gou, Junbo; Chen, Fangfang; Li, Changfu; Zhang, Yansheng

    2016-01-01

    Xanthium strumarium L. is a traditional Chinese herb belonging to the Asteraceae family. The major bioactive components of this plant are sesquiterpene lactones (STLs), which include the xanthanolides. To date, the biogenesis of xanthanolides, especially their downstream pathway, remains largely unknown. In X. strumarium, xanthanolides primarily accumulate in its glandular trichomes. To identify putative gene candidates involved in the biosynthesis of xanthanolides, three X. strumarium transcriptomes, which were derived from the young leaves of two different cultivars and the purified glandular trichomes from one of the cultivars, were constructed in this study. In total, 157 million clean reads were generated and assembled into 91,861 unigenes, of which 59,858 unigenes were successfully annotated. All the genes coding for known enzymes in the upstream pathway to the biosynthesis of xanthanolides were present in the X. strumarium transcriptomes. From a comparative analysis of the X. strumarium transcriptomes, this study identified a number of gene candidates that are putatively involved in the downstream pathway to the synthesis of xanthanolides, such as four unigenes encoding CYP71 P450s, 50 unigenes for dehydrogenases, and 27 genes for acetyltransferases. The possible functions of these four CYP71 candidates are extensively discussed. In addition, 116 transcription factors that are highly expressed in X. strumarium glandular trichomes were also identified. Their possible regulatory roles in the biosynthesis of STLs are discussed. The global transcriptomic data for X. strumarium should provide a valuable resource for further research into the biosynthesis of xanthanolides. PMID:27625674

  11. Identification and functional analysis of Penicillium digitatum genes putatively involved in virulence towards citrus fruit.

    PubMed

    López-Pérez, Mario; Ballester, Ana-Rosa; González-Candelas, Luis

    2015-04-01

    The fungus Penicillium digitatum, the causal agent of green mould rot, is the most destructive post-harvest pathogen of citrus fruit in Mediterranean regions. In order to identify P. digitatum genes up-regulated during the infection of oranges that may constitute putative virulence factors, we followed a polymerase chain reaction (PCR)-based suppression subtractive hybridization and cDNA macroarray hybridization approach. The origin of expressed sequence tags (ESTs) was determined by comparison against the available genome sequences of both organisms. Genes coding for fungal proteases and plant cell wall-degrading enzymes represent the largest categories in the subtracted cDNA library. Northern blot analysis of a selection of P. digitatum genes, including those coding for proteases, cell wall-related enzymes, redox homoeostasis and detoxification processes, confirmed their up-regulation at varying time points during the infection process. Agrobacterium tumefaciens-mediated transformation was used to generate knockout mutants for two genes encoding a pectin lyase (Pnl1) and a naphthalene dioxygenase (Ndo1). Two independent P. digitatum Δndo1 mutants were as virulent as the wild-type. However, the two Δpnl1 mutants analysed were less virulent than the parental strain or an ectopic transformant. Together, these results provide a significant advance in our understanding of the putative determinants of the virulence mechanisms of P. digitatum. PMID:25099378

  12. An assemblage of divergent variants of a novel putative closterovirus from American persimmon.

    PubMed

    Ito, Takao; Sato, Akihiko; Suzaki, Koichi

    2015-08-01

    Deep-sequencing analysis of nucleic acids extracted from leaf tissue of an American persimmon (Diospyros virginiana L.) and subsequent-sequencing analyses uncovered at least four distinct closterovirus-like molecules. Two complete genomes of 18,569 and 18,030 nucleotides (nt) and partial genomes of 4,899 and 9,019 nt were determined. The two complete genomes encoded 11 potential open reading frames and the characteristic organization of closteroviruses. Among the four genomes, the putative heat shock protein 70 homolog (HSP70h), RNA-dependent RNA polymerase, and coat protein showed 82-85, 72-91, and 84-87 % amino acid sequence identities, respectively. These results suggested that the four identified viruses could be divergent variants in a single host plant. The phylogenetic tree based on HSP70h showed that their closest relative, although distant, is Olive leaf yellowing-associated virus, a putative unassigned member of the family Closteroviridae. The name Persimmon virus B was proposed for this new virus, representing another unassigned member of the family. PMID:25921465

  13. Protection of Mice from Fatal Measles Encephalitis by Vaccination with Vaccinia Virus Recombinants Encoding Either the Hemagglutinin or the Fusion Protein

    NASA Astrophysics Data System (ADS)

    Drillien, Robert; Spehner, Daniele; Kirn, Andre; Giraudon, Pascale; Buckland, Robin; Wild, Fabian; Lecocq, Jean-Pierre

    1988-02-01

    Vaccinia virus recombinants encoding the hemagglutinin or fusion protein of measles virus have been constructed. Infection of cell cultures with the recombinants led to the synthesis of authentic measles proteins as judged by their electrophoretic mobility, recognition by antibodies, glycosylation, proteolytic cleavage, and presentation on the cell surface. Mice vaccinated with a single dose of the recombinant encoding the hemagglutinin protein developed antibodies capable of both inhibiting hemagglutination activity and neutralizing measles virus, whereas animals vaccinated with the recombinant encoding the fusion protein developed measles neutralizing antibodies. Mice vaccinated with either of the recombinants resisted a normally lethal intracerebral inoculation of a cell-associated measles virus subacute sclerosing panencephalitis strain.

  14. Gravity referenced elevation encoder development

    NASA Astrophysics Data System (ADS)

    Goddard, R. E.

    1993-05-01

    Recent progress in the development of a gravity-sensor-based instrument for determining the elevation angle of DSN antennas is described. The benefits of such a system include the capability to locate the Gravity Referenced Elevation Encoder (GREE) directly on the primary reflector (thus bypassing structural flexure and deformation error sources), anticipated lower maintenance costs compared to the present gimbal encoders, direct replaceability, or supplementation of the present gimbal encoders and the utilization of off-the-shelf components to construct the GREE. This article includes a description of the nominal GREE design. Test results on a laboratory breadboard model are given. Rigid-body dynamics of the GREE are derived and the simulated performance in response to measured antenna vibrations is given.

  15. Gravity referenced elevation encoder development

    NASA Technical Reports Server (NTRS)

    Goddard, R. E.

    1993-01-01

    Recent progress in the development of a gravity-sensor-based instrument for determining the elevation angle of DSN antennas is described. The benefits of such a system include the capability to locate the Gravity Referenced Elevation Encoder (GREE) directly on the primary reflector (thus bypassing structural flexure and deformation error sources), anticipated lower maintenance costs compared to the present gimbal encoders, direct replaceability, or supplementation of the present gimbal encoders and the utilization of off-the-shelf components to construct the GREE. This article includes a description of the nominal GREE design. Test results on a laboratory breadboard model are given. Rigid-body dynamics of the GREE are derived and the simulated performance in response to measured antenna vibrations is given.

  16. Fly Photoreceptors Encode Phase Congruency

    PubMed Central

    Friederich, Uwe; Billings, Stephen A.; Hardie, Roger C.; Juusola, Mikko; Coca, Daniel

    2016-01-01

    More than five decades ago it was postulated that sensory neurons detect and selectively enhance behaviourally relevant features of natural signals. Although we now know that sensory neurons are tuned to efficiently encode natural stimuli, until now it was not clear what statistical features of the stimuli they encode and how. Here we reverse-engineer the neural code of Drosophila photoreceptors and show for the first time that photoreceptors exploit nonlinear dynamics to selectively enhance and encode phase-related features of temporal stimuli, such as local phase congruency, which are invariant to changes in illumination and contrast. We demonstrate that to mitigate for the inherent sensitivity to noise of the local phase congruency measure, the nonlinear coding mechanisms of the fly photoreceptors are tuned to suppress random phase signals, which explains why photoreceptor responses to naturalistic stimuli are significantly different from their responses to white noise stimuli. PMID:27336733

  17. Fly Photoreceptors Encode Phase Congruency.

    PubMed

    Friederich, Uwe; Billings, Stephen A; Hardie, Roger C; Juusola, Mikko; Coca, Daniel

    2016-01-01

    More than five decades ago it was postulated that sensory neurons detect and selectively enhance behaviourally relevant features of natural signals. Although we now know that sensory neurons are tuned to efficiently encode natural stimuli, until now it was not clear what statistical features of the stimuli they encode and how. Here we reverse-engineer the neural code of Drosophila photoreceptors and show for the first time that photoreceptors exploit nonlinear dynamics to selectively enhance and encode phase-related features of temporal stimuli, such as local phase congruency, which are invariant to changes in illumination and contrast. We demonstrate that to mitigate for the inherent sensitivity to noise of the local phase congruency measure, the nonlinear coding mechanisms of the fly photoreceptors are tuned to suppress random phase signals, which explains why photoreceptor responses to naturalistic stimuli are significantly different from their responses to white noise stimuli. PMID:27336733

  18. Nanoscale analysis of caspofungin-induced cell surface remodelling in Candida albicans

    NASA Astrophysics Data System (ADS)

    El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Alsteens, David; Jackson, Desmond N.; Lipke, Peter N.; Dufrêne, Yves F.

    2013-01-01

    The advent of fungal pathogens that are resistant to the classic repertoire of antifungal drugs has increased the need for new therapeutic agents. A prominent example of such a novel compound is caspofungin, known to alter cell wall biogenesis by inhibiting β-1,3-d-glucan synthesis. Although much progress has been made in understanding the mechanism of action of caspofungin, little is known about its influence on the biophysical properties of the fungal cells. Here, we use atomic force microscopy (AFM) to demonstrate that caspofungin induces major remodelling of the cell surface properties of Candida albicans. Caspofungin causes major morphological and structural alterations of the cells, which correlate with a decrease of the cell wall mechanical strength. Moreover, we find that the drug induces the massive exposure of the cell adhesion protein Als1 on the cell surface and leads to increased cell surface hydrophobicity, two features that trigger cell aggregation. This behaviour is not observed in yeast species lacking Als1, demonstrating the key role that the protein plays in determining the aggregation phenotype of C. albicans. The results show that AFM opens up new avenues for understanding the molecular bases of microbe-drug interactions and for developing new therapeutic agents.The advent of fungal pathogens that are resistant to the classic repertoire of antifungal drugs has increased the need for new therapeutic agents. A prominent example of such a novel compound is caspofungin, known to alter cell wall biogenesis by inhibiting β-1,3-d-glucan synthesis. Although much progress has been made in understanding the mechanism of action of caspofungin, little is known about its influence on the biophysical properties of the fungal cells. Here, we use atomic force microscopy (AFM) to demonstrate that caspofungin induces major remodelling of the cell surface properties of Candida albicans. Caspofungin causes major morphological and structural alterations of the

  19. A Comparative Pan-Genome Perspective of Niche-Adaptable Cell-Surface Protein Phenotypes in Lactobacillus rhamnosus

    PubMed Central

    Kant, Ravi; Sigvart-Mattila, Pia; Paulin, Lars; Mecklin, Jukka-Pekka; Saarela, Maria; Palva, Airi; von Ossowski, Ingemar

    2014-01-01

    Lactobacillus rhamnosus is a ubiquitously adaptable Gram-positive bacterium and as a typical commensal can be recovered from various microbe-accessible bodily orifices and cavities. Then again, other isolates are food-borne, with some of these having been long associated with naturally fermented cheeses and yogurts. Additionally, because of perceived health benefits to humans and animals, numerous L. rhamnosus strains have been selected for use as so-called probiotics and are often taken in the form of dietary supplements and functional foods. At the genome level, it is anticipated that certain genetic variances will have provided the niche-related phenotypes that augment the flexible adaptiveness of this species, thus enabling its strains to grow and survive in their respective host environments. For this present study, we considered it functionally informative to examine and catalogue the genotype-phenotype variation existing at the cell surface between different L. rhamnosus strains, with the presumption that this might be relatable to habitat preferences and ecological adaptability. Here, we conducted a pan-genomic study involving 13 genomes from L. rhamnosus isolates with various origins. In using a benchmark strain (gut-adapted L. rhamnosus GG) for our pan-genome comparison, we had focused our efforts on a detailed examination and description of gene products for certain functionally relevant surface-exposed proteins, each of which in effect might also play a part in niche adaptability among the other strains. Perhaps most significantly of the surface protein loci we had analyzed, it would appear that the spaCBA operon (known to encode SpaCBA-called pili having a mucoadhesive phenotype) is a genomic rarity and an uncommon occurrence in L. rhamnosus. However, for any of the so-piliated L. rhamnosus strains, they will likely possess an increased niche-specific fitness, which functionally might presumably be manifested by a protracted transient colonization of

  20. Cell surface polypeptide CshA mediates binding of Streptococcus gordonii to other oral bacteria and to immobilized fibronectin.

    PubMed Central

    McNab, R; Holmes, A R; Clarke, J M; Tannock, G W; Jenkinson, H F

    1996-01-01

    Isogenic mutants of Streptococcus gordonii DL1 (Challis) in which the genes encoding high-molecular-mass cell surface polypeptides CshA and/or CshB were inactivated were deficient in binding to four strains of Actinomyces naeslundii and two strains of Streptococcus oralis. Lactose-sensitive interactions of S. gordonii with A. naeslundii ATCC 12104 and PK606 were associated with expression of cshA but not of cshB. Lactose-insensitive interactions of S. gordonii with A. naeslundii T14V and WVU627, and with S. oralis C104 and 34, were dependent on expression of cshA and cshB. S. gordonii DL1 cells bound to immobilized human fibronectin (Fn), but not to soluble Fn, in a dose-dependent manner, and binding was noninhibitable by heparin. S. gordonii cshA and cshB mutants were also deficient in binding to immobilized human Fn. Antibodies to an NH2-terminal nonrepetitive region (amino acid residues 42 to 886) of recombinant CshA inhibited binding of S. gordonii DL1 cells to A. naeslundii T14V and PK606 and to immobilized Fn. Conversely, antibodies to an amino acid repeat block segment of the COOH-terminal domain (amino acid residues 2026 to 2508) were not inhibitory to adherence. Assays using CshA-specific antibodies revealed that surface expression of CshA was reduced in cshB mutants. The results suggest that CshA acts as a multifunctional adhesin in S. gordonii and that major adhesion-mediating sequences are specified within the nonrepetitive NH2-terminal region of the polypeptide. PMID:8926089

  1. CREST - a large and diverse superfamily of putative transmembrane hydrolases

    PubMed Central

    2011-01-01

    Background A number of membrane-spanning proteins possess enzymatic activity and catalyze important reactions involving proteins, lipids or other substrates located within or near lipid bilayers. Alkaline ceramidases are seven-transmembrane proteins that hydrolyze the amide bond in ceramide to form sphingosine. Recently, a group of putative transmembrane receptors called progestin and adipoQ receptors (PAQRs) were found to be distantly related to alkaline ceramidases, raising the possibility that they may also function as membrane enzymes. Results Using sensitive similarity search methods, we identified statistically significant sequence similarities among several transmembrane protein families including alkaline ceramidases and PAQRs. They were unified into a large and diverse superfamily of putative membrane-bound hydrolases called CREST (alkaline ceramidase, PAQR receptor, Per1, SID-1 and TMEM8). The CREST superfamily embraces a plethora of cellular functions and biochemical activities, including putative lipid-modifying enzymes such as ceramidases and the Per1 family of putative phospholipases involved in lipid remodeling of GPI-anchored proteins, putative hormone receptors, bacterial hemolysins, the TMEM8 family of putative tumor suppressors, and the SID-1 family of putative double-stranded RNA transporters involved in RNA interference. Extensive similarity searches and clustering analysis also revealed several groups of proteins with unknown function in the CREST superfamily. Members of the CREST superfamily share seven predicted core transmembrane segments with several conserved sequence motifs. Conclusions Universal conservation of a set of histidine and aspartate residues across all groups in the CREST superfamily, coupled with independent discoveries of hydrolase activities in alkaline ceramidases and the Per1 family as well as results from previous mutational studies of Per1, suggests that the majority of CREST members are metal-dependent hydrolases

  2. Deletion of PREPL, a Gene Encoding a Putative Serine Oligopeptidase, in Patients with Hypotonia-Cystinuria Syndrome

    PubMed Central

    Jaeken, Jaak; Martens, Kevin; François, Inge; Eyskens, François; Lecointre, Claudine; Derua, Rita; Meulemans, Sandra; Slootstra, Jerry W.; Waelkens, Etienne; Zegher, Francis de; Creemers, John W. M.; Matthijs, Gert

    2006-01-01

    In 11 patients with a recessive congenital disorder, which we refer to as “the hypotonia-cystinuria syndrome,” microdeletion of part of the SLC3A1 and PREPL genes on chromosome 2p21 was found. Patients present with generalized hypotonia at birth, nephrolithiasis, growth hormone deficiency, minor facial dysmorphism, and failure to thrive, followed by hyperphagia and rapid weight gain in late childhood. Since loss-of-function mutations in SLC3A1 are known to cause isolated cystinuria type I, and since the expression of the flanking genes, C2orf34 and PPM1B, was normal, the extended phenotype can be attributed to the deletion of PREPL. PREPL is localized in the cytosol and shows homology with prolyl endopeptidase and oligopeptidase B. Substitution of the predicted catalytic residues (Ser470, Asp556, and His601) by alanines resulted in loss of reactivity with a serine hydrolase-specific probe. In sharp contrast to prolyl oligopeptidase and oligopeptidase B, which require both aminoterminal and carboxyterminal sequences for activity, PREPL activity appears to depend only on the carboxyterminal domain. Taken together, these results suggest that PREPL is a novel oligopeptidase, with unique structural and functional characteristics, involved in hypotonia-cystinuria syndrome. PMID:16385448

  3. [Characterization of a putative S locus encoded receptor protein kinase and its role in self-incompatibility

    SciTech Connect

    Not Available

    1993-01-01

    The serine/threonine protein kinase (SRK) protein was predicted to be similar to the growth factor receptor tyrosine kinases in animals but its amino acid sequence of the catalytic domain is more similar to that of the catalytic domains of protein serine/threonine kinases than to protein tyrosine kinases. We have shown that the SRK protein has intrinsic scrine/threonine kinase activity. We subcloned the protein kinase-homologous domain of the SRK[sub 6] cDNA into the bacterial expression vector pGEX-3X and we have constructed a second plasmid identical to the first except that it carried a conservative mutation that substituted Arg for the Lys[sup 524] codon of SRK6 This lysine corresponds to the ATP-binding site, is essential in protein kinases, and is a common target for site-directed mutagenesis as a means to obtain kinase-defective proteins. Cultures bearing the wild-type and mutant SRK catalytic domains each produced an approximately 64 kD protein that reacted with anti-SRK6 antibodies. Following pulse-labeling with [sup 32]P we found that the wild-type SRK6 protein but not the mutant form was detectably phosphorylated. Phosphoamino acid analysis of the affinity purified [sup 32]p-labeled GST-SRK6 fusion protein demonstrated that SRK was phosphorylated predominantly on semine and to a lesser extent on threonine, but not on tyrosine. Thus, SRK6 is a functional serine/threonine protein kinase.

  4. Characterization of a putative S-locus encoded receptor protein kinase and its role in self-incompatibility. Progress report

    SciTech Connect

    Nasrallah, J.B.

    1994-05-01

    The major results of our research effort include the determination of the S-Receptor Kinase (SRK) gene structure, the demonstration of S-haplotype-associated SRK polymorphisms and possible co-evolution of SRK and SLG, the characterization of the temporal and spatial expression patterns of SRK, and the demonstration that SRK has intrinsic serine/threonine kinase activity. Our results have indicated that SLG originated from an SRK-like gene by a gene duplication event and suggested a possible molecular basis for leaky S haplotypes. The data have allowed us to develop a model of self-incompatibility based on the interaction of SRK and SLG and the activation of SRK in response to self-pollination. More generally, the information that we have obtained is potentially relevant to understanding mechanisms of signalling inplants. Thus, the interaction of membrane-based receptor protein kinases with secreted forms of their extracellular domains may represent a generalized mechanism by which receptors signal across the plant cell wall.

  5. Characterization of a Pinus pinaster cDNA encoding an auxin up-regulated putative peroxidase in roots.

    PubMed

    Charvet-Candela, V; Hitchin, S; Reddy, M S; Cournoyer, B; Marmeisse, R; Gay, G

    2002-03-01

    As part of a study to identify host plant genes regulated by fungal auxin during ectomycorrhiza formation, we differentially screened a cDNA library constructed from roots of auxin-treated Pinus pinaster (Ait.) Sol. seedlings. We identified three cDNAs up-regulated by auxin. Sequence analysis of one of these cDNAs, PpPrx75, revealed the presence of an open reading frame of 216 amino acids with the characteristic consensus sequences of plant peroxidases. The deduced amino acid sequence showed homology with Arabidopsis thaliana (L.) Heynh., Arachis hypogaea L. and Stylosanthes humilis HBK cationic peroxidases. Amino acid sequence identities in the conserved domains of plant peroxidases ranged from 60 to 100%. In PpPrx75, there are five cysteine residues and one histidine residue that are found at conserved positions among other peroxidases. A potential glycosylation site (NTS) is present in the deduced sequence. Phylogenetic analysis showed that PpPrx75 is closely related to two A. thaliana peroxidases. The PpPrx75 cDNA was induced by active auxins, ethylene, abscisic acid and quercetin, a flavonoid possibly involved in plant-microorganism interactions. Transcript accumulation was detected within 3 h following root induction by auxin, and the amount of mRNA increased over the following 24 h. The protein synthesis inhibitor cycloheximide did not inhibit indole-3-acetic acid-induced transcript accumulation, suggesting that PpPrx75 induction is a primary (direct) response to auxin. This cDNA can be used to study expression of an auxin-regulated peroxidase during ectomycorrhiza formation. PMID:11874719

  6. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 1 2012-10-01 2012-10-01 false EAS Encoder. 11.32 Section 11.32 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL EMERGENCY ALERT SYSTEM (EAS) Equipment Requirements § 11.32 EAS Encoder. (a) EAS Encoders must at a minimum be capable of encoding the EAS protocol described in § 11.31 and providing the EAS...

  7. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 1 2014-10-01 2014-10-01 false EAS Encoder. 11.32 Section 11.32 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL EMERGENCY ALERT SYSTEM (EAS) Equipment Requirements § 11.32 EAS Encoder. (a) EAS Encoders must at a minimum be capable of encoding the EAS protocol described in § 11.31 and providing the EAS...

  8. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 1 2011-10-01 2011-10-01 false EAS Encoder. 11.32 Section 11.32 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL EMERGENCY ALERT SYSTEM (EAS) Equipment Requirements § 11.32 EAS Encoder. (a) EAS Encoders must at a minimum be capable of encoding the EAS protocol described in § 11.31 and providing the EAS...

  9. Comparison of putative circulating cancer stem cell detection between the hepatic portal system and peripheral blood in colorectal cancer patients

    PubMed Central

    Park, Byung Soo; Jung, Seok Yun; Kwon, Sang Mo; Bae, Jae Ho; Lee, Sun Min; Shin, Dong Hoon

    2014-01-01

    Purpose The present pilot study was conducted to detect putative cancer stem cell (CSC) from the hepatic portal system and peripheral blood in the colorectal cancer patients and to compare them to healthy donor and diverticulitis patients. Methods Laboratory study was performed to identify the expression of cell surface markers, epithelial cell adhesion molecule (EpCAM), cytokeratin (CK) 18, CK20, CD44, and CD133, on several colon cancer cell lines. Clinical pilot study was conducted to detect putative circulating CSC as EpCAM+CD133+ cell in colorectal cancer (n = 10), diverticulitis (n = 5), and four healthy donors, by using flow cytometry. Blood was drawn from the hepatic portal system and peripheral vein. Results On laboratory study, EpCAM was expressed in whole colon cancer cell lines, and CD44 and CD133 were simultaneously expressed in 50% of the cell lines with stemness phenotype, but CK18 and CK20 were not expressed in most of the cell lines. On clinical study, the mean EpCAM+CD133+ cell counts of 11.6/105 in the hepatic portal system were somewhat lower than 15.4/105 in peripheral vein (P = 0.241). As for diverticulitis patients, EpCAM+CD133+ cells were also detected to have steeper dropped to near zero, after the surgery. Conclusion The numbers of putative CSC were not statistically different between the detection sites of the portal vein and peripheral vein in the colon cancer patients. Therefore, we may not have benefitted by getting the cells from the hepatic portal system. In addition, the CD133+EpCAM+ cells in the colon cancer patients might contain normal stem cells from cancer inflammation similar to diverticulitis. PMID:25368848

  10. An AFM-based pit-measuring method for indirect measurements of cell-surface membrane vesicles

    SciTech Connect

    Zhang, Xiaojun; Chen, Yuan; Chen, Yong

    2014-03-28

    Highlights: • Air drying induced the transformation of cell-surface membrane vesicles into pits. • An AFM-based pit-measuring method was developed to measure cell-surface vesicles. • Our method detected at least two populations of cell-surface membrane vesicles. - Abstract: Circulating membrane vesicles, which are shed from many cell types, have multiple functions and have been correlated with many diseases. Although circulating membrane vesicles have been extensively characterized, the status of cell-surface membrane vesicles prior to their release is less understood due to the lack of effective measurement methods. Recently, as a powerful, micro- or nano-scale imaging tool, atomic force microscopy (AFM) has been applied in measuring circulating membrane vesicles. However, it seems very difficult for AFM to directly image/identify and measure cell-bound membrane vesicles due to the similarity of surface morphology between membrane vesicles and cell surfaces. Therefore, until now no AFM studies on cell-surface membrane vesicles have been reported. In this study, we found that air drying can induce the transformation of most cell-surface membrane vesicles into pits that are more readily detectable by AFM. Based on this, we developed an AFM-based pit-measuring method and, for the first time, used AFM to indirectly measure cell-surface membrane vesicles on cultured endothelial cells. Using this approach, we observed and quantitatively measured at least two populations of cell-surface membrane vesicles, a nanoscale population (<500 nm in diameter peaking at ∼250 nm) and a microscale population (from 500 nm to ∼2 μm peaking at ∼0.8 μm), whereas confocal microscopy only detected the microscale population. The AFM-based pit-measuring method is potentially useful for studying cell-surface membrane vesicles and for investigating the mechanisms of membrane vesicle formation/release.

  11. Roles of regulated internalization in the polarization of cell surface receptors

    PubMed Central

    Tian, Wei; Cao, Youfang; Ismael, Amber; Stone, David

    2016-01-01

    Cell polarization, the generation of cellular asymmetries, is a fundamental biological process. Polarity of different molecules can arise through several mechanisms. Among these, internalization has been shown to play an important role in the polarization of cell surface receptors. The internalization of cell surface receptors can be upregulated upon ligand binding. Additional regulatory mechanism can downregulate the internalization process. Here we describe a general model, which incorporates these two opposing processes, to study the role of internalization in the establishment of cell polarity. We find that the competition between these two processes is sufficient to induce receptor polarization. Our results show that regulated internalization provides additional regulation on polarization as well. In addition, we discuss applications of our model to the yeast system, which shows the capability and potential of the model. PMID:25570171

  12. Cell-Surface Protein Profiling Identifies Distinctive Markers of Progenitor Cells in Human Skeletal Muscle.

    PubMed

    Uezumi, Akiyoshi; Nakatani, Masashi; Ikemoto-Uezumi, Madoka; Yamamoto, Naoki; Morita, Mitsuhiro; Yamaguchi, Asami; Yamada, Harumoto; Kasai, Takehiro; Masuda, Satoru; Narita, Asako; Miyagoe-Suzuki, Yuko; Takeda, Shin'ichi; Fukada, So-Ichiro; Nishino, Ichizo; Tsuchida, Kunihiro

    2016-08-01

    Skeletal muscle contains two distinct stem/progenitor populations. One is the satellite cell, which acts as a muscle stem cell, and the other is the mesenchymal progenitor, which contributes to muscle pathogeneses such as fat infiltration and fibrosis. Detailed and accurate characterization of these progenitors in humans remains elusive. Here, we performed comprehensive cell-surface protein profiling of the two progenitor populations residing in human skeletal muscle and identified three previously unrecognized markers: CD82 and CD318 for satellite cells and CD201 for mesenchymal progenitors. These markers distinguish myogenic and mesenchymal progenitors, and enable efficient isolation of the two types of progenitors. Functional study revealed that CD82 ensures expansion and preservation of myogenic progenitors by suppressing excessive differentiation, and CD201 signaling favors adipogenesis of mesenchymal progenitors. Thus, cell-surface proteins identified here are not only useful markers but also functionally important molecules, and provide valuable insight into human muscle biology and diseases. PMID:27509136

  13. Endocytosis of cell surface material mediates cell plate formation during plant cytokinesis.

    PubMed

    Dhonukshe, Pankaj; Baluska, Frantisek; Schlicht, Markus; Hlavacka, Andrej; Samaj, Jozef; Friml, Jirí; Gadella, Theodorus W J

    2006-01-01

    Dividing plant cells perform a remarkable task of building a new cell wall within the cytoplasm in a few minutes. A long-standing paradigm claims that this primordial cell wall, known as the cell plate, is generated by delivery of newly synthesized material from Golgi apparatus-originated secretory vesicles. Here, we show that, in diverse plant species, cell surface material, including plasma membrane proteins, cell wall components, and exogenously applied endocytic tracers, is rapidly delivered to the forming cell plate. Importantly, this occurs even when de novo protein synthesis is blocked. In addition, cytokinesis-specific syntaxin KNOLLE as well as plasma membrane (PM) resident proteins localize to endosomes that fuse to initiate the cell plate. The rate of endocytosis is strongly enhanced during cell plate formation, and its genetic or pharmacological inhibition leads to cytokinesis defects. Our results reveal that endocytic delivery of cell surface material significantly contributes to cell plate formation during plant cytokinesis. PMID:16399085

  14. Cell-surface nucleolin is sequestered into EPEC microcolonies and may play a role during infection.

    PubMed

    Dean, Paul; Kenny, Brendan

    2011-06-01

    Nucleolin is a prominent nucleolar protein that is mobilized into the cytoplasm during infection by enteropathogenic Escherichia coli (EPEC). Nucleolin also exists at low levels at the cell surface of eukaryotic cells and here we show that upon infection of an intestinal cell model, EPEC recruits and subsequently sequesters cell-surface EGFP-nucleolin into extracellularly located bacterial microcolonies. The recruitment of nucleolin was evident around bacteria within the centre of the microcolonies that were not directly associated with actin-based pedestals. Incubation of host intestinal cells with different ligands that specifically bind nucleolin impaired the ability of EPEC to disrupt epithelial barrier function but did not inhibit bacterial attachment or other effector-driven processes such as pedestal formation or microvilli effacement. Taken together, this work suggests that EPEC exploits two spatially distinct pools of nucleolin during the infection process. PMID:21436219

  15. Applications of yeast cell-surface display in bio-refinery.

    PubMed

    Kondo, Akihiko; Tanaka, Tsutomu; Hasunuma, Tomohisa; Ogino, Chiaki

    2010-11-01

    The dependency on depleting natural resources is a challenge for energy security that can be potentially answered by bioenergy. Bioenergy is derived from starchy and lignocellulosic biomass in the form of bioethanol or from vegetable oils in the form of biodiesel fuel. The acid and enzymatic methods have been developed for the hydrolysis of biomass and for transesterifiaction of plant oils. However, acid hydrolysis results in the production of unnatural compounds which has adverse effects on yeast fermentation. Recent advancements in the yeast cell surface engineering developed strategies to genetically immobilize amylolytic, cellulolytic and xylanolytic enzymes on yeast cell surface for the production of fuel ethanol from biomass. This review gives an insight in to the recent technological developments in the production of bioenergy, i.e, bioethanol using surface engineered yeast. PMID:21171959

  16. A Multichannel Biosensor for Rapid Determination of Cell Surface Glycomic Signatures

    PubMed Central

    2015-01-01

    Cell surface glycosylation serves a fundamental role in dictating cell and tissue behavior. Cell surface glycomes differ significantly, presenting viable biomarkers for identifying cell types and their states. Glycoprofiling is a challenging task, however, due to the complexity of the constituent glycans. We report here a rapid and effective sensor for surface-based cell differentiation that uses a three-channel sensor produced by noncovalent conjugation of a functionalized gold nanoparticle (AuNP) and fluorescent proteins. Wild-type and glycomutant mammalian cells were effectively stratified using fluorescence signatures obtained from a single sensor element. Blinded unknowns generated from the tested cell types were identified with high accuracy (44 out of 48 samples), validating the robustness of the multichannel sensor. Notably, this selectivity-based high-throughput sensor differentiated between cells, employing a nondestructive protocol that required only a single well of a microplate for detection. PMID:26405691

  17. Contribution of Cell Surface Hydrophobicity in the Resistance of Staphylococcus aureus against Antimicrobial Agents.

    PubMed

    Lather, Puja; Mohanty, A K; Jha, Pankaj; Garsa, Anita Kumari

    2016-01-01

    Staphylococcus aureus is found in a wide variety of habitats, including human skin, where many strains are commensals that may be clinically significant or contaminants of food. To determine the physiological characteristics of resistant strain of Staphylococcus aureus against pediocin, a class IIa bacteriocin, a resistant strain was compared with wild type in order to investigate the contribution of hydrophobicity to this resistance. Additional clumping of resistant strain relative to wild type in light microscopy was considered as an elementary evidence of resistance attainment. A delay in log phase attainment was observed in resistant strain compared to the wild type strain. A significant increase in cell surface hydrophobicity was detected for resistant strain in both hexadecane and xylene indicating the contribution of cell surface hydrophobicity as adaptive reaction against antimicrobial agents. PMID:26966577

  18. Spatiotemporal regulation of chemical reaction kinetics of cell surface molecules by active remodeling of cortical actin

    NASA Astrophysics Data System (ADS)

    Bhattacharyya, Bhaswati; Chaudhuri, Abhishek; Gowrishankar, Kripa; Mayor, Satyajit; Rao, Madan

    2010-03-01

    Cell surface proteins such as lipid tethered GPI-anchored proteins and Ras-proteins are distributed as monomers and nanoclusters on the surface of living cells. Recent work from our laboratory suggests that the spatial distribution and dynamics of formation and breakup of these nanoclusters is controlled by the active remodeling dynamics of the underlying cortical actin. To explain these observations, we propose a novel mechanism of nanoclustering, involving the transient binding to and advection along constitutively occuring ``asters'' of cortical actin. Here we study the consequences of such active actin based clustering, in the context of chemical reactions involving conformational changes of cell surface proteins. We find that active remodeling of cortical actin, can give rise to a dramatic increase in the reaction efficiency and output levels. In general, such actin driven clustering of membrane proteins could be a cellular mechanism to spatiotemporally regulate and amplify local chemical reaction rates, in the context of signalling and endocytosis.

  19. Contribution of Cell Surface Hydrophobicity in the Resistance of Staphylococcus aureus against Antimicrobial Agents

    PubMed Central

    Lather, Puja; Mohanty, A. K.; Jha, Pankaj; Garsa, Anita Kumari

    2016-01-01

    Staphylococcus aureus is found in a wide variety of habitats, including human skin, where many strains are commensals that may be clinically significant or contaminants of food. To determine the physiological characteristics of resistant strain of Staphylococcus aureus against pediocin, a class IIa bacteriocin, a resistant strain was compared with wild type in order to investigate the contribution of hydrophobicity to this resistance. Additional clumping of resistant strain relative to wild type in light microscopy was considered as an elementary evidence of resistance attainment. A delay in log phase attainment was observed in resistant strain compared to the wild type strain. A significant increase in cell surface hydrophobicity was detected for resistant strain in both hexadecane and xylene indicating the contribution of cell surface hydrophobicity as adaptive reaction against antimicrobial agents. PMID:26966577

  20. Human and rat mast cell high-affinity immunoglobulin E receptors: Characterization of putative. alpha. -chain gene products

    SciTech Connect

    Shimizu, Akira; Benfey, P.N.; Leder, P. ); Tepler, I. Brigham and Women's Hospital, Boston, MA ); Berenstein, E.H.; Siraganian, R.P. )

    1988-03-01

    The authors have cloned and determined the entire nucleotide sequence of cDNAs corresponding to the putative {alpha} subunits of the human and rat mast cell high-affinity IgE receptors. Both human and rat cDNAs encode an NH{sub 2}-terminal signal peptide, two immunoglobulin-like extracellular domains (encoded by discrete exons), a hydrophobic transmembrane region, and a positively charged cytoplasmic tail. The human and rat {alpha} subunits share an overall homology with one another and the immunoglobulin gene family, suggesting that they arose from a common ancestral gene and continue to share structural homology with their ligands. In addition, the rat gene is transcribed into at least three distinct forms, each of which yields a somewhat different coding sequence.

  1. How Infants Encode Spatial Extent

    ERIC Educational Resources Information Center

    Duffy, Sean; Huttenlocher, Janellen; Levine, Susan; Duffy, Renee

    2005-01-01

    This study explores how infants encode an object's spatial extent. We habituated 6.5-month-old infants to a dowel inside a container and then tested whether they dishabituate to a change in absolute size when the relation between dowel and container is held constant (by altering the size of both container and dowel) and when the relation changes…

  2. Shaft encoder presents digital output

    NASA Technical Reports Server (NTRS)

    Hillis, D. A.

    1966-01-01

    Circuits that include compensation circuitry time a capacitance relative to a reference voltage so that a digital presentation occurs that is representative of the positional condition of the mechanical shaft being monitored. This circuitry may be employed in multiples to furnish binary encoding of a number of rotating devices simultaneously.

  3. Encoding Standards for Linguistic Corpora.

    ERIC Educational Resources Information Center

    Ide, Nancy

    The demand for extensive reusability of large language text collections for natural languages processing research requires development of standardized encoding formats. Such formats must be capable of representing different kinds of information across the spectrum of text types and languages, capable of representing different levels of…

  4. Genome-wide analysis of putative peroxiredoxin in unicellular and filamentous cyanobacteria

    PubMed Central

    2012-01-01

    Background Cyanobacteria are photoautotrophic prokaryotes with wide variations in genome sizes and ecological habitats. Peroxiredoxin (PRX) is an important protein that plays essential roles in protecting own cells against reactive oxygen species (ROS). PRXs have been identified from mammals, fungi and higher plants. However, knowledge on cyanobacterial PRXs still remains obscure. With the availability of 37 sequenced cyanobacterial genomes, we performed a comprehensive comparative analysis of PRXs and explored their diversity, distribution, domain structure and evolution. Results Overall 244 putative prx genes were identified, which were abundant in filamentous diazotrophic cyanobacteria, Acaryochloris marina MBIC 11017, and unicellular cyanobacteria inhabiting freshwater and hot-springs, while poor in all Prochlorococcus and marine Synechococcus strains. Among these putative genes, 25 open reading frames (ORFs) encoding hypothetical proteins were identified as prx gene family members and the others were already annotated as prx genes. All 244 putative PRXs were classified into five major subfamilies (1-Cys, 2-Cys, BCP, PRX5_like, and PRX-like) according to their domain structures. The catalytic motifs of the cyanobacterial PRXs were similar to those of eukaryotic PRXs and highly conserved in all but the PRX-like subfamily. Classical motif (CXXC) of thioredoxin was detected in protein sequences from the PRX-like subfamily. Phylogenetic tree constructed of catalytic domains coincided well with the domain structures of PRXs and the phylogenies based on 16s rRNA. Conclusions The distribution of genes encoding PRXs in different unicellular and filamentous cyanobacteria especially those sub-families like PRX-like or 1-Cys PRX correlate with the genome size, eco-physiology, and physiological properties of the organisms. Cyanobacterial and eukaryotic PRXs share similar conserved motifs, indicating that cyanobacteria adopt similar catalytic mechanisms as eukaryotes. All

  5. The yeast tumor suppressor homologue Sro7p is required for targeting of the sodium pumping ATPase to the cell surface.

    PubMed

    Wadskog, Ingrid; Forsmark, Annabelle; Rossi, Guendalina; Konopka, Catherine; Oyen, Mattias; Goksör, Mattias; Ronne, Hans; Brennwald, Patrick; Adler, Lennart

    2006-12-01

    The SRO7/SOP1 encoded tumor suppressor homologue of Saccharomyces cerevisiae is required for maintenance of ion homeostasis in cells exposed to NaCl stress. Here we show that the NaCl sensitivity of the sro7Delta mutant is due to defective sorting of Ena1p, the main sodium pump in yeast. On exposure of sro7Delta mutants to NaCl stress, Ena1p fails to be targeted to the cell surface, but is instead routed to the vacuole for degradation via the multivesicular endosome pathway. SRO7-deficient mutants accumulate post-Golgi vesicles at high salinity, in agreement with a previously described role for Sro7p in late exocytosis. However, Ena1p is not sorted into these post-Golgi vesicles, in contrast to what is observed for the vesicles that accumulate when exocytosis is blocked in sec6-4 mutants at high salinity. These observations imply that Sro7p has a previously unrecognized role for sorting of specific proteins into the exocytic pathway. Screening for multicopy suppressors identified RSN1, encoding a transmembrane protein of unknown function. Overexpression of RSN1 restores NaCl tolerance of sro7Delta mutants by retargeting Ena1p to the plasma membrane. We propose a model in which blocked exocytic sorting in sro7Delta mutants, gives rise to quality control-mediated routing of Ena1p to the vacuole. PMID:17005914

  6. Role of cell surface oligosaccharides of mouse mammary tumor cell lines in cancer metastasis.

    PubMed

    Zhao, Yunxue; Li, Jing; Wang, Jingjian; Xing, Yanli; Geng, Meiyu

    2007-06-01

    Malignant transformation is associated with changes in the glycosylation of cell surface proteins and lipids. In tumor cells, alterations in cellular glycosylation may play a key role in their metastatic behaviour. In the present study, we have assessed the relationship between cell surface oligosaccharides and the metastasis ability of mouse mammary tumor cell lines 67NR and 4TO7. The cell surface oligosaccharides have been analyzed using specific binding assays with some plant lectins and the metastasis ability has been studied using transwell migration and invasion assays. In addition, we investigated the role of terminal sialic acids in the metastatic potential (cell adhesion on fibronectin, cell migration and invasion) in the 4TO7 cells on treatment with neuraminidase. The cell lines used in study have different metastasis abilities in vivo - the 67NR form primary tumors, but no tumor cells are detectable in any distant tissues, while cells of the 4TO7 line are able to spread to lung. In vitro metastasis experiments have revealed higher ability of adhesion, cell migration and invasion in the 4TO7 cells than the 67NR cells. Specific lectins binding assays show that the 4TO7 cells expressed more high-mannose type, multi-antennary complex-type N-glycans, beta-1,6-GlcNAc-branching, alpha-2,6-linked sialic acids, N-acetylgalactosamine and galactosyl(beta-1,3)-N-acetylgalactosamine. Removal of sialic acids on treatment with neuraminidase decreases adhesion, but increases the migration and has shown no significant change in the invasion ability of the 4TO7 cells. The study suggests that the sialic acids are not crucial for the cell migration and invasion in the 4TO7 cells. The findings provide the new insights in understanding the role of cell surface oligosaccharides in cancer metastasis. PMID:17650582

  7. Ultrastructural and biochemical studies of two dynamically expressed cell surface determinants on Candida albicans.

    PubMed Central

    Brawner, D L; Cutler, J E

    1986-01-01

    Variability in the expression of two different cell surface carbohydrate determinants was examined with two agglutinating immunoglobulin M monoclonal antibodies (H9 and C6) and immunoelectron microscopy during growth of three strains of Candida albicans. A single strain of Candida parapsilosis did not express either antigen at any time during growth. Antigens were detected on the surface of C. albicans by agglutination tests with either H9 or C6 over a 48-h growth period. The difference in specificities of the monoclonal antibodies was demonstrated by Ouchterlony double-diffusion tests with solubilized antigens and by variabilities in the reactivity of the agglutinins among yeast strains. The antigenic determinants were isolated by specific immunoprecipitation and protease digestion and characterized by methods including high-pressure liquid chromatography, gas-liquid chromatography, and mass spectroscopy with both chemical and electron ionization. These determinants both contain mannose and glucose. In the case of antigen H9, an additional carbohydrate was detected with gas chromatography and mass spectroscopy. The location of antigens on individual cells was determined by indirect labeling of the determinants, first reacting cells with H9 or C6 followed by goat anti-mouse antibody conjugated with 20-nm colloidal gold particles. Transmission electron microscopy was used to examine cells. The antigens that were reactive with the monoclonal antibodies were associated with a flocculent surface layer. Expression of this layer and expression of the antigens is a dynamic process which is growth phase and strain dependent. The antigens were not expressed on very young cells and disappeared from the cell surface of most C. albicans strains with age. The use of monoclonal antibody to cell surface determinants may allow characterization of cell surface antigens of C. albicans and be helpful in establishing receptors which mediate adherence. Images PMID:3510174

  8. Modeling the Excess Cell Surface Stored in a Complex Morphology of Bleb-Like Protrusions.

    PubMed

    Kapustina, Maryna; Tsygankov, Denis; Zhao, Jia; Wessler, Timothy; Yang, Xiaofeng; Chen, Alex; Roach, Nathan; Elston, Timothy C; Wang, Qi; Jacobson, Ken; Forest, M Gregory

    2016-03-01

    Cells transition from spread to rounded morphologies in diverse physiological contexts including mitosis and mesenchymal-to-amoeboid transitions. When these drastic shape changes occur rapidly, cell volume and surface area are approximately conserved. Consequently, the rounded cells are suddenly presented with a several-fold excess of cell surface whose area far exceeds that of a smooth sphere enclosing the cell volume. This excess is stored in a population of bleb-like protrusions (BLiPs), whose size distribution is shown by electron micrographs to be skewed. We introduce three complementary models of rounded cell morphologies with a prescribed excess surface area. A 2D Hamiltonian model provides a mechanistic description of how discrete attachment points between the cell surface and cortex together with surface bending energy can generate a morphology that satisfies a prescribed excess area and BLiP number density. A 3D random seed-and-growth model simulates efficient packing of BLiPs over a primary rounded shape, demonstrating a pathway for skewed BLiP size distributions that recapitulate 3D morphologies. Finally, a phase field model (2D and 3D) posits energy-based constitutive laws for the cell membrane, nematic F-actin cortex, interior cytosol, and external aqueous medium. The cell surface is equipped with a spontaneous curvature function, a proxy for the cell surface-cortex couple, that is a priori unknown, which the model "learns" from the thin section transmission electron micrograph image (2D) or the "seed and growth" model image (3D). Converged phase field simulations predict self-consistent amplitudes and spatial localization of pressure and stress throughout the cell for any posited stationary morphology target and cell compartment constitutive properties. The models form a general framework for future studies of cell morphological dynamics in a variety of biological contexts. PMID:27015526

  9. Evolutionary Forces Shaping the Golgi Glycosylation Machinery: Why Cell Surface Glycans Are Universal to Living Cells

    PubMed Central

    Varki, Ajit

    2011-01-01

    Despite more than 3 billion years since the origin of life on earth, the powerful forces of biological evolution seem to have failed to generate any living cell that is devoid of a dense and complex array of cell surface glycans. Thus, cell surface glycans seem to be as essential for life as having a DNA genetic code, diverse RNAs, structural/functional proteins, lipid-based membranes, and metabolites that mediate energy flux and signaling. The likely reasons for this apparently universal law of biology are considered here, and include the fact that glycans have the greatest potential for generating diversity, and thus evading recognition by pathogens. This may also explain why in striking contrast to the genetic code, glycans show widely divergent patterns between taxa. On the other hand, glycans have also been coopted for myriad intrinsic functions, which can vary in their importance for organismal survival. In keeping with these considerations, a significant percentage of the genes in the typical genome are dedicated to the generation and/or turnover of glycans. Among eukaryotes, the Golgi is the subcellular organelle that serves to generate much of the diversity of cell surface glycans, carrying out various glycan modifications of glycoconjugates that transit through the Golgi, en route to the cell surface or extracellular destinations. Here I present an overview of general considerations regarding the selective forces shaping evolution of the Golgi glycosylation machinery, and then briefly discuss the common types of variations seen in each major class of glycans, finally focusing on sialic acids as an extreme example of evolutionary glycan diversity generated by the Golgi. Future studies need to address both the phylogenetic diversity the Golgi and the molecular mechanisms for its rapid responses to intrinsic and environmental stimuli. PMID:21525513

  10. Heparan Sulfate Proteoglycans Mediate Factor XIIa Binding to the Cell Surface*

    PubMed Central

    Wujak, Lukasz; Didiasova, Miroslava; Zakrzewicz, Dariusz; Frey, Helena; Schaefer, Liliana; Wygrecka, Malgorzata

    2015-01-01

    Hageman factor (FXIIa) initiates the intrinsic coagulation pathway and triggers the kallikrein-kinin and the complement systems. In addition, it functions as a growth factor by expressing promitogenic activities toward several cell types. FXIIa binds to the cell surface via a number of structurally unrelated surface receptors; however, the underlying mechanisms are not yet fully understood. Here, we demonstrate that FXIIa utilizes cell membrane-bound glycosaminoglycans to interact with the cell surface of human lung fibroblasts (HLF). The combination of enzymatic, inhibitory, and overexpression approaches identified a heparan sulfate (HS) component of proteoglycans as an important determinant of the FXIIa binding capacity of HLF. Moreover, cell-free assays and competition experiments revealed preferential binding of FXIIa to HS and heparin over dextran sulfate, dermatan sulfate, and chondroitin sulfate A and C. Finally, we demonstrate that fibroblasts isolated from the lungs of the patients suffering from idiopathic pulmonary fibrosis (IPF) exhibit enhanced FXIIa binding capacity. Increased sulfation of HS resulting from elevated HS 6-O-sulfotransferase-1 expression in IPF HLF accounted, in part, for this phenomenon. Application of RNA interference technology and inhibitors of intracellular sulfation revealed the cooperative action of cell surface-associated HS and urokinase-type plasminogen activator receptor in the accumulation of FXIIa on the cell surface of IPF HLF. Moreover, FXIIa stimulated IPF HLF migration, which was abrogated by pretreatment of cells with heparinase I. Collectively, our study uncovers a novel role of HS-type glycosaminoglycans in a local accumulation of FXIIa on the cell membrane. The enhanced association of FXIIa with IPF HLF suggests its contribution to fibrogenesis. PMID:25589788

  11. The mycotoxin deoxynivalenol inhibits the cell surface expression of activation markers in human macrophages.

    PubMed

    Waché, Yann J; Hbabi-Haddioui, Laila; Guzylack-Piriou, Laurence; Belkhelfa, Haouaria; Roques, Christine; Oswald, Isabelle P

    2009-08-21

    Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. It exhibits several toxic effects including impaired growth and immune dysregulation. Macrophages play pivotal role in the host defense; upon activation, they express several specific cell surface receptors that are important in adhesion and cell signaling. Several studies have demonstrated that DON can affect macrophages, however, very few data are available concerning the effect of DON on human macrophages, and the effect on macrophage cell surface receptors is unknown. In the present study, human blood monocytes, differentiated in vitro into macrophages, were activated with IFN-gamma, in the presence or absence of low concentrations of DON. The expression of CD11c, CD13, CD14, CD18, CD33, CD35, CD54, CD119 and HLA-DP/DQ/DR was analyzed by flow cytometry. As expected, macrophage activation by IFN-gamma upregulated the expression of CD54, CD14, CD119 and HLA-DP/DQ/DR. Incubation with DON decrease the cell surface expression of these activation markers in a dose-dependent manner. When cells were treated with 5muM DON, the mean fluorescence intensity measured for the expression of these receptors was the same as that observed in non-activated macrophages. This inhibitory effect of DON was only observed when the mycotoxin was applied before the activation signal. Taken together, our results suggest that low concentration of DON alter macrophage activation as measured by the expression of cell surface markers. This may have implications for human health when consuming DON contaminated feed. PMID:19549553

  12. Modeling the Excess Cell Surface Stored in a Complex Morphology of Bleb-Like Protrusions

    PubMed Central

    Wessler, Timothy; Yang, Xiaofeng; Chen, Alex; Roach, Nathan; Elston, Timothy C.; Wang, Qi; Jacobson, Ken; Forest, M. Gregory

    2016-01-01

    Cells transition from spread to rounded morphologies in diverse physiological contexts including mitosis and mesenchymal-to-amoeboid transitions. When these drastic shape changes occur rapidly, cell volume and surface area are approximately conserved. Consequently, the rounded cells are suddenly presented with a several-fold excess of cell surface whose area far exceeds that of a smooth sphere enclosing the cell volume. This excess is stored in a population of bleb-like protrusions (BLiPs), whose size distribution is shown by electron micrographs to be skewed. We introduce three complementary models of rounded cell morphologies with a prescribed excess surface area. A 2D Hamiltonian model provides a mechanistic description of how discrete attachment points between the cell surface and cortex together with surface bending energy can generate a morphology that satisfies a prescribed excess area and BLiP number density. A 3D random seed-and-growth model simulates efficient packing of BLiPs over a primary rounded shape, demonstrating a pathway for skewed BLiP size distributions that recapitulate 3D morphologies. Finally, a phase field model (2D and 3D) posits energy-based constitutive laws for the cell membrane, nematic F-actin cortex, interior cytosol, and external aqueous medium. The cell surface is equipped with a spontaneous curvature function, a proxy for the cell surface-cortex couple, that is a priori unknown, which the model “learns” from the thin section transmission electron micrograph image (2D) or the “seed and growth” model image (3D). Converged phase field simulations predict self-consistent amplitudes and spatial localization of pressure and stress throughout the cell for any posited stationary morphology target and cell compartment constitutive properties. The models form a general framework for future studies of cell morphological dynamics in a variety of biological contexts. PMID:27015526

  13. Pyocyanin facilitates extracellular DNA binding to Pseudomonas aeruginosa influencing cell surface properties and aggregation.

    PubMed

    Das, Theerthankar; Kutty, Samuel K; Kumar, Naresh; Manefield, Mike

    2013-01-01

    Pyocyanin is an electrochemically active metabolite produced by the human pathogen Pseudomonas aeruginosa. It is a recognized virulence factor and is involved in a variety of significant biological activities including gene expression, maintaining fitness of bacterial cells and biofilm formation. It is also recognized as an electron shuttle for bacterial respiration and as an antibacterial and antifungal agent. eDNA has also been demonstrated to be a major component in establishing P. aeruginosa biofilms. In this study we discovered that production of pyocyanin influences the binding of eDNA to P. aeruginosa PA14 cells, mediated through intercalation of pyocyanin with eDNA. P. aeruginosa cell surface properties including cell size (hydrodynamic diameter), hydrophobicity and attractive surface energies were influenced by eDNA in the presence of pyocyanin, affecting physico-chemical interactions and promoting aggregation. A ΔphzA-G PA14 mutant, deficient in pyocynain production, could not bind with eDNA resulting in a reduction in hydrodynamic diameter, a decrease in hydrophobicity, repulsive physico-chemical interactions and reduction in aggregation in comparison to the wildtype strain. Removal of eDNA by DNase I treatment on the PA14 wildtype strain resulted in significant reduction in aggregation, cell surface hydrophobicity and size and an increase in repulsive physico-chemical interactions, similar to the level of the ΔphzA-G mutant. The cell surface properties of the ΔphzA-G mutant were not affected by DNase I treatment. Based on these findings we propose that pyocyanin intercalation with eDNA promotes cell-to-cell interactions in P. aeruginosa cells by influencing their cell surface properties and physico-chemical interactions. PMID:23505483

  14. Tumor suppressor protein SMAR1 modulates the roughness of cell surface: combined AFM and SEM study

    PubMed Central

    2009-01-01

    Background Imaging tools such as scanning electron microscope (SEM) and atomic force microscope (AFM) can be used to produce high-resolution topographic images of biomedical specimens and hence are well suited for imaging alterations in cell morphology. We have studied the correlation of SMAR1 expression with cell surface smoothness in cell lines as well as in different grades of human breast cancer and mouse tumor sections. Methods We validated knockdown and overexpression of SMAR1 using RT-PCR as well as Western blotting in human embryonic kidney (HEK) 293, human breast cancer (MCF-7) and mouse melanoma (B16F1) cell lines. The samples were then processed for cell surface roughness studies using atomic force microscopy (AFM) and scanning electron microscopy (SEM). The same samples were used for microarray analysis as well. Tumors sections from control and SMAR1 treated mice as well as tissues sections from different grades of human breast cancer on poly L-lysine coated slides were used for AFM and SEM studies. Results Tumor sections from mice injected with melanoma cells showed pronounced surface roughness. In contrast, tumor sections obtained from nude mice that were first injected with melanoma cells followed by repeated injections of SMAR1-P44 peptide, exhibited relatively smoother surface profile. Interestingly, human breast cancer tissue sections that showed reduced SMAR1 expression exhibited increased surface roughness compared to the adjacent normal breast tissue. Our AFM data establishes that treatment of cells with SMAR1-P44 results into increase in cytoskeletal volume that is supported by comparative gene expression data showing an increase in the expression of specific cytoskeletal proteins compared to the control cells. Altogether, these findings indicate that tumor suppressor function of SMAR1 might be exhibited through smoothening of cell surface by regulating expression of cell surface proteins. Conclusion Tumor suppressor protein SMAR1 might be

  15. THY-1 Cell Surface Antigen (CD90) Has an Important Role in the Initial Stage of Human Cytomegalovirus Infection.

    PubMed

    Li, Qingxue; Wilkie, Adrian R; Weller, Melodie; Liu, Xueqiao; Cohen, Jeffrey I

    2015-07-01

    Human cytomegalovirus (HCMV) infects about 50% of the US population, is the leading infectious cause of birth defects, and is considered the most important infectious agent in transplant recipients. The virus infects many cell types in vivo and in vitro. While previous studies have identified several cellular proteins that may function at early steps of infection in a cell type dependent manner, the mechanism of virus entry is still poorly understood. Using a computational biology approach, correlating gene expression with virus infectivity in 54 cell lines, we identified THY-1 as a putative host determinant for HCMV infection in these cells. With a series of loss-of-function, gain-of-function and protein-protein interaction analyses, we found that THY-1 mediates HCMV infection at the entry step and is important for infection that occurs at a low m.o.i. THY-1 antibody that bound to the cell surface blocked HCMV during the initial 60 minutes of infection in a dose-dependent manner. Down-regulation of THY-1 with siRNA impaired infectivity occurred during the initial 60 minutes of inoculation. Both THY-1 antibody and siRNA inhibited HCMV-induced activation of the PI3-K/Akt pathway required for entry. Soluble THY-1 protein blocked HCMV infection during, but not after, virus internalization. Expression of exogenous THY-1 enhanced entry in cells expressing low levels of the protein. THY-1 interacted with HCMV gB and gH and may form a complex important for entry. However, since gB and gH have previously been shown to interact, it is uncertain if THY-1 directly binds to both of these proteins. Prior observations that THY-1 (a) interacts with αVβ3 integrin and recruits paxillin (implicated in HCMV entry), (b) regulates leukocyte extravasation (critical for HCMV viremia), and (c) is expressed on many cells targeted for HCMV infection including epithelial and endothelial cells, fibroblast, and CD34+/CD38- stem cells, all support a role for THY-1 as an HCMV entry mediator in

  16. THY-1 Cell Surface Antigen (CD90) Has an Important Role in the Initial Stage of Human Cytomegalovirus Infection

    PubMed Central

    Li, Qingxue; Wilkie, Adrian R.; Weller, Melodie; Liu, Xueqiao; Cohen, Jeffrey I.

    2015-01-01

    Human cytomegalovirus (HCMV) infects about 50% of the US population, is the leading infectious cause of birth defects, and is considered the most important infectious agent in transplant recipients. The virus infects many cell types in vivo and in vitro. While previous studies have identified several cellular proteins that may function at early steps of infection in a cell type dependent manner, the mechanism of virus entry is still poorly understood. Using a computational biology approach, correlating gene expression with virus infectivity in 54 cell lines, we identified THY-1 as a putative host determinant for HCMV infection in these cells. With a series of loss-of-function, gain-of-function and protein-protein interaction analyses, we found that THY-1 mediates HCMV infection at the entry step and is important for infection that occurs at a low m.o.i. THY-1 antibody that bound to the cell surface blocked HCMV during the initial 60 minutes of infection in a dose-dependent manner. Down-regulation of THY-1 with siRNA impaired infectivity occurred during the initial 60 minutes of inoculation. Both THY-1 antibody and siRNA inhibited HCMV-induced activation of the PI3-K/Akt pathway required for entry. Soluble THY-1 protein blocked HCMV infection during, but not after, virus internalization. Expression of exogenous THY-1 enhanced entry in cells expressing low levels of the protein. THY-1 interacted with HCMV gB and gH and may form a complex important for entry. However, since gB and gH have previously been shown to interact, it is uncertain if THY-1 directly binds to both of these proteins. Prior observations that THY-1 (a) interacts with αVβ3 integrin and recruits paxillin (implicated in HCMV entry), (b) regulates leukocyte extravasation (critical for HCMV viremia), and (c) is expressed on many cells targeted for HCMV infection including epithelial and endothelial cells, fibroblast, and CD34+/CD38- stem cells, all support a role for THY-1 as an HCMV entry mediator in

  17. An Entirely Cell-based System to Generate Single-Chain Antibodies Against Cell Surface Receptors

    PubMed Central

    Lipes, Barbara D.; Chen, Yu-Hsun; Ma, HongZheng; Staats, Herman F.; Kenan, Daniel J.; Gunn, Michael Dee

    2008-01-01

    Summary The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen (Ag). Traditionally, the generation of single chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high throughput screening of arrayed phage clones, and characterization of recombinant single chain variable regions (scFvs). This strategy was used to generate a panel of single chain Abs specific for the innate immunity receptor Toll-like receptor 2 (TLR2). Once generated, individual scFvs were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination. PMID:18455737

  18. Detection of CXCR4 receptors on cell surface using a fluorescent metal nanoshell

    NASA Astrophysics Data System (ADS)

    Zhang, Jian; Fu, Yi; Li, Ge; Zhao, Richard Y.; Lakowicz, Joseph R.

    2011-01-01

    Fluorescence cell imaging can be used for disease diagnosis and cellular signal transduction. Using a metal nanoshell as molecular imaging agent, we develop a cellular model system to detect CXCR4 chemokine receptor on T-lymphatic cell surface. These metal nanoshells are observed to express enhanced emission intensity and shortened lifetimes due to the near-field interactions. They are covalently bound with anti-CXCR4 monoclonal antibodies for immunoreactions with the target sites of the CXCR4 receptors on the CEM-SS cells. The fluorescence intensity and lifetime cell images are recorded with a time-resolved confocal microscopy. As expected, the emission signals from the metal nanoshells are clearly isolated from the cellular autofluorescence due to strong intensities and distinctive lifetimes. The number of emission spots on the single cell image is estimated by direct count to the emission signals. Analyzing a pool of cell images, a maximal count number is obtained in a range of 200+/-50. Because there is an average of ~6000 binding sites on the cell surface, we estimate that one emission spot from the metal nanoshell may represent ~30 CXCR5 receptors. In addition, the CXCR4 receptors are estimated to distribute on ~70% area of the cell surface.

  19. Specific tracheal migration is mediated by complementary expression of cell surface proteins

    PubMed Central

    Boube, Muriel; Martin-Bermudo, Maria D.; Brown, Nicholas H.; Casanova, Jordi

    2001-01-01

    Migration of the Drosophila tracheal cells relies on cues provided by nearby cells; however, little is known about how these signals specify a migratory path. Here we investigate the role of cell surface proteins in the definition of such a pathway. We have found that the PS1 integrin is required in the tracheal cells of the visceral branch, whereas the PS2 integrin is required in the visceral mesoderm; both integrins are necessary for the spreading of the visceral branch over its substratum. This is the first identification of a cell surface molecule with expression restricted to a subset of tracheal cells that all migrate in a given direction. We have also found that expression of PS1 in the visceral branch is regulated by the genes that direct tracheal cell migration, showing that integrin expression is part of the cell-fate program that they specify. These results support a model in which signal transduction determines the tracheal migratory pathways by regulating the expression of cell surface proteins, which in turn interact with surface molecules on the surrounding cell population. PMID:11410535

  20. Lactobacillus fermentum AGR1487 cell surface structures and supernatant increase paracellular permeability through different pathways.

    PubMed

    Sengupta, Ranjita; Anderson, Rachel C; Altermann, Eric; McNabb, Warren C; Ganesh, Siva; Armstrong, Kelly M; Moughan, Paul J; Roy, Nicole C

    2015-08-01

    Lactobacillus fermentum is commonly found in food products, and some strains are known to have beneficial effects on human health. However, our previous research indicated that L. fermentum AGR1487 decreases in vitro intestinal barrier integrity. The hypothesis was that cell surface structures of AGR1487 are responsible for the observed in vitro effect. AGR1487 was compared to another human oral L. fermentum strain, AGR1485, which does not cause the same effect. The examination of phenotypic traits associated with the composition of cell surface structures showed that compared to AGR1485, AGR1487 had a smaller genome, utilized different sugars, and had greater tolerance to acid and bile. The effect of the two strains on intestinal barrier integrity was determined using two independent measures of paracellular permeability of the intestinal epithelial Caco-2 cell line. The transepithelial electrical resistance (TEER) assay specifically measures ion permeability, whereas the mannitol flux assay measures the passage of uncharged molecules. Both live and UV-inactivated AGR1487 decreased TEER across Caco-2 cells implicating the cell surfaces structures in the effect. However, only live AGR1487, and not UV-inactivated AGR1487, increased the rate of passage of mannitol, implying that a secreted component(s) is responsible for this effect. These differences in barrier integrity results are likely due to the TEER and mannitol flux assays measuring different characteristics of the epithelial barrier, and therefore imply that there are multiple mechanisms involved in the effect of AGR1487 on barrier integrity. PMID:25943073

  1. GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export.

    PubMed

    Zhang, Maoxiang; Davis, Jason E; Li, Chunman; Gao, Jie; Huang, Wei; Lambert, Nevin A; Terry, Alvin V; Wu, Guangyu

    2016-01-01

    Molecular mechanisms governing the anterograde trafficking of nascent G protein-coupled receptors (GPCRs) are poorly understood. Here, we have studied the regulation of cell surface transport of α2-adrenergic receptors (α2-ARs) by GGA3 (Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding protein 3), a multidomain clathrin adaptor protein that sorts cargo proteins at the trans-Golgi network (TGN) to the endosome/lysosome pathway. By using an inducible system, we demonstrated that GGA3 knockdown significantly inhibited the cell surface expression of newly synthesized α2B-AR without altering overall receptor synthesis and internalization. The receptors were arrested in the TGN. Furthermore, GGA3 knockdown attenuated α2B-AR-mediated signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) activation and cyclic AMP (cAMP) inhibition. More interestingly, GGA3 physically interacted with α2B-AR, and the interaction sites were identified as the triple Arg motif in the third intracellular loop of the receptor and the acidic motif EDWE in the VHS domain of GGA3. In contrast, α2A-AR did not interact with GGA3 and its cell surface export and signaling were not affected by GGA3 knockdown. These data reveal a novel function of GGA3 in export trafficking of a GPCR that is mediated via a specific interaction with the receptor. PMID:26811329

  2. Cell-surface GRP78 facilitates colorectal cancer cell migration and invasion.

    PubMed

    Li, Zongwei; Zhang, Lichao; Zhao, Yarui; Li, Hanqing; Xiao, Hong; Fu, Rong; Zhao, Chao; Wu, Haili; Li, Zhuoyu

    2013-05-01

    Glucose regulated protein 78 (GRP78) is predominantly located in the endoplasmic reticulum as a molecular chaperone. It has also been found on the membranes of some cancer cells, acting as a receptor for a wide variety of ligands. However, its presence on colorectal cancer (CRC) cell surface and its role in CRC metastatic progression remain elusive. Here we reported that GRP78 was predominantly present in the form of clustering aggregates on CRC cell surfaces, and its surface abundance was strongly correlated with CRC differentiation stage. Interestingly, we observed that cell-surface GRP78 had an interaction with the ECM adhesion molecule β1-integrin and was involved in cell-matrix adhesion through regulation of focal adhesion kinase (FAK). Moreover, the present data also implicated that surface GRP78 promoted the cell invasion process, and this effect was at least partly mediated through its association with uPA-uPAR protease system. Together, our data suggests that surface GRP78 promotes CRC cell migration and invasion by regulating cell-matrix adhesion and ECM degradation, which is independent of its signaling receptor function. PMID:23485528

  3. Construction of a food-grade cell surface display system for Lactobacillus casei.

    PubMed

    Qin, Jiayang; Wang, Xiuwen; Kong, Jian; Ma, Cuiqing; Xu, Ping

    2014-01-01

    In this study, a food-grade cell surface display host/vector system for Lactobacillus casei was constructed. The food-grade host L. casei Q-5 was a lactose-deficient derivative of L. casei ATCC 334 obtained by plasmid elimination. The food-grade cell surface display vector was constructed based on safe DNA elements from lactic acid bacteria containing the following: pSH71 replicon from Lactococcus lactis, lactose metabolism genes from L. casei ATCC 334 as complementation markers, and surface layer protein gene from Lactobacillus acidophilus ATCC 4356 for cell surface display. The feasibility of the new host/vector system was verified by the expression of green fluorescent protein (GFP) on L. casei. Laser scanning confocal microscopy and immunofluorescence analysis using anti-GFP antibody confirmed that GFP was anchored on the surface of the recombinant cells. The stability of recombinant L. casei cells in artificial gastrointestinal conditions was verified, which is beneficial for oral vaccination applications. These results indicate that the food-grade host/vector system can be an excellent antigen delivery vehicle in oral vaccine construction. PMID:24598012

  4. Lipoprotein lipase enhances binding of lipoproteins to heparan sulfate on cell surfaces and extracellular matrix.

    PubMed Central

    Eisenberg, S; Sehayek, E; Olivecrona, T; Vlodavsky, I

    1992-01-01

    Lipoprotein lipase enhances binding at 4 degrees C of human plasma lipoproteins (chylomicrons, VLDL, intermediate density lipoprotein, LDL, and HDL3) to cultured fibroblasts and hepG-2 cells and to extracellular matrix. Heparinase treatment of cells and matrix reduces the lipoprotein lipase enhanced binding by 90-95%. Lipoprotein lipase causes only a minimal effect on the binding of lipoproteins to heparan sulfate deficient mutant Chinese hamster ovary cells while it promotes binding to wild type cells that is abolished after heparinase treatment. With 125I-LDL, lipoprotein lipase also enhances uptake and proteolytic degradation at 37 degrees C by normal human skin fibroblasts but has no effect in heparinase-treated normal cells or in LDL receptor-negative fibroblasts. These observations prove that lipoprotein lipase causes, predominantly, binding of lipoproteins to heparan sulfate at cell surfaces and in extracellular matrix rather than to receptors. This interaction brings the lipoproteins into close proximity with cell surfaces and may promote metabolic events that occur at the cell surface, including facilitated transfer to cellular receptors. Images PMID:1430223

  5. Progress in detecting cell-surface protein receptors: the erythropoietin receptor example.

    PubMed

    Elliott, Steve; Sinclair, Angus; Collins, Helen; Rice, Linda; Jelkmann, Wolfgang

    2014-02-01

    Testing for the presence of specific cell-surface receptors (such as EGFR or HER2) on tumor cells is an integral part of cancer care in terms of treatment decisions and prognosis. Understanding the strengths and limitations of these tests is important because inaccurate results may occur if procedures designed to prevent false-negative or false-positive outcomes are not employed. This review discusses tests commonly used to identify and characterize cell-surface receptors, such as the erythropoietin receptor (EpoR). First, a summary is provided on the biology of the Epo/EpoR system, describing how EpoR is expressed on erythrocytic progenitors and precursors in the bone marrow where it mediates red blood cell production in response to Epo. Second, studies are described that investigated whether erythropoiesis-stimulating agents could stimulate tumor progression in cancer patients and whether EpoR is expressed and functional on tumor cells or on endothelial cells. The methods used in these studies included immunohistochemistry, Northern blotting, Western blotting, and binding assays. This review summarizes the strengths and limitations of these methods. Critically analyzing data from tests for cell-surface receptors such as EpoR requires understanding the techniques utilized and demonstrating that results are consistent with current knowledge about receptor biology. PMID:24337485

  6. Development of novel cell surface display in Corynebacterium glutamicum using porin.

    PubMed

    Tateno, Toshihiro; Hatada, Kazuki; Tanaka, Tsutomu; Fukuda, Hideki; Kondo, Akihiko

    2009-09-01

    We have developed a novel cell surface display in Corynebacterium glutamicum using porin proteins as anchor proteins. Porins are localized at C. glutamicum mycolic acid layer and exist as a hexamer. We used alpha-amylase from Streptococcus bovis 148 (AmyA) as a model protein to be displayed on the C. glutamicum cell surface. AmyA was fused to the C terminus of the porins PorB, PorC, or PorH. Expression vectors using fused proteins under the control of the cspB promoter were constructed and introduced into the C. glutamicum Cm strain. Immunostaining microscopy and flow cytometric analysis revealed that PorB-AmyA, PorC-AmyA, and PorH-AmyA were displayed on the C. glutamicum cell surface. AmyA activity was only detected in the cell fraction of C. glutamicum cells that displayed AmyA fused to PorB, PorC or PorH and AmyA activity was not detected in the supernatants of C. glutamicum culture broths after 72 h cultivation. Thus, we have demonstrated that C. glutamicum porins are very efficient anchor proteins for protein display in C. glutamicum. PMID:19430772

  7. Effect of Direct Electric Current on the Cell Surface Properties of Phenol-Degrading Bacteria

    PubMed Central

    Luo, Qishi; Wang, Hui; Zhang, Xihui; Qian, Yi

    2005-01-01

    The change in cell surface properties in the presence of electric currents is of critical concern when the potential to manipulate bacterial movement with electric fields is evaluated. In this study, the effects of different direct electric currents on the cell surface properties involved in bacterial adhesion were investigated by using a mixed phenol-degrading bacterial culture in the exponential growth phase. The traits investigated were surface hydrophobicity (measured by adherence to n-octane), net surface electrostatic charge (determined by measurement of the zeta potential), and the cell surface shape and polymers (determined by scanning electron microscope analysis). The results showed that a lower current (less than 20 mA) induced no significant changes in the surface properties of phenol-degrading bacteria, that an electric current of 20 mA could increase the surface hydrophobicity and flatten the cell shape, and that a higher current (40 mA) could increase the surface extracellular substances and the net negative surface electrostatic charge. The results also revealed that the electric current effects on cell hydrophobicity varied with the suspending medium. We suggest that an electric current greater than 20 mA is not suitable for use in manipulation of the movement of the phenol-degrading bacteria, although such a current might favor the electrophoretic movement of the bacterial species. PMID:15640217

  8. Cell surface nucleolin antagonist causes endothelial cell apoptosis and normalization of tumor vasculature.

    PubMed

    Fogal, Valentina; Sugahara, Kazuki N; Ruoslahti, Erkki; Christian, Sven

    2009-01-01

    Nucleolin is specifically transported to the surface of proliferating endothelial cells in vitro and in vivo. In contrast to its well defined functions in the nucleus and cytoplasm, the function of cell surface nucleolin is poorly defined. We have previously identified the nucleolin-binding antibody NCL3 that specifically binds to cell surface nucleolin on angiogenic blood vessels in vivo and is internalized into the cell. Here, we show that NCL3 inhibits endothelial tube formation in vitro as well as angiogenesis in the matrigel plaque assay and subcutaneous tumor models in vivo. Intriguingly, the specific targeting of proliferating endothelial cells by NCL3 in subcutaneous tumor models leads to the normalization of the tumor vasculature and as a result to an increase in tumor oxygenation. Treatment of endothelial cells with anti-nucleolin antibody NCL3 leads to a decrease of mRNA levels of the anti-apoptotic molecule Bcl-2 and as a consequence induces endothelial cell apoptosis as evidenced by PARP cleavage. These data reveal a novel mode of action for anti-angiogenic therapy and identify cell surface nucleolin as a novel target for combinatorial chemotherapy. PMID:19225898

  9. Yeast cell surface display for lipase whole cell catalyst and its applications

    SciTech Connect

    Liu, Yun; Zhang, Rui; Lian, Zhongshuai; Wang, Shihui; Wright, Aaron T.

    2014-08-01

    The cell surface display technique allows for the expression of target proteins or peptides on the microbial cell surface by fusing an appropriate protein as an anchoring motif. Yeast display systems, such as Pichia pastoris, Yarowia lipolytica and Saccharomyces cerevisiae, are ideal, alternative and extensive display systems with the advantage of simple genetic manipulation and post-translational modification of expressed heterologous proteins. Engineered yeasts show high performance characteristics and variant utilizations. Herein, we comprehensively summarize the variant factors affecting lipase whole cell catalyst activity and display efficiency, including the structure and size of target proteins, screening anchor proteins, type and chain length of linkers, and the appropriate matching rules among the above-mentioned display units. Furthermore, we also address novel approaches to enhance stability and activity of recombinant lipases, such as VHb gene co-expression, multi-enzyme co-display technique, and the micro-environmental interference and self-assembly techniques. Finally, we represent the variety of applications of whole cell surface displayed lipases on yeast cells in non-aqueous phases, including synthesis of esters, PUFA enrichment, resolution of chiral drugs, organic synthesis and biofuels. We demonstrate that the lipase surface display technique is a powerful tool for functionalizing yeasts to serve as whole cell catalysts, and increasing interest is providing an impetus for broad application of this technique.

  10. Small-molecule labeling of live cell surfaces for three-dimensional super-resolution microscopy.

    PubMed

    Lee, Marissa K; Rai, Prabin; Williams, Jarrod; Twieg, Robert J; Moerner, W E

    2014-10-01

    Precise imaging of the cell surface of fluorescently labeled bacteria requires super-resolution methods because the size-scale of these cells is on the order of the diffraction limit. In this work, we present a photocontrollable small-molecule rhodamine spirolactam emitter suitable for non-toxic and specific labeling of the outer surface of cells for three-dimensional (3D) super-resolution (SR) imaging. Conventional rhodamine spirolactams photoswitch to the emitting form with UV light; however, these wavelengths can damage cells. We extended photoswitching to visible wavelengths >400 nm by iterative synthesis and spectroscopic characterization to optimize the substitution on the spirolactam. Further, an N-hydroxysuccinimide-functionalized derivative enabled covalent labeling of amines on the surface of live Caulobacter crescentus cells. Resulting 3D SR reconstructions of the labeled cell surface reveal uniform and specific sampling with thousands of localizations per cell and excellent localization precision in x, y, and z. The distribution of cell stalk lengths (a sub-diffraction-sized cellular structure) was quantified for a mixed population of cells. Pulse-chase experiments identified sites of cell surface growth. Covalent labeling with the optimized rhodamine spirolactam label provides a general strategy to study the surfaces of living cells with high specificity and resolution down to 10-20 nm. PMID:25222297

  11. Small-Molecule Labeling of Live Cell Surfaces for Three-Dimensional Super-Resolution Microscopy

    PubMed Central

    2015-01-01

    Precise imaging of the cell surface of fluorescently labeled bacteria requires super-resolution methods because the size-scale of these cells is on the order of the diffraction limit. In this work, we present a photocontrollable small-molecule rhodamine spirolactam emitter suitable for non-toxic and specific labeling of the outer surface of cells for three-dimensional (3D) super-resolution (SR) imaging. Conventional rhodamine spirolactams photoswitch to the emitting form with UV light; however, these wavelengths can damage cells. We extended photoswitching to visible wavelengths >400 nm by iterative synthesis and spectroscopic characterization to optimize the substitution on the spirolactam. Further, an N-hydroxysuccinimide-functionalized derivative enabled covalent labeling of amines on the surface of live Caulobacter crescentus cells. Resulting 3D SR reconstructions of the labeled cell surface reveal uniform and specific sampling with thousands of localizations per cell and excellent localization precision in x, y, and z. The distribution of cell stalk lengths (a sub-diffraction-sized cellular structure) was quantified for a mixed population of cells. Pulse-chase experiments identified sites of cell surface growth. Covalent labeling with the optimized rhodamine spirolactam label provides a general strategy to study the surfaces of living cells with high specificity and resolution down to 10–20 nm. PMID:25222297

  12. A putative hybrid swarm within Oonopsis foliosa (Asteraceae: Astereae)

    USGS Publications Warehouse

    Hughes, J.F.; Brown, G.K.

    2004-01-01

    Oo??nopsis foliosa var. foliosa and var. monocephala are endemic to short-grass steppe of southeastern Colorado and until recently were considered geographically disjunct. The only known qualitative feature separating these 2 varieties is floral head type; var. foliosa has radiate heads, whereas var. monocephala heads are discoid. Sympatry between these varieties is restricted to a small area in which a range of parental types and intermediate head morphologies is observed. We used distribution mapping, morphometric analyses, chromosome cytology, and pollen stainability to characterize the sympatric zone. Morphometrics confirms that the only discrete difference between var. foliosa and var. monocephala is radiate versus discoid heads, respectively. The outer florets of putative hybrid individuals ranged from conspicuously elongated yet radially symmetric disc-floret corollas, to elongated radially asymmetric bilabiate- or deeply cleft corollas, to stunted ray florets with appendages remnant of corolla lobes. Chromosome cytology of pollen mother cells from both putative parental varieties and a series of intermediate morphological types collected at the sympatric zone reveal evidence of translocation heterozygosity. Pollen stainability shows no significant differences in viability between the parental varieties and putative hybrids. The restricted distribution of putative hybrids to a narrow zone of sympatry between the parental types and the presence of meiotic chromosome-pairing anomalies in these intermediate plants are consistent with a hybrid origin. The high stainability of putative-hybrid pollen adds to a growing body of evidence that hybrids are not universally unfit.

  13. Molecular cloning and properties of a full-length putative thyroid hormone receptor coactivator.

    PubMed

    Takeshita, A; Yen, P M; Misiti, S; Cardona, G R; Liu, Y; Chin, W W

    1996-08-01

    Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate target gene transcription. The conserved carboxy-terminal region of the ligand-binding domain (AF-2) has been thought to play a critical role in mediating ligand-dependent transactivation by the interaction with coactivator(s). Using bacterially-expressed TR as a probe, far-Western-based expression cDNA library screening identified cDNAs that encode, in part, the recently reported partial steroid receptor coactivator-1 (SRC-1) sequence. Additional work, including 5' RACE, has characterized a full-length cDNA that encodes a approximately 160 kD protein as a putative thyroid hormone receptor coactivator (F-SRC-1). In vitro binding studies show that F-SRC-1 binds to a variety of nuclear hormone receptors in a ligand-dependent manner, along with TBP and TFIIB, suggesting that F-SRC-1 may play a role as a bridging molecule between nuclear hormone receptors and general transcription factors. Interestingly, AF-2 mutants also retain ligand-dependent interaction with F-SRC-1. Although F-SRC-1 recognizes the ligand-induced conformational changes of nuclear hormone receptors, our observations suggest that F-SRC-1 may bind directly with subregion(s) in nuclear hormone receptors other than the AF-2 region. PMID:8754792

  14. Homogalacturonan synthesis in Arabidopsis thaliana requires a Golgi-localized protein with a putative methyltransferase domain.

    PubMed

    Mouille, Grégory; Ralet, Marie-Christine; Cavelier, Céline; Eland, Cathlene; Effroy, Delphine; Hématy, Kian; McCartney, Lesley; Truong, Hoai Nam; Gaudon, Virginie; Thibault, Jean-François; Marchant, Alan; Höfte, Herman

    2007-05-01

    Pectins are a family of complex cell-wall polysaccharides, the biosynthesis of which remains poorly understood. We identified dwarf mutants with reduced cell adhesion at a novel locus, QUASIMODO2 (QUA2). qua2-1 showed a 50% reduction in homogalacturonan (HG) content compared with the wild type, without affecting other cell-wall polysaccharides. The remaining HG in qua2-1 showed an unaltered degree of methylesterification. Positional cloning and GFP fusions showed that QUA2, consistent with a role in HG synthesis, encodes a Golgi-localized protein. In contrast to QUA1, another Golgi-localized protein required for HG-synthesis, QUA2 does not show sequence similarity to glycosyltransferases, but instead contains a putative methyltransferase (MT) domain. The Arabidopsis genome encodes 29 QUA2-related proteins. Interestingly, the transcript profiles of QUA1 and QUA2 are correlated and other pairs of QUA1 and QUA2 homologues with correlated transcript profiles can be identified. Together, the results lead to the hypothesis that QUA2 is a pectin MT, and that polymerization and methylation of homogalacturonan are interdependent reactions. PMID:17425712

  15. Cell Surface Glycan Alterations in Epithelial Mesenchymal Transition Process of Huh7 Hepatocellular Carcinoma Cell

    PubMed Central

    Kang, Xiaonan; Sun, Chun; Jiang, Kai; Huang, Li; Lu, Yu; Sui, Jingzhe; Qin, Xue; Liu, Yinkun

    2013-01-01

    Background and Objective Due to recurrence and metastasis, the mortality of Hepatocellular carcinoma (HCC) is high. It is well known that the epithelial mesenchymal transition (EMT) and glycan of cell surface glycoproteins play pivotal roles in tumor metastasis. The goal of this study was to identify HCC metastasis related differential glycan pattern and their enzymatic basis using a HGF induced EMT model. Methodology HGF was used to induce HCC EMT model. Lectin microarray was used to detect the expression of cell surface glycan and the difference was validated by lectin blot and fluorescence cell lectin-immunochemistry. The mRNA expression levels of glycotransferases were determined by qRT-PCR. Results After HGF treatment, the Huh7 cell lost epithelial characteristics and obtained mesenchymal markers. These changes demonstrated that HGF could induce a typical cell model of EMT. Lectin microarray analysis identified a decreased affinity in seven lectins ACL, BPL, JAC, MPL, PHA-E, SNA, and SBA to the glycan of cell surface glycoproteins. This implied that glycan containing T/Tn-antigen, NA2 and bisecting GlcNAc, Siaα2-6Gal/GalNAc, terminal α or βGalNAc structures were reduced. The binding ability of thirteen lectins, AAL, LCA, LTL, ConA, NML, NPL, DBA, HAL, PTL II, WFL, ECL, GSL II and PHA-L to glycan were elevated, and a definite indication that glycan containing terminal αFuc and ± Sia-Le, core fucose, α-man, gal-β(α) GalNAc, β1,6 GlcNAc branching and tetraantennary complex oligosaccharides structures were increased. These results were further validated by lectin blot and fluorescence cell lectin-immunochemistry. Furthermore, the mRNA expression level of Mgat3 decreased while that of Mgat5, FucT8 and β3GalT5 increased. Therefore, cell surface glycan alterations in the EMT process may coincide with the expression of glycosyltransferase. Conclusions The findings of this study systematically clarify the alterations of cell surface glycan in cancer EMT, and

  16. EST mining identifies proteins putatively secreted by the anthracnose pathogen Colletotrichum truncatum

    PubMed Central

    2011-01-01

    Background Colletotrichum truncatum is a haploid, hemibiotrophic, ascomycete fungal pathogen that causes anthracnose disease on many economically important leguminous crops. This pathogen exploits sequential biotrophic- and necrotrophic- infection strategies to colonize the host. Transition from biotrophy to a destructive necrotrophic phase called the biotrophy-necrotrophy switch is critical in symptom development. C. truncatum likely secretes an arsenal of proteins that are implicated in maintaining a compatible interaction with its host. Some of them might be transition specific. Results A directional cDNA library was constructed from mRNA isolated from infected Lens culinaris leaflet tissues displaying the biotrophy-necrotrophy switch of C. truncatum and 5000 expressed sequence tags (ESTs) with an average read of > 600 bp from the 5-prime end were generated. Nearly 39% of the ESTs were predicted to encode proteins of fungal origin and among these, 162 ESTs were predicted to contain N-terminal signal peptides (SPs) in their deduced open reading frames (ORFs). The 162 sequences could be assembled into 122 tentative unigenes comprising 32 contigs and 90 singletons. Sequence analyses of unigenes revealed four potential groups: hydrolases, cell envelope associated proteins (CEAPs), candidate effectors and other proteins. Eleven candidate effector genes were identified based on features common to characterized fungal effectors, i.e. they encode small, soluble (lack of transmembrane domain), cysteine-rich proteins with a putative SP. For a selected subset of CEAPs and candidate effectors, semiquantitative RT-PCR showed that these transcripts were either expressed constitutively in both in vitro and in planta or induced during plant infection. Using potato virus X (PVX) based transient expression assays, we showed that one of the candidate effectors, i. e. contig 8 that encodes a cerato-platanin (CP) domain containing protein, unlike CP proteins from other fungal

  17. The involvement of cell surface nucleolin in the initiation of CCR6 signaling in human hepatocellular carcinoma.

    PubMed

    Qiu, Wei; Wang, Guangyi; Sun, Xiaodong; Ye, Junfeng; Wei, Feng; Shi, Xiaoju; Lv, Guoyue

    2015-03-01

    In recent years, the chemokine CC receptor 6 (CCR6) and its ligand CCL20 were reported to play an essential role in hepatocellular carcinoma (HCC). However, the role of cell surface nucleolin in the CCR6 pathway of HCC is not well featured. Using immunohistochemistry, Western blotting, siRNA, wound healing and transwell assay, we investigated the relationships of cell surface nucleolin and CCR6 signaling in HCC. In the present study, our findings identified that cell surface nucleolin and CCR6 protein were stained in most of HCC tissues (64, 68 %, respectively) and differently expressed in HCC cell lines; meanwhile, both expression has an association with advanced stage, lymph node metastasis and poor 5-year prognosis. According to in vitro assays, we found that the silencing of either cell surface nucleolin or CCR6 inhibited the protein expression of p-ERK, p-AKT, MMP2, MMP9 and ICAM-1 in the CCL20-stimulated HCCLM6 cells. Functional analysis revealed that cell surface nucleolin or CCR6 silencing significantly hampered HCCLM6 cell motility and invasiveness ability, when compared with control. In conclusion, this work suggests that cell surface nucleolin participates in the initiation of CCR6 pathway and biological behaviors of HCC, leading to HCC cell adhesion, migration and invasive behavior. In the clinical practice, cell surface nucleolin and CCR6 are recommended to predict poor prognosis and be used as a useful target for HCC patients. PMID:25698534

  18. HinT proteins and their putative interaction partners in Mollicutes and Chlamydiaceae

    PubMed Central

    Hopfe, Miriam; Hegemann, Johannes H; Henrich, Birgit

    2005-01-01

    Background HinT proteins are found in prokaryotes and eukaryotes and belong to the superfamily of HIT proteins, which are characterized by an histidine-triad sequence motif. While the eukaryotic variants hydrolyze AMP derivates and modulate transcription, the function of prokaryotic HinT proteins is less clearly defined. In Mycoplasma hominis, HinT is concomitantly expressed with the proteins P60 and P80, two domains of a surface exposed membrane complex, and in addition interacts with the P80 moiety. Results An cluster of hitABL genes, similar to that of M. hominis was found in M. pulmonis, M. mycoides subspecies mycoides SC, M. mobile and Mesoplasma florum. RT-PCR analyses provided evidence that the P80, P60 and HinT homologues of M. pulmonis were polycistronically organized, suggesting a genetic and physical interaction between the proteins encoded by these genes in these species. While the hit loci of M. pneumoniae and M. genitalium encoded, in addition to HinT, a protein with several transmembrane segments, the hit locus of Ureaplasma parvum encoded a pore-forming protein, UU270, a P60 homologue, UU271, HinT, UU272, and a membrane protein of unknown function, UU273. Although a full-length mRNA spanning the four genes was not detected, amplification of all intergenic regions from the center of UU270 to the end of UU273 by RT-PCR may be indicative of a common, but unstable mRNA. In Chlamydiaceae the hit gene is flanked upstream by a gene predicted to encode a metal dependent hydrolase and downstream by a gene putatively encoding a protein with ARM-repeats, which are known to be involved in protein-protein interactions. In RT-PCR analyses of C. pneumoniae, regions comprising only two genes, Cp265/Cp266 and Cp266/Cp267 were able to be amplified. In contrast to this in vivo interaction analysis using the yeast two-hybrid system and in vitro immune co-precipitation revealed an interaction between Cp267, which contains the ARM repeats, Cp265, the predicted hydrolase

  19. Monolithic-integrated microlaser encoder.

    PubMed

    Sawada, R; Higurashi, E; Ito, T; Ohguchi, O; Tsubamoto, M

    1999-11-20

    We have developed an extremely small integrated microencoder whose sides are less than 1 mm long. It is 1/100 the size of conventional encoders. This microencoder consists of a laser diode, monolithic photodiodes, and fluorinated polyimide waveguides with total internal reflection mirrors. The instrument can measure the relative displacement between a grating scale and the encoder with a resolution of the order of 0.01 microm; it can also determine the direction in which the scale is moving. By using the two beams that were emitted from the two etched mirrors of the laser diode, by monolithic integration of the waveguide and photodiodes, and by fabrication of a step at the edge of the waveguide, we were able to eliminate conventional bulky optical components such as the beam splitter, the quarter-wavelength plate, bulky mirrors, and bulky photodetectors. PMID:18324228

  20. The PsychENCODE project

    PubMed Central

    Akbarian, Schahram; Liu, Chunyu; Knowles, James A; Vaccarino, Flora M; Farnham, Peggy J; Crawford, Gregory E; Jaffe, Andrew E; Pinto, Dalila; Dracheva, Stella; Geschwind, Daniel H; Mill, Jonathan; Nairn, Angus C; Abyzov, Alexej; Pochareddy, Sirisha; Prabhakar, Shyam; Weissman, Sherman; Sullivan, Patrick F; State, Matthew W; Weng, Zhiping; Peters, Mette A; White, Kevin P; Gerstein, Mark B; Senthil, Geetha; Lehner, Thomas; Sklar, Pamela; Sestan, Nenad

    2015-01-01

    Recent research on disparate psychiatric disorders has implicated rare variants in genes involved in global gene regulation and chromatin modification, as well as many common variants located primarily in regulatory regions of the genome. Understanding precisely how these variants contribute to disease will require a deeper appreciation for the mechanisms of gene regulation in the developing and adult human brain. The PsychENCODE project aims to produce a public resource of multidimensional genomic data using tissue- and cell type–specific samples from approximately 1,000 phenotypically well-characterized, high-quality healthy and disease-affected human post-mortem brains, as well as functionally characterize disease-associated regulatory elements and variants in model systems. We are beginning with a focus on autism spectrum disorder, bipolar disorder and schizophrenia, and expect that this knowledge will apply to a wide variety of psychiatric disorders. This paper outlines the motivation and design of PsychENCODE. PMID:26605881

  1. Exopolysaccharide Production and Ropy Phenotype Are Determined by Two Gene Clusters in Putative Probiotic Strain Lactobacillus paraplantarum BGCG11

    PubMed Central

    Zivkovic, Milica; Miljkovic, Marija; Ruas-Madiedo, Patricia; Strahinic, Ivana; Tolinacki, Maja; Golic, Natasa

    2014-01-01

    Lactobacillus paraplantarum BGCG11, a putative probiotic strain isolated from a soft, white, artisanal cheese, produces a high-molecular-weight heteropolysaccharide, exopolysaccharide (EPS)-CG11, responsible for the ropy phenotype and immunomodulatory activity of the strain. In this study, a 26.4-kb region originating from the pCG1 plasmid, previously shown to be responsible for the production of EPS-CG11 and a ropy phenotype, was cloned, sequenced, and functionally characterized. In this region 16 putative open reading frames (ORFs), encoding enzymes for the production of EPS-CG11, were organized in specific loci involved in the biosynthesis of the repeat unit, polymerization, export, regulation, and chain length determination. Interestingly, downstream of the eps gene cluster, a putative transposase gene was identified, followed by an additional rfb gene cluster containing the rfbACBD genes, the ones most probably responsible for dTDP-l-rhamnose biosynthesis. The functional analysis showed that the production of the high-molecular-weight fraction of EPS-CG11 was absent in two knockout mutants, one in the eps and the other in the rfb gene cluster, as confirmed by size exclusion chromatography analysis. Therefore, both eps and rfb genes clusters are prerequisites for the production of high-molecular-weight EPS-CG11 and for the ropy phenotype of strain L. paraplantarum BGCG11. PMID:25527533

  2. Nonconjugative Plasmids Encoding Sulfanilamide Resistance

    PubMed Central

    Mitsuhashi, Susumu; Inoue, Kunio; Inoue, Matsuhisa

    1977-01-01

    Nonconjugative plasmids encoding sulfanilamide (Sa) resistance were demonstrated at a high frequency in Shigella and Escherichia coli strains resistant to sulfanilamide. These Sa plasmids were all compatible with the standard plasmids used in compatibility testing. The sizes of seven Sa plasmids were measured by electron microscopy and ranged from 1.79 to 2.08 μm, corresponding to 3.5 to 3.9 megadaltons. Images PMID:334067

  3. Cell surface adhesiveness of mouse sarcoma lines evaluated by latex particle adherence assay: correlation with growth behavior and electrophoretic mobility.

    PubMed

    Bubeník, J; Jandlová, T; Suhajová, E; Malkovský, M

    1979-01-01

    Using the latex particle adherence assay and five mouse sarcoma cell lines of the identical origin, etiology and genotype but differing in malignancy we attempted to correlate the degree of cell surface adhesiveness with growth behavior and electrophoretic mobility of cells. Higher tumorigenicity of four of the cell lines (Mc11--Mc14) was associated with lower cell surface adhesiveness and, conversely, lower malignancy of the fifth line (Mc15) with higher cell surface adhesiveness. No simple correlation or causal relationship was found among the electrophoretic mobility of the lines and other cellular characteristics. PMID:522921

  4. Characterization of a Spontaneous Nonmagnetic Mutant of Magnetospirillum gryphiswaldense Reveals a Large Deletion Comprising a Putative Magnetosome Island

    PubMed Central

    Schübbe, Sabrina; Kube, Michael; Scheffel, André; Wawer, Cathrin; Heyen, Udo; Meyerdierks, Anke; Madkour, Mohamed H.; Mayer, Frank; Reinhardt, Richard; Schüler, Dirk

    2003-01-01

    Frequent spontaneous loss of the magnetic phenotype was observed in stationary-phase cultures of the magnetotactic bacterium Magnetospirillum gryphiswaldense MSR-1. A nonmagnetic mutant, designated strain MSR-1B, was isolated and characterized. The mutant lacked any structures resembling magnetosome crystals as well as internal membrane vesicles. The growth of strain MSR-1B was impaired under all growth conditions tested, and the uptake and accumulation of iron were drastically reduced under iron-replete conditions. A large chromosomal deletion of approximately 80 kb was identified in strain MSR-1B, which comprised both the entire mamAB and mamDC clusters as well as further putative operons encoding a number of magnetosome-associated proteins. A bacterial artificial chromosome clone partially covering the deleted region was isolated from the genomic library of wild-type M. gryphiswaldense. Sequence analysis of this fragment revealed that all previously identified mam genes were closely linked with genes encoding other magnetosome-associated proteins within less than 35 kb. In addition, this region was remarkably rich in insertion elements and harbored a considerable number of unknown gene families which appeared to be specific for magnetotactic bacteria. Overall, these findings suggest the existence of a putative large magnetosome island in M. gryphiswaldense and other magnetotactic bacteria. PMID:13129949

  5. Characterization of putative class II bacteriocins identified from a non-bacteriocin-producing strain Lactobacillus casei ATCC 334.

    PubMed

    Kuo, Yang-Cheng; Liu, Cheng-Feng; Lin, Jhao-Fen; Li, An-Chieh; Lo, Ta-Chun; Lin, Thy-Hou

    2013-01-01

    Several putative class II bacteriocin-like genes were identified in Lactobacillus casei ATCC 334, all of which might encode peptides with a double-glycine leader. Six peptides encoded by these genes were heterologously expressed in Escherichia coli and then partially purified in order to test their bacteriocin activity. The results revealed that the mature LSEI_2163 peptide was a class IId bacteriocin that exhibited antimicrobial activity against some lactobacilli and several Listeria species. Similarly, mature LSEI_2386 was a putative pheromone peptide that also had significant bacteriocin activity against several Listeria species. The activities of both peptides tolerated 121°C for 30 min but not treatment with proteinase K or trypsin. The two Cys residues located at positions 4 and 24 in the mature LSEI_2163 peptide were shown by mass spectrometry to form a disulfide bridge, which was required for optimal antibacterial activity. However, replacement of one or both Cys with Ser would cause significant reduction of the antibacterial activity, the reduction being greater when only one of the Cys residues (C4S) was replaced than when both (C4S/C24S) were replaced. PMID:22688903

  6. Lessons from modENCODE.

    PubMed

    Brown, James B; Celniker, Susan E

    2015-01-01

    The modENCODE (Model Organism Encyclopedia of DNA Elements) Consortium aimed to map functional elements-including transcripts, chromatin marks, regulatory factor binding sites, and origins of DNA replication-in the model organisms Drosophila melanogaster and Caenorhabditis elegans. During its five-year span, the consortium conducted more than 2,000 genome-wide assays in developmentally staged animals, dissected tissues, and homogeneous cell lines. Analysis of these data sets provided foundational insights into genome, epigenome, and transcriptome structure and the evolutionary turnover of regulatory pathways. These studies facilitated a comparative analysis with similar data types produced by the ENCODE Consortium for human cells. Genome organization differs drastically in these distant species, and yet quantitative relationships among chromatin state, transcription, and cotranscriptional RNA processing are deeply conserved. Of the many biological discoveries of the modENCODE Consortium, we highlight insights that emerged from integrative studies. We focus on operational and scientific lessons that may aid future projects of similar scale or aims in other, emerging model systems. PMID:26133010

  7. Vector Encoding in Biochemical Networks

    NASA Astrophysics Data System (ADS)

    Potter, Garrett; Sun, Bo

    Encoding of environmental cues via biochemical signaling pathways is of vital importance in the transmission of information for cells in a network. The current literature assumes a single cell state is used to encode information, however, recent research suggests the optimal strategy utilizes a vector of cell states sampled at various time points. To elucidate the optimal sampling strategy for vector encoding, we take an information theoretic approach and determine the mutual information of the calcium signaling dynamics obtained from fibroblast cells perturbed with different concentrations of ATP. Specifically, we analyze the sampling strategies under the cases of fixed and non-fixed vector dimension as well as the efficiency of these strategies. Our results show that sampling with greater frequency is optimal in the case of non-fixed vector dimension but that, in general, a lower sampling frequency is best from both a fixed vector dimension and efficiency standpoint. Further, we find the use of a simple modified Ornstein-Uhlenbeck process as a model qualitatively captures many of our experimental results suggesting that sampling in biochemical networks is based on a few basic components.

  8. Flavobacterium johnsoniae GldK, GldL, GldM, and SprA Are Required for Secretion of the Cell Surface Gliding Motility Adhesins SprB and RemA

    PubMed Central

    Shrivastava, Abhishek; Johnston, Joseph J.; van Baaren, Jessica M.

    2013-01-01

    Flavobacterium johnsoniae cells move rapidly over surfaces by gliding motility. Gliding results from the movement of adhesins such as SprB and RemA along the cell surface. These adhesins are delivered to the cell surface by a Bacteroidetes-specific secretion system referred to as the type IX secretion system (T9SS). GldN, SprE, SprF, and SprT are involved in secretion by this system. Here we demonstrate that GldK, GldL, GldM, and SprA are each also involved in secretion. Nonpolar deletions of gldK, gldL, or gldM resulted in the absence of gliding motility and in T9SS defects. The mutant cells produced SprB and RemA proteins but failed to secrete them to the cell surface. The mutants were resistant to phages that use SprB or RemA as a receptor, and they failed to attach to glass, presumably because of the absence of cell surface adhesins. Deletion of sprA resulted in similar but slightly less dramatic phenotypes. sprA mutant cells failed to secrete SprB and RemA, but cells remained susceptible to some phages and retained some limited ability to glide. The phenotype of the sprA mutant was similar to those previously described for sprE and sprT mutants. SprA, SprE, and SprT are needed for secretion of SprB and RemA but may not be needed for secretion of other proteins targeted to the T9SS. Genetic and molecular experiments demonstrate that gldK, gldL, gldM, and gldN form an operon and suggest that the proteins encoded by these genes may interact to form part of the F. johnsoniae T9SS. PMID:23667240

  9. Putative excitatory and putative inhibitory inputs are localised in different dendritic domains in a Drosophila flight motoneuron.

    PubMed

    Kuehn, Claudia; Duch, Carsten

    2013-03-01

    Input-output computations of individual neurons may be affected by the three-dimensional structure of their dendrites and by the location of input synapses on specific parts of their dendrites. However, only a few examples exist of dendritic architecture which can be related to behaviorally relevant computations of a neuron. By combining genetic, immunohistochemical and confocal laser scanning methods this study estimates the location of the spike-initiating zone and the dendritic distribution patterns of putative synaptic inputs on an individually identified Drosophila flight motorneuron, MN5. MN5 is a monopolar neuron with > 4,000 dendritic branches. The site of spike initiation was estimated by mapping sodium channel immunolabel onto geometric reconstructions of MN5. Maps of putative excitatory cholinergic and of putative inhibitory GABAergic inputs on MN5 dendrites were created by charting tagged Dα7 nicotinic acetylcholine receptors and Rdl GABAA receptors onto MN5 dendritic surface reconstructions. Although these methods provide only an estimate of putative input synapse distributions, the data indicate that inhibitory and excitatory synapses were located preferentially on different dendritic domains of MN5 and, thus, computed mostly separately. Most putative inhibitory inputs were close to spike initiation, which was consistent with sharp inhibition, as predicted previously based on recordings of motoneuron firing patterns during flight. By contrast, highest densities of putative excitatory inputs at more distant dendritic regions were consistent with the prediction that, in response to different power demands during flight, tonic excitatory drive to flight motoneuron dendrites must be smoothly translated into different tonic firing frequencies. PMID:23279094

  10. Cell-surface expression of Aspergillus saitoi-derived functional α-1,2-mannosidase on Yarrowia lipolytica for glycan remodeling.

    PubMed

    Moon, Hye Yun; Van, Trinh Luu; Cheon, Seon Ah; Choo, Jinho; Kim, Jeong-Yoon; Kang, Hyun Ah

    2013-08-01

    Expression of proteins on the surface of yeast has a wide range of applications, such as development of live vaccines, screening of antibody libraries, and use as whole-cell biocatalysts. The hemiascomycetes yeast Yarrowia lipolytica has been raised as a potential host for heterologous expression of recombinant proteins. In this study, we report the expression of Aspergillus saitoi α-1,2-mannosidase, encoded by the msdS gene, on the cell surface of Y. lipolytica. As the first step to achieve the secretory expression of msdS protein, four different signal sequences-derived from the endogenous Y. lipolytica Lip2 and Xpr2 prepro regions and the heterologous A. niger α-amylase and rice α-amylase signal sequences-were analyzed for their secretion efficiency. It was shown that the YlLip2 prepro sequence was most efficient in directing the secretory expression of msdS in fully N-glycosylated forms. The surface display of msdS was subsequently directed by fusing GPI anchoring motifs derived from Y. lipolytica cell wall proteins, YlCwp1p and YlYwp1p, respectively, to the C-terminus of the Lip2 prepro-msdS protein. The expression of actively functional msdS protein on the cell surface was confirmed by western blot, flow cytometry analysis, along with the α-1,2-mannosidase activity assay using intact Y. lipolytica cells as the enzyme source. Furthermore, the glycoengineered Y. lipolytica Δoch1Δmpo1 strains displaying α-1,2-mannosidase were able to convert Man8GlcNAc2 to Man5GlcNAc2 efficiently on their cell-wall mannoproteins, demonstrating its potential used for glycoengineering in vitro or in vivo. PMID:23990303

  11. Complete sequence of the human mucin MUC4: a putative cell membrane-associated mucin.

    PubMed Central

    Moniaux, N; Nollet, S; Porchet, N; Degand, P; Laine, A; Aubert, J P

    1999-01-01

    The MUC4 gene, which encodes a human epithelial mucin, is expressed in various epithelial tissues, just as well in adult as in poorly differentiated cells in the embryo and fetus. Its N-terminus and central sequences have previously been reported as comprising a 27-residue peptide signal, followed by a large domain varying in length from 3285 to 7285 amino acid residues. The present study establishes the whole coding sequence of MUC4 in which the C-terminus is 1156 amino acid residues long and shares a high degree of similarity with the rat sialomucin complex (SMC). SMC is a heterodimeric glycoprotein complex composed of mucin (ascites sialoglycoprotein 1, ASGP-1) and transmembrane (ASGP-2) subunits. The same organization is found in MUC4, where the presence of a GlyAspProHis proteolytic site may cleave the large precursor into two subunits, MUC4alpha and MUC4beta. Like ASGP-2, which binds the receptor tyrosine kinase p185(neu), MUC4beta possesses two epidermal growth factor-like domains, a transmembrane sequence and a potential phosphorylated site. MUC4, the human homologue of rat SMC, may be a heterodimeric bifunctional cell-surface glycoprotein of 2.12 micrometers. These results confer a new biological role for MUC4 as a ligand for ErbB2 in cell signalling. PMID:10024507

  12. Characterization of Putative Iron Responsive Genes as Species-Specific Indicators of Iron Stress in Thalassiosiroid Diatoms

    PubMed Central

    Whitney, LeAnn P.; Lins, Jeremy J.; Hughes, Margaret P.; Wells, Mark L.; Chappell, P. Dreux; Jenkins, Bethany D.

    2011-01-01

    Iron (Fe) availability restricts diatom growth and primary production in large areas of the oceans. It is a challenge to assess the bulk Fe nutritional health of natural diatom populations, since species can differ in their physiological and molecular responses to Fe limitation. We assayed expression of selected genes in diatoms from the Thalassiosira genus to assess their potential utility as species-specific molecular markers to indicate Fe status in natural diatom assemblages. In this study, we compared the expression of the photosynthetic genes encoding ferredoxin (a Fe-requiring protein) and flavodoxin (a Fe-free protein) in culture experiments with Fe replete and Fe stressed Thalassiosira pseudonana (CCMP 1335) isolated from coastal waters and Thalassiosira weissflogii (CCMP 1010) isolated from the open ocean. In T. pseudonana, expression of flavodoxin and ferredoxin genes were not sensitive to Fe status but were found to display diel periodicities. In T. weissflogii, expression of flavodoxin was highly responsive to iron levels and was only detectable when cultures were Fe limited. Flavodoxin genes have been duplicated in most diatoms with available genome data and we show that T. pseudonana has lost its copy related to the Fe-responsive copy in T. weissflogii. We also examined the expression of genes for a putative high affinity, copper (Cu)-dependent Fe uptake system in T. pseudonana. Our results indicate that genes encoding putative Cu transporters, a multi-Cu oxidase, and a Fe reductase are not linked to Fe status. The expression of a second putative Fe reductase increased in Fe limited cultures, but this gene was also highly expressed in Fe replete cultures, indicating it may not be a useful marker in the field. Our findings highlight that Fe metabolism may differ among diatoms even within a genus and show a need to validate responses in different species as part of the development pipeline for genetic markers of Fe status in field populations. PMID

  13. Bartonella henselae AS A PUTATIVE CAUSE OF CONGENITAL CHOLESTASIS

    PubMed Central

    VELHO, Paulo Eduardo Neves Ferreira; BELLOMO-BRANDÃO, Maria Ângela; DRUMMOND, Marina Rovani; MAGALHÃES, Renata Ferreira; HESSEL, Gabriel; BARJAS-CASTRO, Maria de Lourdes; ESCANHOELA, Cecília Amélia Fazzio; NEGRO, Gilda Maria Barbaro DEL; OKAY, Thelma Suely

    2016-01-01

    SUMMARY Severe anemia and cholestatic hepatitis are associated with bartonella infections. A putative vertical Bartonella henselae infection was defined on the basis of ultrastructural and molecular analyses in a three-year-old child with anemia, jaundice and hepatosplenomegaly since birth. Physicians should consider bartonellosis in patients with anemia and hepatitis of unknown origin. PMID:27410916

  14. DIFFERENTIAL GENE EXPRESSION OF PUTATIVE VIRULENCE GENES IN Flavobacterium columnare

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A shot-gun genomic library of the Flavobacterium columnare ALG-530 virulent strain has been constructed and more than 3,000 clones have been sequenced to date (800 contigs). Based on sequence identity with putative known virulence genes from related species, seven genes were selected for differentia...

  15. Developing putative AOPs from high content dataDeveloping putative AOPs from high content dataDeveloping putative AOPs from high content dataDeveloping putative AOPs from high content data

    EPA Science Inventory

    Developing putative AOPs from high content data Shannon M. Bell1,2, Stephen W. Edwards2 1 Oak Ridge Institute for Science and Education 2 Integrated Systems Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development,...

  16. Sulfur Isotope Composition of Putative Primary Troilite in Chondrules

    NASA Technical Reports Server (NTRS)

    Tachibana, Shogo; Huss, Gary R.

    2002-01-01

    Sulfur isotope compositions of putative primary troilites in chondrules from Bishunpur were measured by ion probe. These primary troilites have the same S isotope compositions as matrix troilites and thus appear to be isotopically unfractionated. Additional information is contained in the original extended abstract.

  17. Reinforcing Lipid A Acylation on the Cell Surface of Acinetobacter baumannii Promotes Cationic Antimicrobial Peptide Resistance and Desiccation Survival

    PubMed Central

    Boll, Joseph M.; Tucker, Ashley T.; Klein, Dustin R.; Beltran, Alexander M.; Brodbelt, Jennifer S.; Davies, Bryan W.

    2015-01-01

    ABSTRACT Acinetobacter baumannii is an emerging Gram-negative pathogen found in hospitals and intensive care units. In order to persist in hospital environments, A. baumannii withstands desiccative conditions and can rapidly develop multidrug resistance to conventional antibiotics. Cationic antimicrobial peptides (CAMPs) have served as therapeutic alternatives because they target the conserved lipid A component of the Gram-negative outer membrane to lyse the bacterial cell. However, many Gram-negative pathogenic bacteria, including A. baumannii, fortify their outer membrane with hepta-acylated lipid A to protect the cell from CAMP-dependent cell lysis. Whereas in Escherichia coli and Salmonella, increased production of the outer membrane acyltransferase PagP results in formation of protective hepta-acylated lipid A, which reinforces the lipopolysaccharide portion of the outer membrane barrier, A. baumannii does not carry a gene that encodes a PagP homolog. Instead, A. baumannii has evolved a PagP-independent mechanism to synthesize protective hepta-acylated lipid A. Taking advantage of a recently adapted A. baumannii genetic recombineering system, we characterized two putative acyltransferases in A. baumannii designated LpxLAb (A. baumannii LpxL) and LpxMAb (A. baumannii LpxM), which transfer one and two lauroyl (C12:0) acyl chains, respectively, during lipid A biosynthesis. Hepta-acylation of A. baumannii lipid A promoted resistance to vertebrate and polymyxin CAMPs, which are prescribed as last-resort treatment options. Intriguingly, our analysis also showed that LpxMAb-dependent acylation of lipid A is essential for A. baumannii desiccation survival, a key resistance mechanism for survival in hospital environments. Compounds that inhibit LpxMAb-dependent hepta-acylation of lipid A could act synergistically with CAMPs to provide innovative transmission prevention strategies and treat multidrug-resistant infections. PMID:25991684

  18. Immunomic Screening of Cell Surface Molecules on Undifferentiated Human Dental Pulp Stem Cells.

    PubMed

    Hwang, Hyo-In; Lee, Tae-Hyung; Kang, Kyung-Jung; Ryu, Chun-Jeih; Jang, Young-Joo

    2015-08-15

    Human adult dental pulp tissue is a source of adult stem cells that have a potential to differentiate into various tissues, although the primary cell suspensions cultured from pulp tissue are mixtures of both stem cell and nonstem cell populations with heterogeneous phenotypes and various differentiation efficiencies. Therefore, cell surface protein markers on dental pulp stem cells are critical for detection and purification of stem cell populations. Yet, little is known about the cell surface molecules that are specifically associated with the undifferentiated and progenitor state of human adult dental pulp stem cells (hDPSCs). Presently, cell surface proteins expressed on hDPSCs were assessed by screening surface molecules specifically expressed on dentinogenic progenitors. Using a decoy immunization strategy, a set of monoclonal antibodies (MAbs) was generated against undifferentiated pulp progenitor cells. Forty-five hybridomas produced MAbs that interacted weakly, if at all, to differentiated pulp cells. Of these, 19 MAbs (18 IgG, 1 IgM) recognized surface molecules on undifferentiated hDPSCs. By multicolor flow cytometric analysis, 40%-60% of newly identified MAb-positive cells were demonstrated to be positive for the CD44 and CD90 mesenchymal markers. When MAb-positive cells were sorted from the heterogeneous pulp cell suspension, mineralization efficiency was increased three to five times compared with MAb-negative cells. The results suggest that the decoy immunization is an efficient method for isolation of MAbs against dentinogenic progenitors. These MAbs will be helpful for identification and enrichment of hDPSCs for efficient dentin regeneration. PMID:25919113

  19. Characterization of atrial natriuretic peptide degradation by cell-surface peptidase activity on endothelial cells

    NASA Technical Reports Server (NTRS)

    Frost, S. J.; Whitson, P. A.

    1993-01-01

    Atrial natriuretic peptide (ANP) is a fluid-regulating peptide hormone that promotes vasorelaxation, natriuresis, and diuresis. The mechanisms for the release of ANP and for its clearance from the circulation play important roles in modulating its biological effects. Recently, we have reported that the cell surface of an endothelial cell line, CPA47, could degrade 125I-ANP in the presence of EDTA. In this study, we have characterized this degradation of 125I-ANP. The kinetics of ANP degradation by the surface of CPA47 cells were first order, with a Km of 320 +/- 60 nM and Vmax of 35 +/- 14 pmol of ANP degraded/10 min/10(5) cells at pH 7.4. ANP is degraded by the surface of CPA47 cells over a broad pH range from 7.0-8.5. Potato carboxypeptidase inhibitor and bestatin inhibited 125I-ANP degradation, suggesting that this degradative activity on the surface of CPA47 cells has exopeptidase characteristics. The selectivity of CPA47 cell-surface degradation of ANP was demonstrated when 125I-ANP degradation was inhibited in the presence of neuropeptide Y and angiotensin I and II but not bradykinin, bombesin, endothelin-1, or substance P. The C-terminal amino acids phe26 and tyr28 were deduced to be important for ANP interaction with the cell-surface peptidase(s) based on comparison of the IC50 of various ANP analogues and other natriuretic peptides for the inhibition of ANP degradation. These data suggest that a newly characterized divalent cation-independent exopeptidase(s) that selectively recognizes ANP and some other vasoactive peptides exists on the surface of endothelial cells.

  20. Streptococcus salivarius strains carry either fibrils or fimbriae on the cell surface.

    PubMed Central

    Handley, P S; Carter, P L; Fielding, J

    1984-01-01

    Strains of Streptococcus salivarius were screened by negative staining for the presence of surface structures. Two structural subgroups were found, carrying either fibrils or fimbriae, projecting from the cell surface. Eight strains carried a very dense peritrichous array of fibrils of two distinct lengths. Long fibrils had an average length of 175 nm, and short fibrils had an average length of 95 nm. Two strains carried only long fibrils, one strain carried only short fibrils, and another strain carried a lateral tuft of very prominent fibrils of two lengths, with a fibrillar fuzz covering the remainder of the cell surface. In all the strains in which they were present, the long fibrils were unaffected by protease or trypsin treatment. In contrast, the short fibrils were completely digested by protease and partially removed by trypsin. Neither long nor short fibrils were affected structurally by mild pepsin digestion or by lipase. The Lancefield extraction procedure removed both long and short fibrils. These twelve fibrillar strains were therefore divisible into four structural subgroups. Extracts of all the fibrillar strains reacted with group K antiserum. The second main structural subgroup consisted of nine strains of S. salivarius, all of which carried morphologically identical, flexible fimbriae arranged peritrichously over the cell surface. The fimbriae were structurally distinct from fibrils and measured 0.5 to 1.0 micron long and 3 to 4 nm wide, with an irregular outline and no obvious substructure. There was no obvious reduction in the number of fimbriae after protease or trypsin treatment. Extracts of the fimbriated strains did not react with the group K antiserum. The two serological and structural subgroups could also be distinguished by colony morphology. Images PMID:6197404

  1. Vimentin and desmin possess GlcNAc-binding lectin-like properties on cell surfaces.

    PubMed

    Ise, Hirohiko; Kobayashi, Satoshi; Goto, Mitsuaki; Sato, Takao; Kawakubo, Masatomo; Takahashi, Masafumi; Ikeda, Uichi; Akaike, Toshihiro

    2010-07-01

    Vimentin and desmin are intermediate filament proteins found in various mesenchymal and skeletal muscle cells, respectively. These proteins play an important role in the stabilization of the cytoplasmic architecture. Here, we found, using artificial biomimicking glycopolymers, that vimentin and desmin possess N-acetylglucosamine (GlcNAc)-binding lectin-like properties on the cell surfaces of various vimentin- and desmin-expressing cells such as cardiomyocytes and vascular smooth muscle cells. The rod II domain of these proteins was demonstrated to be localized to the cell surface and to directly bind to the artificial biomimicking GlcNAc-bearing polymer, by confocal laser microscopy and surface plasmon resonance analysis. These glycopolymers strongly interact with lectins and are useful tools for the analysis of lectin-carbohydrate interactions, since glycopolymers binding to lectins can induce the clustering of lectins due to multivalent glycoside ligand binding. Moreover, immunocytochemistry and pull-down assay with His-tagged vimentin-rod II domain protein showed that the vimentin-rod II domain interacts with O-GlcNAc proteins. These results suggest that O-GlcNAc proteins might be one candidate for physiological GlcNAc-bearing ligands with which vimentin and desmin interact. These findings demonstrate a novel function of vimentin and desmin that does not involve stabilization of the cytoplasmic architecture by which these proteins interact with physiological GlcNAc-bearing ligands such as O-GlcNAc proteins on the cell surface through their GlcNAc-binding lectin-like properties. PMID:20332081

  2. Probe for the measurement of cell surface pH in vivo and ex vivo

    PubMed Central

    Anderson, Michael; Moshnikova, Anna; Engelman, Donald M.; Reshetnyak, Yana K.; Andreev, Oleg A.

    2016-01-01

    We have developed a way to measure cell surface pH by positioning a pH-sensitive fluorescent dye, seminaphtharhodafluor (SNARF), conjugated to the pH low insertion peptide (pHLIP). It has been observed that many diseased tissues are acidic and that tumors are especially so. A combination of effects acidifies tumor cell interiors, and cells pump out lactic acid and protons to maintain intracellular pH, acidifying the extracellular space. Overexpression of carbonic anhydrases on cell surfaces further contributes to acidification. Thus, the pH near tumor cell surfaces is expected to be low and to increase with distance from the membrane, so bulk pH measurements will not report surface acidity. Our new surface pH-measurement tool was validated in cancer cells grown in spheroids, in mouse tumor models in vivo, and in excised tumors. We found that the surface pH is sensitive to cell glycolytic activity: the pH decreases in high glucose and increases if glucose is replaced with nonmetabolized deoxyglucose. For highly metastatic cancer cells, the pH measured at the surface was 6.7–6.8, when the surrounding external pH was 7.4. The approach is sensitive enough to detect 0.2–0.3 pH unit changes in vivo in tumors induced by i.p. injection of glucose. The pH at the surfaces of highly metastatic cells within tumors was found to be about 6.1–6.4, whereas in nonmetastatic tumors, it was 6.7–6.9, possibly creating a way to distinguish more aggressive from less aggressive tumors. Other biological roles of surface acidity may be found, now that targeted measurements are possible. PMID:27382181

  3. High Cell Surface Death Receptor Expression Determines Type I Versus Type II Signaling*

    PubMed Central

    Meng, Xue Wei; Peterson, Kevin L.; Dai, Haiming; Schneider, Paula; Lee, Sun-Hee; Zhang, Jin-San; Koenig, Alexander; Bronk, Steve; Billadeau, Daniel D.; Gores, Gregory J.; Kaufmann, Scott H.

    2011-01-01

    Previous studies have suggested that there are two signaling pathways leading from ligation of the Fas receptor to induction of apoptosis. Type I signaling involves Fas ligand-induced recruitment of large amounts of FADD (FAS-associated death domain protein) and procaspase 8, leading to direct activation of caspase 3, whereas type II signaling involves Bid-mediated mitochondrial perturbation to amplify a more modest death receptor-initiated signal. The biochemical basis for this dichotomy has previously been unclear. Here we show that type I cells have a longer half-life for Fas message and express higher amounts of cell surface Fas, explaining the increased recruitment of FADD and subsequent signaling. Moreover, we demonstrate that cells with type II Fas signaling (Jurkat or HCT-15) can signal through a type I pathway upon forced receptor overexpression and that shRNA-mediated Fas down-regulation converts cells with type I signaling (A498) to type II signaling. Importantly, the same cells can exhibit type I signaling for Fas and type II signaling for TRAIL (TNF-α-related apoptosis-inducing ligand), indicating that the choice of signaling pathway is related to the specific receptor, not some other cellular feature. Additional experiments revealed that up-regulation of cell surface death receptor 5 levels by treatment with 7-ethyl-10-hydroxy-camptothecin converted TRAIL signaling in HCT116 cells from type II to type I. Collectively, these results suggest that the type I/type II dichotomy reflects differences in cell surface death receptor expression. PMID:21865165

  4. Probe for the measurement of cell surface pH in vivo and ex vivo.

    PubMed

    Anderson, Michael; Moshnikova, Anna; Engelman, Donald M; Reshetnyak, Yana K; Andreev, Oleg A

    2016-07-19

    We have developed a way to measure cell surface pH by positioning a pH-sensitive fluorescent dye, seminaphtharhodafluor (SNARF), conjugated to the pH low insertion peptide (pHLIP). It has been observed that many diseased tissues are acidic and that tumors are especially so. A combination of effects acidifies tumor cell interiors, and cells pump out lactic acid and protons to maintain intracellular pH, acidifying the extracellular space. Overexpression of carbonic anhydrases on cell surfaces further contributes to acidification. Thus, the pH near tumor cell surfaces is expected to be low and to increase with distance from the membrane, so bulk pH measurements will not report surface acidity. Our new surface pH-measurement tool was validated in cancer cells grown in spheroids, in mouse tumor models in vivo, and in excised tumors. We found that the surface pH is sensitive to cell glycolytic activity: the pH decreases in high glucose and increases if glucose is replaced with nonmetabolized deoxyglucose. For highly metastatic cancer cells, the pH measured at the surface was 6.7-6.8, when the surrounding external pH was 7.4. The approach is sensitive enough to detect 0.2-0.3 pH unit changes in vivo in tumors induced by i.p. injection of glucose. The pH at the surfaces of highly metastatic cells within tumors was found to be about 6.1-6.4, whereas in nonmetastatic tumors, it was 6.7-6.9, possibly creating a way to distinguish more aggressive from less aggressive tumors. Other biological roles of surface acidity may be found, now that targeted measurements are possible. PMID:27382181

  5. Ex-vivo tissue classification of cell surface receptor concentrations using kinetic modeling

    NASA Astrophysics Data System (ADS)

    Sinha, Lagnojita; Wang, Yu; Yang, Cynthia; Khan, Altaz; Liu, Jonathan T.; Tichauer, Kenneth M.

    2015-03-01

    One of the major challenges in the complete resection of cancer is the difficulty of distinctly classifying tumor and healthy tissue. This paper investigates the capability of competing kinetic modeling approaches for identifying different tissue types based on differential cell-surface receptor expressions. These approaches require fresh resected tissues to be stained with a mixture of two probes: one targeted to a cancer specific cell-surface receptor, and another left "untargeted" to account for nonspecific retention of the targeted agent, with subsequent repeated rinsing and imaging of the probe concentrations. Analysis of the results were carried out in simulations and in animal experiments for the cancer target, epidermal growth factor receptor (EGFR), a cell surface receptor overexpressed by many cancers. In the animal experiments, subcutaneous xenografts of human glioma (U251; moderate EGFR) and human epidermoid (A431; high EGFR) tumors, grown in six athymic mice, were excised and stained with an EGFR targeted surface-enhanced Raman scattering nanoparticle (SERS NP) and untargeted SERS NP pair. The salient finding in this study was that significant non-specific retention was observed for the EGFR targeted probe [anti-EGFR antibody labeled with a surface-enhanced Raman scattering (SERS) nanoparticle], but could be corrected for by the equivalent non-specific retention of the untargeted probe (isotype control antibody labeled with a different SERS nanoparticle). Once this non-specific binding was accounted for, the kinetic model was able to predict the expected differences in EGFR concentration among different tissue types: healthy, U251, and A431 in accordance with an ex vivo flow cytometry analysis, successfully classifying different tissue types.

  6. Association of cell surface heparan sulfate proteoglycans of Schwann cells with extracellular matrix proteins.

    PubMed

    Carey, D J; Crumbling, D M; Stahl, R C; Evans, D M

    1990-11-25

    The terminal differentiation of Schwann cells is dependent on contact with basement membrane. The present study was undertaken to investigate the role of cell surface heparan sulfate proteoglycans (HSPGs) in mediating Schwann cell responses to extracellular matrix contact. Phosphatidylinositol-specific phospholipase C-releasable cell surface HSPGs purified from cultures of neonatal rat Schwann cells were subjected to affinity chromatography on immobilized laminin and fibronectin. Binding of the HSPG to both affinity matrices was observed. The strength of the association, however, was sensitive to the ionic strength of the buffer. In 0.1 M Tris-HCl, HSPG binding was essentially irreversible whereas in physiological ionic strength buffer (e.g. 0.142 M NaCl, 10 mM Tris), weaker binding was detected as a delay in elution of the HSPG from the affinity columns. Further studies of HSPG-laminin binding suggested that the binding was mediated by the glycosaminoglycan chains of the proteoglycans. Results of equilibrium gel filtration chromatography provided additional evidence for a reversible association of the HSPG and laminin with a Kd of approximately 1 x 10(-6) M. When Schwann cells were plated on plastic dishes coated with laminin, the cells attached and extended long slender processes. Inclusion of heparin, but not chondroitin sulfate, in the assay medium resulted in partial inhibition of process extension, but at concentrations of heparin which were higher than that needed to disrupt laminin-HSPG association in vitro. Addition of anti-integrin receptor antibodies resulted in more extensive inhibition of laminin-dependent process extension. Anti-integrin antibodies plus heparin essentially totally inhibited laminin-dependent process extension. These results demonstrate that cell surface HSPGs are capable of reversible association with extracellular matrix molecules and suggest that HSPG-laminin interactions play a role in laminin-dependent Schwann cell spreading. PMID

  7. Enterovirus 71 Uses Cell Surface Heparan Sulfate Glycosaminoglycan as an Attachment Receptor

    PubMed Central

    Tan, Chee Wah; Poh, Chit Laa; Sam, I-Ching

    2013-01-01

    Enterovirus 71 (EV-71) infections are usually associated with mild hand, foot, and mouth disease in young children but have been reported to cause severe neurological complications with high mortality rates. To date, four EV-71 receptors have been identified, but inhibition of these receptors by antagonists did not completely abolish EV-71 infection, implying that there is an as yet undiscovered receptor(s). Since EV-71 has a wide range of tissue tropisms, we hypothesize that EV-71 infections may be facilitated by using receptors that are widely expressed in all cell types, such as heparan sulfate. In this study, heparin, polysulfated dextran sulfate, and suramin were found to significantly prevent EV-71 infection. Heparin inhibited infection by all the EV-71 strains tested, including those with a single-passage history. Neutralization of the cell surface anionic charge by polycationic poly-d-lysine and blockage of heparan sulfate by an anti-heparan sulfate peptide also inhibited EV-71 infection. Interference with heparan sulfate biosynthesis either by sodium chlorate treatment or through transient knockdown of N-deacetylase/N-sulfotransferase-1 and exostosin-1 expression reduced EV-71 infection in RD cells. Enzymatic removal of cell surface heparan sulfate by heparinase I/II/III inhibited EV-71 infection. Furthermore, the level of EV-71 attachment to CHO cell lines that are variably deficient in cell surface glycosaminoglycans was significantly lower than that to wild-type CHO cells. Direct binding of EV-71 particles to heparin-Sepharose columns under physiological salt conditions was demonstrated. We conclude that EV-71 infection requires initial binding to heparan sulfate as an attachment receptor. PMID:23097443

  8. Cell surface glycopeptides from human intestinal epithelial cell lines derived from normal colon and colon adenocarcinomas

    SciTech Connect

    Youakim, A.; Herscovics, A.

    1985-11-01

    The cell surface glycopeptides from an epithelial cell line (CCL 239) derived from normal human colon were compared with those from three cell lines (HCT-8R, HCT-15, and CaCo-2) derived independently from human colonic adenocarcinomas. Cells were incubated with D-(2-TH)mannose or L-(5,6-TH)fucose for 24 h and treated with trypsin to release cell surface components which were then digested exhaustively with Pronase and fractionated on Bio-Gel P-6 before and after treatment with endo-beta-N-acetylglucosaminidase H. The most noticeable difference between the labeled glycopeptides from the tumor and CCL 239 cells was the presence in the former of an endo-beta-N-acetylglucosaminidase H-resistant high molecular weight glycopeptide fraction which was eluted in the void volume of Bio-Gel P-6. This fraction was obtained with both labeled mannose and fucose as precursors. However, acid hydrolysis of this fraction obtained after incubation with (2-TH)mannose revealed that as much as 60-90% of the radioactivity was recovered as fucose. Analysis of the total glycopeptides (cell surface and cell pellet) obtained after incubation with (2-TH)mannose showed that from 40-45% of the radioactivity in the tumor cells and less than 10% of the radioactivity in the CCL 239 cells was recovered as fucose. After incubation of the HCT-8R cells with D-(1,6-TH)glucosamine and L-(1- UC)fucose, strong acid hydrolysis of the labeled glycopeptide fraction excluded from Bio-Gel P-6 produced TH-labeled N-acetylglucosamine and N-acetylgalactosamine.

  9. Transcriptional Profiling of Bipotential Embryonic Liver Cells to Identify Liver Progenitor Cell Surface Markers

    PubMed Central

    Ochsner, Scott A.; Strick-Marchand, Hélène; Qiu, Qiong; Venable, Susan; Dean, Adam; Wilde, Margaret; Weiss, Mary C.; Darlington, Gretchen J.

    2010-01-01

    The ability to purify to homogeneity a population of hepatic progenitor cells from adult liver is critical for their characterization prior to any therapeutic application. As a step in this direction, we have used a bipotential liver cell line from 14 days postcoitum mouse embryonic liver to compile a list of cell surface markers expressed specifically by liver progenitor cells. These cells, known as bipotential mouse embryonic liver (BMEL) cells, proliferate in an undifferentiated state and are capable of differentiating into hepatocyte-like and cholangiocyte-like cells in vitro. Upon transplantation, BMEL cells are capable of differentiating into hepatocytes and cholangiocytes in vivo. Microarray and Gene Ontology (GO) analysis of gene expression in the 9A1 and 14B3 BMEL cell lines grown under proliferating and differentiating conditions was used to identify cell surface markers preferentially expressed in the bipotential undifferentiated state. This analysis revealed that proliferating BMEL cells express many genes involved in cell cycle regulation, whereas differentiation of BMEL cells by cell aggregation causes a switch in gene expression to functions characteristic of mature hepatocytes. In addition, microarray data and protein analysis indicated that the Notch signaling pathway could be involved in maintaining BMEL cells in an undifferentiated stem cell state. Using GO annotation, a list of cell surface markers preferentially expressed on undifferentiated BMEL cells was generated. One marker, Cd24a, is specifically expressed on progenitor oval cells in livers of diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate-treated animals. We therefore consider Cd24a expression a candidate molecule for purification of hepatic progenitor cells. PMID:17641245

  10. Calcium influences sensitivity to growth inhibition induced by a cell surface sialoglycopeptide

    NASA Technical Reports Server (NTRS)

    Betz, N. A.; Fattaey, H. K.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    While studies concerning mitogenic factors have been an important area of research for many years, much less is understood about the mechanisms of action of cell surface growth inhibitors. We have purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) which can reversibly inhibit the proliferation of diverse cell types. The studies discussed in this article show that three mouse keratinocyte cell lines exhibit sixty-fold greater sensitivity than other fibroblasts and epithelial-like cells to CeReS-18-induced growth inhibition. Growth inhibition induced by CeReS-18 treatment is a reversible process, and the three mouse keratinocyte cell lines exhibited either single or multiple cell cycle arrest points, although a predominantly G0/G1 cell cycle arrest point was exhibited in Swiss 3T3 fibroblasts. The sensitivity of the mouse keratinocyte cell lines to CeReS-18-induced growth inhibition was not affected by the degree of tumorigenic progression in the cell lines and was not due to differences in CeReS-18 binding affinity or number of cell surface receptors per cell. However, the sensitivity of both murine fibroblasts and keratinocytes could be altered by changing the extracellular calcium concentration, such that increased extracellular calcium concentrations resulted in decreased sensitivity to CeReS-18-induced proliferation inhibition. Thus the increased sensitivity of the murine keratinocyte cell lines to CeReS-18 could be ascribed to the low calcium concentration used in their propagation. Studies are currently under way investigating the role of calcium in CeReS-18-induced growth arrest. The CeReS-18 may serve as a very useful tool to study negative growth control and the signal transduction events associated with cell cycling.

  11. Cell surface characteristics of Lactobacillus casei subsp. casei, Lactobacillus paracasei subsp. paracasei, and Lactobacillus rhamnosus strains.

    PubMed Central

    Pelletier, C; Bouley, C; Cayuela, C; Bouttier, S; Bourlioux, P; Bellon-Fontaine, M N

    1997-01-01

    Hydrophilic and electrostatic cell surface properties of eight Lactobacillus strains were characterized by using the microbial adhesion to solvents method and microelectrophoresis, respectively. All strains appeared relatively hydrophilic. The strong microbial adhesion to chloroform, an acidic solvent, in comparison with microbial adhesion to hexadecane, an apolar n-alkane, demonstrated the particularity of lactobacilli to have an important electron donor and basic character and consequently their potential ability to generate Lewis acid-base interactions with a support. Regardless of their electrophoretic mobility (EM), strains were in general slightly negatively charged at alkaline pH. A pH-dependent behavior concerning cell surface charges was observed. The EM decreased progressively with more acidic pHs for the L. casei subsp. casei and L. paracasei subsp. paracasei strains until the isoelectric point (IEP), i.e., the pH value for which the EM is zero. On the other hand, the EM for the L. rhamnosus strains was stable from pH 8 to pH 3 to 4, at which point there was a shift near the IEP. Both L. casei subsp. casei and L. paracasei subsp. paracasei strains were characterized by an IEP of around 4, whereas L. rhamnosus strains possessed a markedly lower IEP of 2. The present study showed that the cell surface physicochemical properties of lactobacilli seem to be, at least in part and under certain experimental conditions, particular to the bacterial species. Such differences detected between species are likely to be accompanied by some particular changes in cell wall chemical composition. PMID:9143109

  12. Oxygen Modulates Human Decidual Natural Killer Cell Surface Receptor Expression and Interactions with Trophoblasts1

    PubMed Central

    Wallace, Alison E.; Goulwara, Sonu S.; Whitley, Guy S.; Cartwright, Judith E.

    2014-01-01

    Decidual natural killer (dNK) cells have been shown to both promote and inhibit trophoblast behavior important for decidual remodeling in pregnancy and have a distinct phenotype compared to peripheral blood NK cells. We investigated whether different levels of oxygen tension, mimicking the physiological conditions of the decidua in early pregnancy, altered cell surface receptor expression and activity of dNK cells and their interactions with trophoblast. dNK cells were isolated from terminated first-trimester pregnancies and cultured in oxygen tensions of 3%, 10%, and 21% for 24 h. Cell surface receptor expression was examined by flow cytometry, and the effects of secreted factors in conditioned medium (CM) on the trophoblast cell line SGHPL-4 were assessed in vitro. SGHPL-4 cells treated with dNK cell CM incubated in oxygen tensions of 10% were significantly more invasive (P < 0.05) and formed endothelial-like networks to a greater extent (P < 0.05) than SGHPL-4 cells treated with dNK cell CM incubated in oxygen tensions of 3% or 21%. After 24 h, a lower percentage of dNK cells expressed CD56 at 21% oxygen (P < 0.05), and an increased percentage of dNK cells expressed NKG2D at 10% oxygen (P < 0.05) compared to other oxygen tensions, with large patient variation. This study demonstrates dNK cell phenotype and secreted factors are modulated by oxygen tension, which induces changes in trophoblast invasion and endovascular-like differentiation. Alterations in dNK cell surface receptor expression and secreted factors at different oxygen tensions may represent regulation of function within the decidua during the first trimester of pregnancy. PMID:25232021

  13. Kinin B1 receptor homo-oligomerization is required for receptor trafficking to the cell surface.

    PubMed

    Sandén, Caroline; Leeb-Lundberg, L M Fredrik

    2013-01-01

    The kinin B1 receptor (B1R) is a G protein-coupled receptor with pro-inflammatory activity that is latent in healthy tissues but induced by tissue insult. Here, we investigated if B1R homo-oligomerization is a possible mechanism regulating the presentation of this receptor at the level of maturation and trafficking to the cell surface. To this end, we used HEK293 cells stably expressing N-terminal FLAG and HA epitope-tagged wild-type human B1R and an N-terminal receptor fragment, B1stop135, which terminates at the C-terminal end of the third transmembrane domain and has previously been shown to oligomerize with B1R. Receptors were monitored by immunoblotting and immunoprecipitation, receptor function by agonist binding and agonist-promoted phosphoinositide hydrolysis, and receptor trafficking by confocal immunofluorescence microscopy. When expressed alone, B1R is core N-glycosylated and forms oligomers localized intracellularly and on the cell surface. B1stop135 also exists as core N-glycosylated oligomers but is localized exclusively intracellularly. When co-expressed, B1stop135 prevents specifically B1R homo-oligomerization by forming nonfunctional B1R-B1stop135 hetero-oligomers, retains B1R intracellularly at least in part in the endoplasmatic reticulum (ER), increases calnexin binding to the receptor, and increases receptor degradation. We conclude that B1R homo-oligomerization is necessary for B1R maturation and trafficking to the cell surface. Modulating this mechanism may be a novel therapeutic avenue in inflammatory disease. PMID:23201435

  14. Short-chain ceramides depress integrin cell surface expression and function in colorectal cancer cells.

    PubMed

    Morad, Samy A F; Bridges, Lance C; Almeida Larrea, Alex D; Mayen, Anthony L; MacDougall, Matthew R; Davis, Traci S; Kester, Mark; Cabot, Myles C

    2016-07-01

    Colorectal cancer (CRC) is highly metastatic, significantly so to liver, a characteristic that embodies one of the most challenging aspects of treatment. The integrin family of cell-cell and cell-matrix adhesion receptors plays a central role in migration and invasion, functions that underlie metastatic potential. In the present work we sought to determine the impact of ceramide, which plays a key modulatory role in cancer suppression, on integrin cell surface expression and function in CRC cells in order to reveal possible ceramide-centric effects on tumor cell motility. Human CRC cells LoVo, HT-29, and HCT-116 were employed, which represent lines established from primary and metastatic sites. A cell-permeable, short-chain analog, C6-ceramide, was used as ceramide mimic. Exposure of cells to C6-ceramide (24 h) promoted a dose-dependent (2.5-10 µM) decrease in the expression of cell surface β1 and β4 integrin subunits in all cell lines; at 10 µM C6-ceramide, the decreases ranged from 30 to 50% of the control. Expression of cell surface αVβ6 integrin, which is associated with advanced invasion in CRC, was also suppressed by C6-ceramide. Decreases in integrin expression translated to diminished cellular adhesion, 50% of the control at 5 µM C6-ceramide, and markedly reduced cellular migration, approximately 30-40% of the control in all cell lines. Physicochemical examination revealed potent efficacy of nano-formulated C6-ceramide, but inferior activity of dihydro-C6-ceramide and L-C6-ceramide, compared to the unsaturated counterpart and the natural d-enantiomer, respectively. These studies demonstrate novel actions of ceramides that may have application in suppression of tumor metastasis, in addition to their known tumor suppressor effects. PMID:27045476

  15. Characterization of the Cell Surface Properties of Drinking Water Pathogens by Microbial Adhesion to Hydrocarbon and Electrophoretic Mobility Measurements

    EPA Science Inventory

    The surface characteristics of microbial cells directly influence their mobility and behavior within aqueous environments. The cell surface hydrophobicity (CSH) and electrophoretic mobility (EPM) of microbial cells impact a number of interactions and processes including aggregati...

  16. Cell surface alteration in Epstein-Barr virus-transformed cells from patients with extreme insulin resistance

    SciTech Connect

    Gorden, D.L.; Robert, A.; Moncada, V.Y.; Taylor, S.I.; Muehlhauser, J.C.; Carpentier, J.L. )

    1990-08-01

    An abnormality was detected in the morphology of the cell surface of Epstein-Barr virus-transformed lymphocytes of patients with genetic forms of insulin resistance. In cells from two patients with leprechaunism and two patients with type A extreme insulin resistance, scanning electron microscopy demonstrated a decrease in the percentage of the cell surface occupied by microvilli in cells from the patients with leprechaunism and type A insulin resistance compared with control cells. When cells from a healthy control subject and one of the patients with leprechaunism (Lep/Ark-1) were incubated with {sup 125}I-labeled insulin, there was a decrease in the percentage of {sup 125}I-insulin associated with microvilli on the cell surface. Thus, the decreased localization of insulin receptors with the microvillous region of the cell surface was in proportion to the decrease in microvilli.

  17. Inhibition of experimental ascending urinary tract infection by an epithelial cell-surface receptor analogue

    NASA Astrophysics Data System (ADS)

    Edén, C. Svanborg; Freter, R.; Hagberg, L.; Hull, R.; Hull, S.; Leffler, H.; Schoolnik, G.

    1982-08-01

    It has been shown that the establishment of urinary tract infection by Escherichia coli is dependent on attachment of the bacteria to epithelial cells1-4. The attachment involves specific epithelial cell receptors, which have been characterized as glycolipids5-10. Reversible binding to cell-surface mannosides may also be important4,11-13. This suggests an approach to the treatment of infections-that of blocking bacterial attachment with cell membrane receptor analogues. Using E. coli mutants lacking one or other of the two binding specificities (glycolipid and mannose), we show here that glycolipid analogues can block in vitro adhesion and in vivo urinary tract infection.

  18. Selectivity of Ligand-Receptor Interactions between Nanoparticle and Cell Surfaces

    NASA Astrophysics Data System (ADS)

    Wang, Shihu; Dormidontova, Elena E.

    2012-12-01

    Selectivity of interactions between nanoparticles functionalized by tethered ligands and cell surfaces with different densities of receptors plays an essential role in biorecognition and its implementation in nanobiomedicine. We show that the onset of nanoparticle adsorption has a universal character for a range of nanoparticles: the onset receptor density decreases exponentially with the energy of ligand-receptor binding and inversely with the ligand density. We demonstrate that a bimodal tether distribution, which permits shielding ligands by longer nonfunctional tethers, leads to extra loss of entropy at the adsorption onset, enhancing the selectivity.

  19. Microassay for measurement of binding of radiolabelled ligands to cell surface molecules.

    PubMed

    Woof, J M; Burton, D R

    1988-07-22

    An improved technique for measuring the binding of radiolabelled ligands to cell surface molecules has been developed by modification of a procedure using centrifugation through a water-immiscible oil to separate free and cell-bound ligand. It maximises the percentage of ligand bound since cell-bound and free ligand can be separated easily and reproducibly even when very small reaction volumes are used. This permits low levels of ligand radiolabelling and relatively low numbers of cells to be used. PMID:2840465

  20. Analysis of cell surface alterations in Legionella pneumophila cells treated with human apolipoprotein E.

    PubMed

    Palusinska-Szysz, Marta; Zdybicka-Barabas, Agnieszka; Cytryńska, Małgorzata; Wdowiak-Wróbel, Sylwia; Chmiel, Elżbieta; Gruszecki, Wiesław I

    2015-03-01

    Binding of human apolipoprotein E (apoE) to Legionella pneumophila lipopolysaccharide was analysed at the molecular level by Fourier-transform infrared spectroscopy, thereby providing biophysical evidence for apoE-L. pneumophila lipopolysaccharide interaction. Atomic force microscopy imaging of apoE-exposed L. pneumophila cells revealed alterations in the bacterial cell surface topography and nanomechanical properties in comparison with control bacteria. The changes induced by apoE binding to lipopolysaccharide on the surface of L. pneumophila cells may participate in: (1) impeding the penetration of host cells by the bacteria; (2) suppression of pathogen intracellular growth and eventually; and (3) inhibition of the development of infection. PMID:25176171

  1. The distribution of cell surface proteins on spreading cells. Comparison of theory with experiment.

    PubMed Central

    Goldstein, B; Wiegel, F W

    1988-01-01

    Bretscher (1983) has shown that on uniformly spread giant HeLa cells, the receptors for low density lipoprotein (LDL) and transferrin are concentrated toward the periphery of the cells. To explain these nonuniform distributions, he proposed that on giant HeLa cells, recycling receptors return to the cell surface at the cell's leading edge. Since the distribution of coated pits on these cells is uniform, Bretscher and Thomson (1983) proposed that there is a bulk membrane flow toward the cell centers. Here we present a mathematical model that allows us to predict the distribution of cell surface proteins on a thin circular cell, when exocytosis occurs at the cell periphery and endocytosis occurs uniformly over the cell surface. We show that on such a cell, a bulk membrane flow will be generated, whose average velocity is zero at the cell center and increases linearly with the distance from the cell center. Our model predicts that proteins that aggregate in coated pits will have concentrations that are maximal at the cell periphery. We fit our theory to the data of Bretscher and Thomson (1983) on the distribution of ferritin receptors for the following cases: the receptors move by diffusion alone; they move by bulk membrane flow alone; they move by a combination of diffusion and bulk membrane flow. From our fits we show that tau m greater than 3.5 tau p, where tau m and tau p are the lifetimes of the membrane and the ferritin receptor on the cell surface, and that tau pD less than 6.9 X 10(-7) cm2, where D is the ferritin receptor diffusion coefficient. Surprisingly, we obtain the best fits to the data when we neglect membrane flow. Our model predicts that for proteins that are excluded from coated pits, the protein concentration will be Gaussian, being maximal at the cell center and decreasing with the distance from the cell center. If on giant HeLa cells a protein with such a distribution could be found, it would strongly support Bretcher's proposal that there is an

  2. Roles for glycosylation of cell surface receptors involved in cellular immune recognition.

    PubMed

    Rudd, P M; Wormald, M R; Stanfield, R L; Huang, M; Mattsson, N; Speir, J A; DiGennaro, J A; Fetrow, J S; Dwek, R A; Wilson, I A

    1999-10-22

    The majority of cell surface receptors involved in antigen recognition by T cells and in the orchestration of the subsequent cell signalling events are glycoproteins. The length of a typical N-linked sugar is comparable with that of an immunoglobulin domain (30 A). Thus, by virtue of their size alone, oligosaccharides may be expected to play a significant role in the functions and properties of the cell surface proteins to which they are attached. A databank of oligosaccharide structures has been constructed from NMR and crystallographic data to aid in the interpretation of crystal structures of glycoproteins. As unambiguous electron density can usually only be assigned to the glycan cores, the remainder of the sugar is then modelled into the crystal lattice by superimposing the appropriate oligosaccharide from the database. This approach provides insights into the roles that glycosylation might play in cell surface receptors, by providing models that delineate potential close packing interactions on the cell surface. It has been proposed that the specific recognition of antigen by T cells results in the formation of an immunological synapse between the T cell and the antigen-presenting cell. The cell adhesion glycoproteins, such as CD2 and CD48, help to form a cell junction, providing a molecular spacer between opposing cells. The oligosaccharides located on the membrane proximal domains of CD2 and CD48 provide a scaffold to orient the binding faces, which leads to increased affinity. In the next step, recruitment of the peptide major histocompatibility complex (pMHC) by the T-cell receptors (TCRs) requires mobility on the membrane surface. The TCR sugars are located such that they could prevent non-specific aggregation. Importantly, the sugars limit the possible geometry and spacing of TCR/MHC clusters which precede cell signalling. We postulate that, in the final stage, the sugars could play a general role in controlling the assembly and stabilisation of the

  3. Hall effect encoding of brushless dc motors

    NASA Technical Reports Server (NTRS)

    Berard, C. A.; Furia, T. J.; Goldberg, E. A.; Greene, R. C.

    1970-01-01

    Encoding mechanism integral to the motor and using the permanent magnets embedded in the rotor eliminates the need for external devices to encode information relating the position and velocity of the rotating member.

  4. Novel optical encoder for harsh environments

    NASA Astrophysics Data System (ADS)

    Kress, Bernard; Mueller, Ulrich; Brac-de-la-Perriere, Vincent

    2014-09-01

    We are presenting a new optical encoder architecture for shaft encoding, both in incremental and absolute modes. This encoder is based on a diffractive optics technology platform. We have developed various disk based rotary diffractive encoders previously. This encoder is different in the way it is not a disk composed of successive gratings or computer generated holograms, but rather composed of a single element placed on the shaft. It is thus best suited for hollow shaft or end of shaft applications such as in encoder controlled electrical motors. This new architecture aims at solving some of the problems encountered with previous implementations of diffractive encoders such as disk wobble, disk to shaft centering and also encoding in harsh environments.

  5. A new heterogeneous family of telomerically encoded Cryptosporidium proteins

    PubMed Central

    Bouzid, Maha; Hunter, Paul R; McDonald, Vincent; Elwin, Kristin; Chalmers, Rachel M; Tyler, Kevin M

    2013-01-01

    Cryptosporidiosis is predominantly caused by two closely related species of protozoan parasites the zoonotic Cryptosporidium parvum and anthroponotic Cryptosporidium hominis which diverge phenotypically in respect to host range and virulence. Using comparative genomics we identified two genes displaying overt heterogeneity between species. Although initial work suggested both were species specific, Cops-1 for C. parvum and Chos-1 for C. hominis, subsequent study identified an abridged ortholog of Cops-1 in C. hominis. Cops-1 and Chos-1 showed limited, but significant, similarity to each other and share common features: (i) telomeric location: Cops-1 is the last gene on chromosome 2, whilst Chos-1 is the first gene on chromosome 5, (ii) encode circa 50-kDa secreted proteins with isoelectric points above 10, (iii) are serine rich, and (iv) contain internal nucleotide repeats. Importantly, Cops-1 sequence contains specific SNPs with good discriminatory power useful epidemiologically. C. parvum-infected patient sera recognized a 50-kDa protein in antigen preparations of C. parvum but not C. hominis, consistent with Cops-1 being antigenic for patients. Interestingly, anti-Cops-1 monoclonal antibody (9E1) stained oocyst content and sporozoite surface of C. parvum only. This study provides a new example of protozoan telomeres as rapidly evolving contingency loci encoding putative virulence factors. PMID:23467513

  6. A functional calcium-transporting ATPase encoded by chlorella viruses.

    PubMed

    Bonza, Maria Cristina; Martin, Holger; Kang, Ming; Lewis, Gentry; Greiner, Timo; Giacometti, Sonia; Van Etten, James L; De Michelis, Maria Ida; Thiel, Gerhard; Moroni, Anna

    2010-10-01

    Calcium-transporting ATPases (Ca(2+) pumps) are major players in maintaining calcium homeostasis in the cell and have been detected in all cellular organisms. Here, we report the identification of two putative Ca(2+) pumps, M535L and C785L, encoded by chlorella viruses MT325 and AR158, respectively, and the functional characterization of M535L. Phylogenetic and sequence analyses place the viral proteins in group IIB of P-type ATPases even though they lack a typical feature of this class, a calmodulin-binding domain. A Ca(2+) pump gene is present in 45 of 47 viruses tested and is transcribed during virus infection. Complementation analysis of the triple yeast mutant K616 confirmed that M535L transports calcium ions and, unusually for group IIB pumps, also manganese ions. In vitro assays show basal ATPase activity. This activity is inhibited by vanadate, but, unlike that of other Ca(2+) pumps, is not significantly stimulated by either calcium or manganese. The enzyme forms a (32)P-phosphorylated intermediate, which is inhibited by vanadate and not stimulated by the transported substrate Ca(2+), thus confirming the peculiar properties of this viral pump. To our knowledge this is the first report of a functional P-type Ca(2+)-transporting ATPase encoded by a virus. PMID:20573858

  7. Jam-resistant speech encoding

    NASA Astrophysics Data System (ADS)

    Poole, M. A.; Rifkin, R.

    1983-06-01

    This report describes techniques that provide increased jam resistance for digitized speech. Methods for increasing the jam resistance of pulse code modulated data are analyzed and evaluated in listener tests. Special emphasis is placed on new voice encoding approaches that take advantage of a spread spectrum system with a variable (or multiple)-data-rate/variable (or multiple)-AJ capability. Methods for matching a source to a channel in a jamming environment are investigated. Several techniques that provide about a 4 dB increase in jam resistance have been identified.

  8. Elevated temperature treatment as a novel method for decreasing p57 on the cell surface of Renibacterium salmoninarum.

    PubMed

    Piganelli, J D; Wiens, G D; Kaattari, S L

    1999-04-15

    Renibacterium salmoninarum is a Gram-positive diplo-bacillus and the causative agent of bacterial kidney disease, a prevalent disease of salmonid fish. Virulent isolates of R. salmoninarum have a hydrophobic cell surface and express the 57-58 kDa protein (p57). Here we have investigated parameters which effect cell hydrophobicity and p57 degradation. Incubation of R. salmoninarum cells at 37 degrees C for > 4 h decreased cell surface hydrophobicity as measured by the salt aggregation assay, and decreased the amount of cell associated p57. Incubation of cells at lower temperatures (22, 17, 4 or -20 degrees C) for up to 16 h did not reduce hydrophobicity or the amount of cell associated p57. Both the loss of cell surface hydrophobicity and the degradation of p57 were inhibited by pre-incubation with the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Cell surface hydrophobicity was specifically reconstituted by incubation with extracellular protein (ECP) concentrated from culture supernatant and was correlated with the reassociation of p57 onto the bacterial cell surface as determined by western blot and total protein stain analyses. The ability of p57 to reassociate suggests that the bacterial cell surface is not irreversibly modified by the 37 degrees C treatment and that p57 contributes to the hydrophobic nature of R. salmoninarum. In summary, we describe parameters effecting the removal of the p57 virulence factor and suggest the utility of this modification for generating a whole cell vaccine against bacterial kidney disease. PMID:10349550

  9. Time Course of Grammatical Encoding in Agrammatism

    ERIC Educational Resources Information Center

    Lee, Jiyeon

    2011-01-01

    Producing a sentence involves encoding a preverbal message into a grammatical structure by retrieving lexical items and integrating them into a functional (semantic-to-grammatical) structure. Individuals with agrammatism are impaired in this grammatical encoding process. However, it is unclear what aspect of grammatical encoding is impaired and…

  10. Schematic driven layout of Reed Solomon encoders

    NASA Technical Reports Server (NTRS)

    Arave, Kari; Canaris, John; Miles, Lowell; Whitaker, Sterling

    1992-01-01

    Two Reed Solomon error correcting encoders are presented. Schematic driven layout tools were used to create the encoder layouts. Special consideration had to be given to the architecture and logic to provide scalability of the encoder designs. Knowledge gained from these projects was used to create a more flexible schematic driven layout system.

  11. Putative cross-kingdom horizontal gene transfer in sponge (Porifera) mitochondria

    PubMed Central

    Rot, Chagai; Goldfarb, Itay; Ilan, Micha; Huchon, Dorothée

    2006-01-01

    Background The mitochondrial genome of Metazoa is usually a compact molecule without introns. Exceptions to this rule have been reported only in corals and sea anemones (Cnidaria), in which group I introns have been discovered in the cox1 and nad5 genes. Here we show several lines of evidence demonstrating that introns can also be found in the mitochondria of sponges (Porifera). Results A 2,349 bp fragment of the mitochondrial cox1 gene was sequenced from the sponge Tetilla sp. (Spirophorida). This fragment suggests the presence of a 1143 bp intron. Similar to all the cnidarian mitochondrial introns, the putative intron has group I intron characteristics. The intron is present in the cox1 gene and encodes a putative homing endonuclease. In order to establish the distribution of this intron in sponges, the cox1 gene was sequenced from several representatives of the demosponge diversity. The intron was found only in the sponge order Spirophorida. A phylogenetic analysis of the COI protein sequence and of the intron open reading frame suggests that the intron may have been transmitted horizontally from a fungus donor. Conclusion Little is known about sponge-associated fungi, although in the last few years the latter have been frequently isolated from sponges. We suggest that the horizontal gene transfer of a mitochondrial intron was facilitated by a symbiotic relationship between fungus and sponge. Ecological relationships are known to have implications at the genomic level. Here, an ecological relationship between sponge and fungus is suggested based on the genomic analysis. PMID:16972986

  12. A putative genomic island, PGI-1, in Ralstonia solanacearum biovar 2 revealed by subtractive hybridization.

    PubMed

    Stevens, Patricia; van Elsas, Jan Dirk

    2010-10-01

    Ralstonia solanacearum biovar 2, a key bacterial pathogen of potato, has recently established in temperate climate waters. On the basis of isolates obtained from diseased (potato) plants, its genome has been assumed to be virtually clonal, but information on environmental isolates has been lacking. Based on differences in pulsed-field gel electrophoresis patterns, we compared the genomes of two biovar 2 strains with different life histories. Thus, genomic DNA of the novel environmental strain KZR-5 (The Netherlands) was compared to that of reference potato strain 715 (Bangladesh) by suppressive subtractive hybridization. Various strain-specific sequences were found, all being homologous to those found in the genome of reference potato strain 1609. Approximately 20% of these were related to genes involved in recombinational processes. We found a deletion of a 17.6-Kb region, denoted as a putative genomic island PGI-1, in environmental strain KZR-5. The deleted region was, at both extremes, flanked by a composite of two insertion sequence (IS) elements, identified as ISRso2 and ISRso3. The PGI-1 region contained open reading frames that putatively encoded a (p)ppGpp synthetase, a transporter protein, a transcriptional regulator, a cellobiohydrolase, a site-specific integrase/recombinase, a phage-related protein and seven hypothetical proteins. As yet, no phenotype could be assigned to the loss of PGI-1. The ecological behavior of strain KZR-5 was compared to that of reference strain 715. Strain KZR-5 showed enhanced tolerance to 4°C as compared to the reference strain, but was not affected in its virulence on tomato. PMID:20467813

  13. The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover.

    PubMed

    Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

    2016-01-01

    Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca(2+)-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with

  14. The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover

    PubMed Central

    Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

    2016-01-01

    Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca2+-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with

  15. High-density single-molecule analysis of cell surface dynamics in C. elegans embryos

    PubMed Central

    Robin, Francois B.; McFadden, William M.; Yao, Baixue; Munro, Edwin M.

    2014-01-01

    We describe a general, versatile and non-invasive method to image single molecules near the cell surface that can be applied to any GFP-tagged protein in C. elegans embryos. We exploit tunable expression via RNAi and a dynamically exchanging monomer pool to achieve fast continuous single-molecule imaging at optimal densities with signal-to-noise ratios adequate for robust single particle tracking (SPT) analysis. We also introduce and validate a new method called smPReSS that infers exchange rates from quantitative analysis of single molecule photobleaching kinetics, without using SPT. Combining SPT and smPReSS allows spatially and temporally resolved measurements of protein mobility and exchange kinetics. We use these methods (a) to resolve distinct mobility states and spatial variation in exchange rates of the polarity protein Par-6 and (b) to measure spatiotemporal modulation of actin filament assembly and disassembly. The introduction of these methods in a powerful model system offers a promising new avenue to investigate dynamic mechanisms that pattern the embryonic cell surface. PMID:24727651

  16. Research Resource: Androgen Receptor Activity Is Regulated Through the Mobilization of Cell Surface Receptor Networks

    PubMed Central

    Hsiao, Jordy J.; Ng, Brandon H.; Smits, Melinda M.; Martinez, Harryl D.; Jasavala, Rohini J.; Hinkson, Izumi V.; Fermin, Damian; Eng, Jimmy K.; Nesvizhskii, Alexey I.

    2015-01-01

    The aberrant expression of androgen receptor (AR)-dependent transcriptional programs is a defining pathology of the development and progression of prostate cancers. Transcriptional cofactors that bind AR are critical determinants of prostate tumorigenesis. To gain a deeper understanding of the proteins linked to AR-dependent gene transcription, we performed a DNA-affinity chromatography-based proteomic screen designed to identify proteins involved in AR-mediated gene transcription in prostate tumor cells. Functional experiments validated the coregulator roles of known AR-binding proteins in AR-mediated transcription in prostate tumor cells. More importantly, novel coregulatory functions were detected in components of well-established cell surface receptor-dependent signal transduction pathways. Further experimentation demonstrated that components of the TNF, TGF-β, IL receptor, and epidermal growth factor signaling pathways modulated AR-dependent gene transcription and androgen-dependent proliferation in prostate tumor cells. Collectively, our proteomic dataset demonstrates that the cell surface receptor- and AR-dependent pathways are highly integrated, and provides a molecular framework for understanding how disparate signal-transduction pathways can influence AR-dependent transcriptional programs linked to the development and progression of human prostate cancers. PMID:26181434

  17. SNX15 Regulates Cell Surface Recycling of APP and Aβ Generation.

    PubMed

    Feng, Tuancheng; Niu, Mengmeng; Ji, Chengxiang; Gao, Yuehong; Wen, Jing; Bu, Guojun; Xu, Huaxi; Zhang, Yun-Wu

    2016-08-01

    Amyloid-β (Aβ) peptide plays an essential role in the pathogenesis of Alzheimer's disease (AD) and is generated from amyloid-β precursor protein (APP) through sequential proteolytic cleavages by β-site APP cleaving enzyme 1 (BACE1) and γ-secretase. Trafficking dysregulation of APP, BACE1, and γ-secretase may affect Aβ generation and disease pathogenesis. Sorting nexin 15 (SNX15) is known to regulate protein trafficking. Here, we report that SNX15 is abundantly expressed in mouse neurons and astrocytes. In addition, we show that although not affecting the protein levels of APP, BACE1, and γ-secretase components and the activity of BACE1 and γ-secretase, overexpression and downregulation of SNX15 reduce and promote Aβ production, respectively. Furthermore, we find that overexpression of SNX15 increases APP protein levels in cell surface through accelerating APP recycling, whereas downregulation of SNX15 has an opposite effect. Finally, we show that exogenous expression of human SNX15 in the hippocampal dentate gyrus by adeno-associated virus (AAV) infection can significantly reduce Aβ pathology in the hippocampus and improve short-term working memory in the APPswe/PSEN1dE9 double transgenic AD model mice. Together, our results suggest that SNX15 regulates the recycling of APP to cell surface and, thus, its processing for Aβ generation. PMID:26115702

  18. 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

  19. A giant cell surface protein in Synechococcus WH8102 inhibits feeding by a dinoflagellate predator.

    PubMed

    Strom, Suzanne L; Brahamsha, Bianca; Fredrickson, Kerri A; Apple, Jude K; Rodríguez, Andres Gutiérrez

    2012-03-01

    Diverse strains of the marine planktonic cyanobacterium Synechococcus sp. show consistent differences in their susceptibility to predation. We used mutants of Sargasso Sea strain WH8102 (clade III) to test the hypothesis that cell surface proteins play a role in defence against predation by protists. Predation rates by the heterotrophic dinoflagellate Oxyrrhis marina on mutants lacking the giant SwmB protein were always higher (by 1.6 to 3.9×) than those on wild-type WH8102 cells, and equalled predation rates on a clade I strain (CC9311). In contrast, absence of the SwmA protein, which comprises the S-layer (surface layer of the cell envelope that is external to the outer membrane), had no effect on predation by O. marina. Reductions in predation rate were not due to dissolved substances in Synechococcus cultures, and could not be accounted for by variations in cell hydrophobicity. We hypothesize that SwmB defends Synechococcus WH8102 by interfering with attachment of dinoflagellate prey capture organelles or cell surface receptors. Giant proteins are predicted in the genomes of multiple Synechococcus isolates, suggesting that this defence strategy may be more general. Strategies for resisting predation will contribute to the differential competitive success of different Synechococcus groups, and to the diversity of natural picophytoplankton assemblages. PMID:22103339

  20. LRP-1–CD44, a New Cell Surface Complex Regulating Tumor Cell Adhesion

    PubMed Central

    Perrot, Gwenn; Langlois, Benoit; Devy, Jérôme; Jeanne, Albin; Verzeaux, Laurie; Almagro, Sébastien; Sartelet, Hervé; Hachet, Cathy; Schneider, Christophe; Sick, Emilie; David, Marion; Khrestchatisky, Michel; Emonard, Hervé; Martiny, Laurent

    2012-01-01

    The low-density lipoprotein receptor-related protein 1 (LRP-1) is a large endocytic receptor mediating the clearance of various molecules from the extracellular matrix. In the field of cancer, LRP-1-mediated endocytosis was first associated with antitumor properties. However, recent results suggested that LRP-1 may coordinate the adhesion-deadhesion balance in malignant cells to support tumor progression. Here, we observed that LRP-1 silencing or RAP (receptor-associated protein) treatment led to accumulation of CD44 at the tumor cell surface. Moreover, we evidenced a tight interaction between CD44 and LRP-1, not exclusively localized in lipid rafts. Overexpression of LRP-1-derived minireceptors indicated that the fourth ligand-binding cluster of LRP-1 is required to bind CD44. Labeling of CD44 with EEA1 and LAMP-1 showed that internalized CD44 is routed through early endosomes toward lysosomes in a LRP-1-dependent pathway. LRP-1-mediated internalization of CD44 was highly reduced under hyperosmotic conditions but poorly affected by membrane cholesterol depletion, revealing that it proceeds mostly via clathrin-coated pits. Finally, we demonstrated that CD44 silencing abolishes RAP-induced tumor cell attachment, revealing that cell surface accumulation of CD44 under LRP-1 blockade is mainly responsible for the stimulation of tumor cell adhesion. Altogether, our data shed light on the LRP-1-mediated internalization of CD44 that appeared critical to define the adhesive properties of tumor cells. PMID:22711991

  1. Protein Kinase C Regulates the Cell Surface Activity of Endothelin-Converting Enzyme-1.

    PubMed

    Smith, A Ian; Lew, Rebecca A; Thomas, Walter G; Tochon-Danguy, Nathalie

    2006-09-01

    The potent vasoconstrictor endothelin is a 21 amino acid peptide whose principal physiological function is to regulate vascular tone. The generation of endothelin is crucially dependent on the local presence and activity of endothelin converting enzyme-1 (ECE-1) expressed on the surface of vascular endothelial cells. In this study, we have shown in endothelial cells that the enzyme is phosphorylated, and that phosphorylation is increased by phorbol ester stimulation of protein kinase C (PKC). Furthermore, by monitoring specific ECE-1 activity on the surface of live cells, we also show that following PKC activation, enzyme activity is significantly increased at the cell surface, where it is positioned to catalyse the generation of active endothelin. We believe this novel finding is unprecedented for a peptide processing enzyme. Indeed, this new knowledge regarding the control of endothelin production by regulating ECE-1 activity at the cell surface opens up a new area of endothelin biology and will provide novel insights into the physiology and pathophysiology of endothelin and endothelin-associated diseases. In addition, the information generated in these studies may provide valuable new insights into potential extra- and intracellular targets for the pharmacological and perhaps even therapeutic regulation of endothelin production and thus vascular tone. PMID:19617920

  2. A dual phosphorylation switch controls 14-3-3-dependent cell surface expression of TASK-1

    PubMed Central

    Kilisch, Markus; Lytovchenko, Olga; Arakel, Eric C.; Bertinetti, Daniela; Schwappach, Blanche

    2016-01-01

    ABSTRACT The transport of the K+ channels TASK-1 and TASK-3 (also known as KCNK3 and KCNK9, respectively) to the cell surface is controlled by the binding of 14-3-3 proteins to a trafficking control region at the extreme C-terminus of the channels. The current model proposes that phosphorylation-dependent binding of 14-3-3 sterically masks a COPI-binding motif. However, the direct effects of phosphorylation on COPI binding and on the binding parameters of 14-3-3 isoforms are still unknown. We find that phosphorylation of the trafficking control region prevents COPI binding even in the absence of 14-3-3, and we present a quantitative analysis of the binding of all human 14-3-3 isoforms to the trafficking control regions of TASK-1 and TASK-3. Surprisingly, the affinities of 14-3-3 proteins for TASK-1 are two orders of magnitude lower than for TASK-3. Furthermore, we find that phosphorylation of a second serine residue in the C-terminus of TASK-1 inhibits 14-3-3 binding. Thus, phosphorylation of the trafficking control region can stimulate or inhibit transport of TASK-1 to the cell surface depending on the target serine residue. Our findings indicate that control of TASK-1 trafficking by COPI, kinases, phosphatases and 14-3-3 proteins is highly dynamic. PMID:26743085

  3. Serotonin-induced down-regulation of cell surface serotonin transporter

    PubMed Central

    Jørgensen, Trine Nygaard; Christensen, Peter Møller; Gether, Ulrik

    2014-01-01