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Sample records for encoding putative cell-surface

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

    PubMed Central

    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. An Agrobacterium gene involved in tumorigenesis encodes an outer membrane protein exposed on the bacterial cell surface.

    PubMed

    Jia, Y H; Li, L P; Hou, Q M; Pan, Shen Q

    2002-02-01

    A gene designated as aopB was identified which was involved in tumorigenesis of Agrobacterium tumefaciens. aopB is located on the circular chromosome as a single copy. This gene shares high homology with ropB, a Rhizobium leguminosarum gene encoding an outer membrane protein. A transposon mutant CGI1 containing a gfp-tagged transposon insertion at aopB caused attenuated tumors on plants when inoculated at a low cell concentration (5x10(7) cells/ml). The mutation did not affect the bacterial growth on different media. A broad host range plasmid containing the wild type aopB could restore the tumor formation ability of CGI1 to the wild type level. When both aopB-gfp and aopB-phoA fusions were used to study the aopB gene expression, we found that the aopB gene was inducible by acidic pH but not by plant phenolic compound acetosyringone. aopB encodes a putative protein of 218 amino acids with a predicted molecular weight of 22.8 kDa. TnphoA transposon mutagenesis of aopB, subcellular fractionation and whole cell ELISA experiments indicated that AopB is an outer membrane protein exposed on the bacterial cell surface. It appeared that AopB was exclusively present in the outer membrane and not in other fractions. The vir gene induction assays showed that the aopB gene was not required for the expression of the Ti plasmid encoded vir genes that are essential for tumorigenesis. The C-terminal half of AopB is slightly homologous to some of the bacterial porin proteins and some of plant dehydrins. The role of AopB in Agrobacterium-plant interaction is discussed. PMID:11891052

  3. 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

  4. 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}.

  5. Zipper encodes a putative integral membrane protein required for normal axon patterning during Drosophila neurogenesis.

    PubMed Central

    Zhao, D B; Côté, S; Jähnig, F; Haller, J; Jäckle, H

    1988-01-01

    During the development of the central nervous system, Drosophila embryo axons become organized in a stereo-typed fasciculation pattern. We have found that the zipper (zip) gene, initially identified on the basis of a defective larval cuticle in zip mutant embryos, is possibly involved in the establishment or maintenance of the axon pattern during the late stages of neurogenesis. The zip wild-type gene is expressed in the developing nervous system. It codes for a putative integral membrane protein. Both the molecular features of zipper and its biological effect in the nervous system of mutants suggest that zipper is an essential component for cell surface interactions involved in axon patterning, and that the cuticle phenotype of zip mutants is dependent on the primary defects observed in the nervous system. Images PMID:3402433

  6. 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

  7. CD44 in Differentiated Embryonic Stem Cells: Surface Expression and Transcripts Encoding Multiple Variants

    PubMed Central

    Haegel, Hélène; Dierich, Andrée

    1994-01-01

    Expression of the surface-adhesion molecule CD44 was investigated during the in vitro differentiation of the embryonic stem (ES) cell line D3. By immunofluorescence analysis, totipotent, undifferentiated ES cells did not show surface expression of CD44, although two transcripts of approximately 1.6 and 3.3 kb were detected on Northern blots. Following 1 week of differentiation in either suspension or substrate-attached cultures, CD44 appeared on the surface of some D3 cells, and synthesis of an additional 4.5 kb mRNA species was detected on Northern blots. At this stage, at least three distinct transcripts encoding CD44 variants were induced within the cultures, resulting from alternative splicing of additional exons in the variable domains of CD44. From PCR analysis, they all appeared to contain the variable exon v10, and two of them in addition contained v6. Taken together, these results suggest that CD44 may play a role in cell migration and adhesion in the early development of the mouse embryo. PMID:7542511

  8. Saccharomyces Cerevisiae Hoc1, a Suppressor of Pkc1, Encodes a Putative Glycosyltransferase

    PubMed Central

    Neiman, A. M.; Mhaiskar, V.; Manus, V.; Galibert, F.; Dean, N.

    1997-01-01

    The Saccharomyces cerevisiae gene PKC1 encodes a protein kinase C isozyme that regulates cell wall synthesis. Here we describe the characterization of HOC1, a gene identified by its ability to suppress the cell lysis phenotype of pkc1-371 cells. The HOC1 gene (Homologous to OCH1) is predicted to encode a type II integral membrane protein that strongly resembles Och1p, an α-1,6-mannosyltransferase. Immunofluorescence studies localized Hoc1p to the Golgi apparatus. While overexpression of HOC1 rescued the pkc1-371 temperature-sensitive cell lysis phenotype, disruption of HOC1 lowered the restrictive temperature of the pkc1-371 allele. Disruption of HOC1 also resulted in hypersensitivity to Calcofluor White and hygromycin B, phenotypes characteristic of defects in cell wall integrity and protein glycosylation, respectively. The function of HOC1 appears to be distinct from that of OCH1. Taken together, these results suggest that HOC1 encodes a Golgi-localized putative mannosyltransferase required for the proper construction of the cell wall. PMID:9055074

  9. A putative peroxidase cDNA from turnip and analysis of the encoded protein sequence.

    PubMed

    Romero-Gómez, S; Duarte-Vázquez, M A; García-Almendárez, B E; Mayorga-Martínez, L; Cervantes-Avilés, O; Regalado, C

    2008-12-01

    A putative peroxidase cDNA was isolated from turnip roots (Brassica napus L. var. purple top white globe) by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Total RNA extracted from mature turnip roots was used as a template for RT-PCR, using a degenerated primer designed to amplify the highly conserved distal motif of plant peroxidases. The resulting partial sequence was used to design the rest of the specific primers for 5' and 3' RACE. Two cDNA fragments were purified, sequenced, and aligned with the partial sequence from RT-PCR, and a complete overlapping sequence was obtained and labeled as BbPA (Genbank Accession No. AY423440, named as podC). The full length cDNA is 1167bp long and contains a 1077bp open reading frame (ORF) encoding a 358 deduced amino acid peroxidase polypeptide. The putative peroxidase (BnPA) showed a calculated Mr of 34kDa, and isoelectric point (pI) of 4.5, with no significant identity with other reported turnip peroxidases. Sequence alignment showed that only three peroxidases have a significant identity with BnPA namely AtP29a (84%), and AtPA2 (81%) from Arabidopsis thaliana, and HRPA2 (82%) from horseradish (Armoracia rusticana). Work is in progress to clone this gene into an adequate host to study the specific role and possible biotechnological applications of this alternative peroxidase source. PMID:18686036

  10. The adnAB Locus, Encoding a Putative Helicase-Nuclease Activity, Is Essential in Streptomyces

    PubMed Central

    Zhang, Lingli; Nguyen, Hoang Chuong; Chipot, Ludovic; Piotrowski, Emilie; Bertrand, Claire

    2014-01-01

    Homologous recombination is a crucial mechanism that repairs a wide range of DNA lesions, including the most deleterious ones, double-strand breaks (DSBs). This multistep process is initiated by the resection of the broken DNA ends by a multisubunit helicase-nuclease complex exemplified by Escherichia coli RecBCD, Bacillus subtilis AddAB, and newly discovered Mycobacterium tuberculosis AdnAB. Here we show that in Streptomyces, neither recBCD nor addAB homologues could be detected. The only putative helicase-nuclease-encoding genes identified were homologous to M. tuberculosis adnAB genes. These genes are conserved as a single copy in all sequenced genomes of Streptomyces. The disruption of adnAB in Streptomyces ambofaciens and Streptomyces coelicolor could not be achieved unless an ectopic copy was provided, indicating that adnAB is essential for growth. Both adnA and adnB genes were shown to be inducible in response to DNA damage (mitomycin C) and to be independently transcribed. Introduction of S. ambofaciens adnAB genes in an E. coli recB mutant restored viability and resistance to UV light, suggesting that Streptomyces AdnAB could be a functional homologue of RecBCD and be involved in DNA damage resistance. PMID:24837284

  11. A human gene (DDX10) encoding a putative DEAD-box RNA helicase at 11q22-q23

    SciTech Connect

    Savitsky, K.; Ziv, Y.; Bar-Shira, A.

    1996-04-15

    A human gene encoding a putative RNA helicase, designated DDX10, was identified 400 kb telomeric to the ataxia-telangiectasia gene at chromosome 11q22-q23. The predicted amino acid sequence shows very high similarity to a subgroup of DEAD-box RNA helicases involved in ribosome biogenesis. This novel gene encodes a 3.2-kb transcript in a variety of human tissues. A processed pseudogene of DDX10 was detected at chromosome 9q21-q22. We observed a rare trinucleotide repeat length polymorphism within the coding sequence of DDX10. 39 refs., 5 figs.

  12. Us3 kinase encoded by herpes simplex virus 1 mediates downregulation of cell surface major histocompatibility complex class I and evasion of CD8+ T cells.

    PubMed

    Imai, Takahiko; Koyanagi, Naoto; Ogawa, Ryo; Shindo, Keiko; Suenaga, Tadahiro; Sato, Ayuko; Arii, Jun; Kato, Akihisa; Kiyono, Hiroshi; Arase, Hisashi; Kawaguchi, Yasushi

    2013-01-01

    Detection and elimination of virus-infected cells by CD8(+) cytotoxic T lymphocytes (CTLs) depends on recognition of virus-derived peptides presented by major histocompatibility complex class I (MHC-I) molecules on the surface of infected cells. In the present study, we showed that inactivation of the activity of viral kinase Us3 encoded by herpes simplex virus 1 (HSV-1), the etiologic agent of several human diseases and a member of the alphaherpesvirinae, significantly increased cell surface expression of MHC-I, thereby augmenting CTL recognition of infected cells in vitro. Overexpression of Us3 by itself had no effect on cell surface expression of MHC-I and Us3 was not able to phosphorylate MHC-I in vitro, suggesting that Us3 indirectly downregulated cell surface expression of MHC-I in infected cells. We also showed that inactivation of Us3 kinase activity induced significantly more HSV-1-specific CD8(+) T cells in mice. Interestingly, depletion of CD8(+) T cells in mice significantly increased replication of a recombinant virus encoding a kinase-dead mutant of Us3, but had no effect on replication of a recombinant virus in which the kinase-dead mutation was repaired. These results indicated that Us3 kinase activity is required for efficient downregulation of cell surface expression of MHC-I and mediates evasion of HSV-1-specific CD8(+) T cells. Our results also raised the possibility that evasion of HSV-1-specific CD8(+) T cells by HSV-1 Us3-mediated inhibition of MHC-I antigen presentation might in part contribute to viral replication in vivo.

  13. HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern in the yeast Saccharomyces cerevisiae.

    PubMed Central

    Yabe, T; Yamada-Okabe, T; Kasahara, S; Furuichi, Y; Nakajima, T; Ichishima, E; Arisawa, M; Yamada-Okabe, H

    1996-01-01

    We previously isolated the Saccharomyces cerevisiae HKR1 gene that confers on S. cerevisiae cells resistance to HM-1 killer toxin secreted by Hansenula mrakii (S. Kasahara, H. Yamada, T. Mio, Y. Shiratori, C. Miyamoto, T. Yabe, T. Nakajima, E. Ichishima, and Y. Furuichi, J. Bacteriol. 176:1488-1499, 1994). HKR1 encodes a type 1 membrane protein that contains a calcium-binding consensus sequence (EF hand motif) in the cytoplasmic domain. Although the null mutation of HKR1 is lethal, disruption of the 3' part of the coding region, which would result in deletion of the cytoplasmic domain of Hkr1p, did not affect the viability of yeast cells. This partial disruption of HKR1 significantly reduced beta-1,3-glucan synthase activity and the amount of beta-1,3-glucan in the cell wall and altered the axial budding pattern of haploid cells. Neither chitin synthase activity nor chitin content was significantly affected in the cells harboring the partially disrupted HKR1 allele. Immunofluorescence microscopy with an antibody raised against Hkr1p expressed in Escherichia coli revealed that Hkr1p was predominantly localized on the cell surface. The cell surface localization of Hkr1p required the N-terminal signal sequence because the C-terminal half of Hkr1p was detected uniformly in the cells. These results demonstrate that HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern and suggest that bud site assembly is somehow related to beta-glucan synthesis in S. cerevisiae. PMID:8550469

  14. 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...

  15. Raspberry leaf blotch virus, a putative new member of the genus Emaravirus, encodes a novel genomic RNA.

    PubMed

    McGavin, Wendy J; Mitchell, Carolyn; Cock, Peter J A; Wright, Kathryn M; MacFarlane, Stuart A

    2012-02-01

    A new, segmented, negative-strand RNA virus with morphological and sequence similarities to other viruses in the genus Emaravirus was discovered in raspberry plants exhibiting symptoms of leaf blotch disorder, a disease previously attributed to the eriophyid raspberry leaf and bud mite (Phyllocoptes gracilis). The virus, tentatively named raspberry leaf blotch virus (RLBV), has five RNAs that each potentially encode a single protein on the complementary strand. RNAs 1, 2 and 3 encode, respectively, a putative RNA-dependent RNA polymerase, a glycoprotein precursor and the nucleocapsid. RNA4 encodes a protein with sequence similarity to proteins of unknown function that are encoded by the genomes of other emaraviruses. When expressed transiently in plants fused to green or red fluorescent protein, the RLBV P4 protein localized to the peripheral cell membrane and to punctate spots in the cell wall. These spots co-localized with GFP-tagged tobacco mosaic virus 30K cell-to-cell movement protein, which is itself known to associate with plasmodesmata. These results suggest that the P4 protein may be a movement protein for RLBV. The fifth RLBV RNA, encoding the P5 protein, is unique among the sequenced emaraviruses. The amino acid sequence of the P5 protein does not suggest any potential function; however, when expressed as a GFP fusion, it localized as small aggregates in the cytoplasm near to the periphery of the cell.

  16. Isolation and sequencing of cDNA clones encoding ethylene-induced putative peroxidases from cucumber cotyledons.

    PubMed

    Morgens, P H; Callahan, A M; Dunn, L J; Abeles, F B

    1990-05-01

    A cDNA library from ethephon-treated cucumber cotyledons (Cucumis sativus L. cv. Poinsett 76) was constructed. Two cDNA clones encoding putative peroxidases were isolated by means of a synthetic probe based on a partial amino acid sequence of a 33 kDa cationic peroxidase that had been previously shown to be induced by ethylene. DNA sequencing indicates that the two clones were derived from two closely related RNA species that are related to published plant peroxidase sequences. Southern analysis indicates that there are 1-5 copies in a haploid genome of a gene homologous to the cDNA clones. The deduced amino acid sequences are homologous with a tobacco (55% sequence identity), a horseradish (53%), a turnip (45%), and a potato (41%) peroxidase. The cloned sequences do not encode the 33 kDa peroxidase from which the original synthetic probe was been derived, but rather other putative peroxidases. An increase in the level of mRNA is evident by 3 hours after ethephon or ethylene treatment and plateaus by 15 hours. PMID:2102850

  17. 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

  18. 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

  19. The Saccharomyces cerevisiae ACR3 gene encodes a putative membrane protein involved in arsenite transport.

    PubMed

    Wysocki, R; Bobrowicz, P; Ułaszewski, S

    1997-11-28

    The cluster of three genes, ACR1, ACR2, and ACR3, previously was shown to confer arsenical resistance in Saccharomyces cerevisiae. The overexpression of ACR3 induced high level arsenite resistance. The presence of ACR3 together with ACR2 on a multicopy plasmid was conducive to increased arsenate resistance. The function of ACR3 gene has now been investigated. Amino acid sequence analysis of Acr3p showed that this hypothetical protein has hydrophobic character with 10 putative transmembrane spans and is probably located in yeast plasma membrane. We constructed the acr3 null mutation. The resulting disruptants were 5-fold more sensitive to arsenate and arsenite than wild-type cells. The acr3 disruptants showed wild-type sensitivity to antimony, tellurite, cadmium, and phenylarsine oxide. The mechanism of arsenical resistance was assayed by transport experiments using radioactive arsenite. We did not observe any significant differences in the accumulation of 76AsO33- in wild-type cells, acr1 and acr3 disruptants. However, the high dosage of ACR3 gene resulted in loss of arsenite uptake. These results suggest that arsenite resistance in yeast is mediated by an arsenite transporter (Acr3p).

  20. 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.

  1. ald of Mycobacterium tuberculosis encodes both the alanine dehydrogenase and the putative glycine dehydrogenase.

    PubMed

    Giffin, Michelle M; Modesti, Lucia; Raab, Ronald W; Wayne, Lawrence G; Sohaskey, Charles D

    2012-03-01

    The putative glycine dehydrogenase of Mycobacterium tuberculosis catalyzes the reductive amination of glyoxylate to glycine but not the reverse reaction. The enzyme was purified and identified as the previously characterized alanine dehydrogenase. The Ald enzyme was expressed in Escherichia coli and had both pyruvate and glyoxylate aminating activities. The gene, ald, was inactivated in M. tuberculosis, which resulted in the loss of all activities. Both enzyme activities were found associated with the cell and were not detected in the extracellular filtrate. By using an anti-Ald antibody, the protein was localized to the cell membrane, with a smaller fraction in the cytosol. None was detected in the extracellular medium. The ald knockout strain grew without alanine or glycine and was able to utilize glycine but not alanine as a nitrogen source. Transcription of ald was induced when alanine was the sole nitrogen source, and higher levels of Ald enzyme were measured. Ald is proposed to have several functions, including ammonium incorporation and alanine breakdown.

  2. 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.

  3. A Friend virus mutant that overcomes Fv-2rr host resistance encodes a small glycoprotein that dimerizes, is processed to cell surfaces, and specifically activates erythropoietin receptors.

    PubMed Central

    Kozak, S L; Hoatlin, M E; Ferro, F E; Majumdar, M K; Geib, R W; Fox, M T; Kabat, D

    1993-01-01

    The env gene of Friend spleen focus-forming virus (SFFV) encodes a membrane glycoprotein (gp55) that is inefficiently (3 to 5%) processed from the rough endoplasmic reticulum to form a larger dimeric plasma membrane derivative (gp55p). Moreover, the SFFV env glycoprotein associates with erythropoietin receptors (EpoR) to cause proliferation of infected erythroblasts [J.-P. Li, A. D. D'Andrea, H. F. Lodish, and D. Baltimore, Nature (London) 343:762-764, 1990]. Interestingly, the mitogenic effect of SFFV is blocked in mice homozygous for the Fv-2r resistance gene, but mutant SFFVs can overcome this resistance. Recent evidence suggested that these mutants contain partial env deletions that truncate the membrane-proximal extracellular domain of the encoded glycoproteins (M. H. Majumdar, C.-L. Cho, M. T. Fox, K. L. Eckner, S. Kozak, D. Kabat, and R. W. Geib, J. Virol. 66:3652-3660, 1992). Mutant BB6, which encodes a gp42 glycoprotein that has a large deletion in this domain, causes erythroblastosis in DBA/2 (Fv-2s) as well as in congenic D2.R (Fv-2r) mice. Analogous to gp55, gp42 is processed inefficiently as a disulfide-bonded dimer to form cell surface gp42p. Retroviral vectors with SFFV and BB6 env genes have no effect on interleukin 3-dependent BaF3 hematopoietic cells, but they cause growth factor independency of BaF3/EpoR cells, a derivative that contains recombinant EpoR. After binding 125I-Epo to surface EpoR on these factor-independent cells and adding the covalent cross-linking reagent disuccinimidyl suberate, complexes that had immunological properties and sizes demonstrating that they consisted of 125I-Epo-gp55p and 125I-Epo-gp42p were isolated from cell lysates. Contrary to a previous report, SFFV or BB6 env glycoproteins did not promiscuously activate other members of the EpoR superfamily. Although the related env glycoproteins encoded by dualtropic murine leukemia viruses formed detectable complexes with EpoR, strong mitogenic signalling did not ensue

  4. 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

  5. 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

  6. 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

  7. The Fusarium oxysporum gnt2, encoding a putative N-acetylglucosamine transferase, is involved in cell wall architecture and virulence.

    PubMed

    López-Fernández, Loida; Ruiz-Roldán, Carmen; Pareja-Jaime, Yolanda; Prieto, Alicia; Khraiwesh, Husam; Roncero, M Isabel G

    2013-01-01

    With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed co-localization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity. PMID:24416097

  8. 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.

  9. Cloning and characterization of cDNAs encoding equine infectious anemia virus tat and putative Rev proteins.

    PubMed Central

    Stephens, R M; Derse, D; Rice, N R

    1990-01-01

    We isolated and characterized six cDNA clones from an equine infectious anemia virus-infected cell line that displays a Rev-defective phenotype. With the exception of one splice site in one of the clones, all six cDNAs exhibited the same splicing pattern and consisted of four exons. Exon 1 contained the 5' end of the genome; exon 2 contained the tat gene from mid-genome; exon 3 consisted of a small section of env, near the 5' end of the env gene; and exon 4 contained the putative rev open reading frame from the 3' end of the genome. The structures of the cDNAs predict a bicistronic message in which Tat is encoded by exons 1 and 2 and the presumptive Rev protein is encoded by exons 3 and 4. tat translation appears to be initiated at a non-AUG codon within the first 15 codons of exon 1. Equine infectious anemia virus-specific tat activity was expressed in transient transfections with cDNA expression plasmids. The predicted wild-type Rev protein contains 30 env-derived amino acids and 135 rev open reading frame residues. All of the cDNAs had a frameshift in exon 4, leading to a truncated protein and thus providing a plausible explanation for the Rev-defective phenotype of the original cells. We used peptide antisera to detect the faulty protein, thus confirming the cDNA sequence, and to detect the normal protein in productively infected cells. Images PMID:2164593

  10. The ham-2 locus, encoding a putative transmembrane protein, is required for hyphal fusion in Neurospora crassa.

    PubMed Central

    Xiang, Qijun; Rasmussen, Carolyn; Glass, N Louise

    2002-01-01

    Somatic cell fusion is common during organogenesis in multicellular eukaryotes, although the molecular mechanism of cell fusion is poorly understood. In filamentous fungi, somatic cell fusion occurs during vegetative growth. Filamentous fungi grow as multinucleate hyphal tubes that undergo frequent hyphal fusion (anastomosis) during colony expansion, resulting in the formation of a hyphal network. The molecular mechanism of the hyphal fusion process and the role of networked hyphae in the growth and development of these organisms are unexplored questions. We use the filamentous fungus Neurospora crassa as a model to study the molecular mechanism of hyphal fusion. In this study, we identified a deletion mutant that was restricted in its ability to undergo both self-hyphal fusion and fusion with a different individual to form a heterokaryon. This deletion mutant displayed pleiotropic defects, including shortened aerial hyphae, altered conidiation pattern, female sterility, slow growth rate, lack of hyphal fusion, and suppression of vegetative incompatibility. Complementation with a single open reading frame (ORF) within the deletion region in this mutant restored near wild-type growth rates, female fertility, aerial hyphae formation, and hyphal fusion, but not vegetative incompatibility and wild-type conidiation pattern. This ORF, which we named ham-2 (for hyphal anastomosis), encodes a putative transmembrane protein that is highly conserved, but of unknown function among eukaryotes. PMID:11805054

  11. Inactivation of Francisella tularensis Gene Encoding Putative ABC Transporter Has a Pleiotropic Effect upon Production of Various Glycoconjugates.

    PubMed

    Dankova, Vera; Balonova, Lucie; Link, Marek; Straskova, Adela; Sheshko, Valeria; Stulik, Jiri

    2016-02-01

    Francisella tularensis, an intracellular pathogen causing the disease tularemia, utilizes surface glycoconjugates such as lipopolysaccharide, capsule, and capsule-like complex for its protection against inhospitable conditions of the environment. Francisella species also possess a functional glycosylation apparatus by which specific proteins are O-glycosidically modified. We here created a mutant with a nonfunctional FTS_1402 gene encoding for a putative glycan flippase and studied the consequences of its disruption. The mutant strain expressed diminished glycosylation similarly to, but to a lesser extent than, that of the oligosaccharyltransferase-deficient ΔpglA mutant. In contrast to ΔpglA, inactivation of FTS_1402 had a pleiotropic effect, leading to alteration in glycosylation and, importantly, to decrease in lipopolysaccharide, capsule, and/or capsule-like complex production, which were reflected by distinct phenotypes in host-pathogen associated properties and virulence potential of the two mutant strains. Disruption of FTS_1402 resulted in enhanced sensitivity to complement-mediated lysis and reduced virulence in mice that was independent of diminished glycosylation. Importantly, the mutant strain induced a protective immune response against systemic challenge with homologous wild-type FSC200 strain. Targeted disruption of genes shared by multiple metabolic pathways may be considered a novel strategy for constructing effective live, attenuated vaccines. PMID:26815358

  12. 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.

  13. 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

  14. Cloning and sequencing of cDNAs encoding a pathogen-induced putative peroxidase of wheat (Triticum aestivum L.).

    PubMed

    Rebmann, G; Hertig, C; Bull, J; Mauch, F; Dudler, R

    1991-02-01

    We report here the complete amino acid sequence of a pathogen-induced putative peroxidase from wheat (Triticum aestivum L.) as deduced from cDNA clones representing mRNA from leaves infected with the powdery mildew fungus Erysiphe graminis. The protein consists of 312 amino acids, of which the first 22 form a putative signal sequence, and has a calculated pI of 5.7. Sequence comparison revealed that the putative wheat peroxidase is most similar to the turnip (Brassica rapa) peroxidase, with which it shares 57% identical and 13% conserved amino acids.

  15. Cloning and sequencing of cDNAs encoding a pathogen-induced putative peroxidase of wheat (Triticum aestivum L.).

    PubMed

    Rebmann, G; Hertig, C; Bull, J; Mauch, F; Dudler, R

    1991-02-01

    We report here the complete amino acid sequence of a pathogen-induced putative peroxidase from wheat (Triticum aestivum L.) as deduced from cDNA clones representing mRNA from leaves infected with the powdery mildew fungus Erysiphe graminis. The protein consists of 312 amino acids, of which the first 22 form a putative signal sequence, and has a calculated pI of 5.7. Sequence comparison revealed that the putative wheat peroxidase is most similar to the turnip (Brassica rapa) peroxidase, with which it shares 57% identical and 13% conserved amino acids. PMID:1893103

  16. 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.

  17. 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.

  18. MDS1, a dosage suppressor of an mck1 mutant, encodes a putative yeast homolog of glycogen synthase kinase 3.

    PubMed Central

    Puziss, J W; Hardy, T A; Johnson, R B; Roach, P J; Hieter, P

    1994-01-01

    The yeast gene MCK1 encodes a serine/threonine protein kinase that is thought to function in regulating kinetochore activity and entry into meiosis. Disruption of MCK1 confers a cold-sensitive phenotype, a temperature-sensitive phenotype, and sensitivity to the microtubule-destabilizing drug benomyl and leads to loss of chromosomes during growth on benomyl. A dosage suppression selection was used to identify genes that, when present at high copy number, could suppress the cold-sensitive phenotype of mck1::HIS3 mutant cells. Several unique classes of clones were identified, and one of these, designated MDS1, has been characterized in some detail. Nucleotide sequence data reveal that MDS1 encodes a serine/threonine protein kinase that is highly homologous to the shaggy/zw3 kinase in Drosophila melanogaster and its functional homolog, glycogen synthase kinase 3, in rats. The presence of MDS1 in high copy number rescues both the cold-sensitive and the temperature-sensitive phenotypes, but not the benomyl-sensitive phenotype, associated with the disruption of MCK1. Analysis of strains harboring an mds1 null mutation demonstrates that MDS1 is not essential during normal vegetative growth but appears to be required for meiosis. Finally, in vitro experiments indicate that the proteins encoded by both MCK1 and MDS1 possess protein kinase activity with substrate specificity similar to that of mammalian glycogen synthase kinase 3. Images PMID:8264650

  19. Isolation and structure of a cDNA encoding the B1 (CD20) cell-surface antigen of human B lymphocytes.

    PubMed Central

    Tedder, T F; Streuli, M; Schlossman, S F; Saito, H

    1988-01-01

    The B1 (CD20) molecule is a Mr 33,000 phosphoprotein on the surface of human B lymphocytes that may serve a central role in the humoral immune response by regulating B-cell proliferation and differentiation. In this report, a cDNA clone that encodes the B1 molecule was isolated and the amino acid sequence of B1 was determined. B-cell-specific cDNA clones were selected from a human tonsillar cDNA library by differential hybridization with labeled cDNA derived from either size-fractionated B-cell mRNA or size-fractionated T-cell mRNA. Of the 261 cDNA clones isolated, 3 cross-hybridizing cDNA clones were chosen as potential candidates for encoding B1 based on their selective hybridization to RNA from B1-positive cell lines. The longest clone, pB1-21, contained a 2.8-kilobase insert with an 891-base-pair open reading frame that encodes a protein of 33 kDa. mRNA synthesized from the pB1-21 cDNA clone in vitro was translated into a protein of the same apparent molecular weight as B1. Limited proteinase digestion of the pB1-21 translation product and B1 generated peptides of the same sizes, indicating that the pB1-21 cDNA encodes the B1 molecule. Gel blot analysis indicated that pB1-21 hybridized with two mRNA species of 2.8 and 3.4 kilobases only in B1-positive cell lines. The amino acid sequence deduced from the pB1-21 nucleotide sequence apparently lacks a signal sequence and contains three extensive hydrophobic regions. The deduced B1 amino acid sequence shows no significant homology with other known proteins. Images PMID:2448768

  20. Evolution of Pleopsidium (lichenized Ascomycota) S943 group I introns and the phylogeography of an intron-encoded putative homing endonuclease.

    PubMed

    Reeb, Valérie; Haugen, Peik; Bhattacharya, Debashish; Lutzoni, François

    2007-03-01

    The sporadic distribution of nuclear group I introns among different fungal lineages can be explained by vertical inheritance of the introns followed by successive losses, or horizontal transfers from one lineage to another through intron homing or reverse splicing. Homing is mediated by an intron-encoded homing endonuclease (HE) and recent studies suggest that the introns and their associated HE gene (HEG) follow a recurrent cyclical model of invasion, degeneration, loss, and reinvasion. The purpose of this study was to compare this model to the evolution of HEGs found in the group I intron at position S943 of the nuclear ribosomal DNA of the lichen-forming fungus Pleopsidium. Forty-eight S943 introns were found in the 64 Pleopsidium samples from a worldwide screen, 22 of which contained a full-length HEG that encodes a putative 256-amino acid HE, and 2 contained HE pseudogenes. The HEGs are divided into two closely related types (as are the introns that encode them) that differ by 22.6% in their nucleotide sequences. The evolution of the Pleopsidium intron-HEG element shows strong evidence for a cyclical model of evolution. The intron was likely acquired twice in the genus and then transmitted via two or three interspecific horizontal transfers. Close geographical proximity plays an important role in intron-HEG horizontal transfer because most of these mobile elements were found in Europe. Once acquired in a lineage, the intron-HEG element was also vertically transmitted, and occasionally degenerated or was lost.

  1. Gene ercA, encoding a putative iron-containing alcohol dehydrogenase, is involved in regulation of ethanol utilization in Pseudomonas aeruginosa.

    PubMed

    Hempel, Niels; Görisch, Helmut; Mern, Demissew S

    2013-09-01

    Several two-component regulatory systems are known to be involved in the signal transduction pathway of the ethanol oxidation system in Pseudomonas aeruginosa ATCC 17933. These sensor kinases and response regulators are organized in a hierarchical manner. In addition, a cytoplasmic putative iron-containing alcohol dehydrogenase (Fe-ADH) encoded by ercA (PA1991) has been identified to play an essential role in this regulatory network. The gene ercA (PA1991) is located next to ercS, which encodes a sensor kinase. Inactivation of ercA (PA1991) by insertion of a kanamycin resistance cassette created mutant NH1. NH1 showed poor growth on various alcohols. On ethanol, NH1 grew only with an extremely extended lag phase. During the induction period on ethanol, transcription of structural genes exa and pqqABCDEH, encoding components of initial ethanol oxidation in P. aeruginosa, was drastically reduced in NH1, which indicates the regulatory function of ercA (PA1991). However, transcription in the extremely delayed logarithmic growth phase was comparable to that in the wild type. To date, the involvement of an Fe-ADH in signal transduction processes has not been reported.

  2. A putative cyclic peptide efflux pump encoded by the TOXA gene of the plant-pathogenic fungus Cochliobolus carbonum.

    PubMed

    Pitkin, J W; Panaccione, D G; Walton, J D

    1996-06-01

    Race 1 isolates of Cochliobolus carbonum are pathogenic on certain maize lines due to production of a host-selective cyclic tetrapeptide, HC-toxin. Flanking HTS1, which encodes the central enzyme in HC-toxin biosynthesis, a gene was identified and named TOXA. Like HTS1, TOXA occurred only in isolates of the fungus that make HC-toxin and was present as two linked copies in most toxin-producing isolates. HTS1 and TOXA were transcribed in the opposite orientation and their transcriptional start sites were 386 bp apart. The predicted product of TOXA was a 58 kDa hydrophobic protein with 10-13 membrane-spanning regions. The sequence was highly similar to several members of the major facilitator superfamily that confer resistance to tetracycline, methylenomycin, and other antibiotics. Although it was possible to mutate one copy or the other of TOXA by targeted gene disruption, numerous attempts to disrupt both copies in a single strain were unsuccessful, suggesting that TOXA is an essential gene in strains that synthesize HC-toxin. On the basis of its presence only in HC-toxin-producing strains, its proximity to HTS1 and its predicted amino acid sequence, we propose that TOXA encodes an HC-toxin efflux pump which contributes to self-protection against HC-toxin and/or the secretion of HC-toxin into the extracellular milieu.

  3. 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

  4. 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.

  5. Cloning of a human cDNA encoding a putative nucleotide-binding protein related to Escherichia coli MinD.

    PubMed

    Shahrestanifar, M; Saha, D P; Scala, L A; Basu, A; Howells, R D

    1994-09-30

    A novel human cDNA encoding a putative nucleotide-binding protein (NBP) was obtained by screening a human SHSY5Y neuroblastoma library. The deduced protein contains 320 amino acids (aa) with a M(r) of 34,540. NBP displays sequence similarity with the product of the minD gene from Escherichia coli. MinD is involved in the proper placement of the division septum, and has ATPase activity. NBP and MinD contain consensus nucleotide (nt)-binding domains. The NBP mRNA is approx. 1500 nt in length and is expressed in several human cell lines and in all rat tissues examined, with the highest levels in lung and testis.

  6. Structure, mapping and expression of a growth factor inducible gene encoding a putative nuclear hormonal binding receptor.

    PubMed Central

    Ryseck, R P; Macdonald-Bravo, H; Mattéi, M G; Ruppert, S; Bravo, R

    1989-01-01

    We have characterized a growth factor inducible gene, N10, encoding a nuclear protein of 601 amino acids with a significant similarity to members of the steroid and thyroid hormone receptor families. The gene is rapidly but transiently induced by several mitogens. Immunoprecipitation studies show that the N10 protein is transiently expressed after stimulation of quiescent cells, presenting a half-life of approximately 30 min. The N10 transcription unit is 8 kb in length, split into seven exons. The exon-intron distribution is in general similar to that of other members of the nuclear receptor superfamily, but presents some differences which suggest that N10 belongs to a new family of these molecules. The 5' flanking region contains one DSE which could explain its immediate response to external stimulus. The N10 gene is located in the [F1-F3] region of mouse chromosome 15. Images PMID:2555161

  7. Transient transcription of a putative RNase containing BEN domain encoded in Cotesia plutellae bracovirus induces an immunosuppression of the diamondback moth, Plutella xylostella.

    PubMed

    Park, Bokri; Kim, Yonggyun

    2010-10-01

    A polydnavirus, Cotesia plutellae bracovirus (CpBV), possesses segmented genome located on chromosome(s) of an endoparasitoid wasp, C. plutellae. An episomal viral segment (CpBV-S3) consists of 11,017 bp and encodes two putative open reading frames (ORFs). ORF301 shows amino acid sequence homologies (28-50%) with RNase T2s of various organisms. It also contains BEN domain in C-terminal region. ORF302 is a hypothetical gene, which is also found in other bracoviruses. Both genes were expressed in larvae of Plutella xylostella parasitized by C. plutellae. Their expressions were detected in all tested tissues including hemocyte, fat body, gut, and epidermis. To analyze effects of these genes on the parasitism, the segment of CpBV-S3 was injected to nonparasitized larvae of P. xylostella, in which the two genes were expressed at least for 4 days post-injection. The larvae injected with CpBV-S3 exhibited significant immunosuppression, such as reduction in total hemocyte population and impairment in nodule formation behavior of hemocytes in response to bacterial challenge. Each gene expression in the treated larvae was inhibited by co-injecting respective double strand RNA (dsRNA) specific to each ORF. Injection of dsRNA of ORF301 could rescue the immunosuppression of the viral segment-treated larvae, while dsRNA specific to ORF302 did not. These results suggest that a putative RNase fused with a BEN domain encoded in CpBV-S3 plays a parasitic role in inducing host immunosuppression in the parasitism.

  8. 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.

  9. GintABC1 encodes a putative ABC transporter of the MRP subfamily induced by Cu, Cd, and oxidative stress in Glomus intraradices.

    PubMed

    González-Guerrero, Manuel; Benabdellah, Karim; Valderas, Ascensión; Azcón-Aguilar, Concepción; Ferrol, Nuria

    2010-02-01

    A full-length cDNA sequence putatively encoding an ATP-binding cassette (ABC) transporter (GintABC1) was isolated from the extraradical mycelia of the arbuscular mycorrhizal fungus Glomus intraradices. Bioinformatic analysis of the sequence indicated that GintABC1 encodes a 1513 amino acid polypeptide, containing two six-transmembrane clusters (TMD) intercalated with sequences characteristics of the nucleotide binding domains (NBD) and an extra N-terminus extension (TMD0). GintABC1 presents a predicted TMD0-(TMD-NBD)(2) topology, typical of the multidrug resistance-associated protein subfamily of ABC transporters. Gene expression analyses revealed no difference in the expression levels of GintABC1 in the extra- vs the intraradical mycelia. GintABC1 was up-regulated by Cd and Cu, but not by Zn, suggesting that this transporter might be involved in Cu and Cd detoxification. Paraquat, an oxidative agent, also induced the transcription of GintABC1. These data suggest that redox changes may be involved in the transcriptional regulation of GintABC1 by Cd and Cu.

  10. The helC gene encodes a putative DEAD-box RNA helicase required for development in Dictyostelium discoideum.

    PubMed

    Machesky, L M; Insall, R H; Kay, R R

    1998-05-01

    DEAD-box RNA helicases, defined by the sequence Asp-Glu-Ala-Asp (DEAD, in single-letter amino-acid code), regulate RNA unwinding and secondary structure in an ATP-dependent manner in vitro [1] and control mRNA stability and protein translation. Both yeast and mammals have large families of DEAD-box proteins, many of unknown function. We have disrupted a Dictyostelium discoideum gene, helC, which encodes helicase C, a member of the DEAD-box family of RNA helicases that shows strong homology to the product of the essential Saccharomyces cerevisiae gene dbp5 [2] and to related helicases in mouse and Schizosaccharomyces pombe. The HelC protein also shows weaker homology to the translation initiation factor elF-4a. Other DEAD-box-containing proteins, which are less closely related to HelC, have been implicated in developmental roles in Drosophila [3] and Xenopus laevis; one example is the Xenopus Vasa-like protein (XVLP) [4-6]. In Drosophila and Xenopus, Vasa and XVLP, respectively, are required for the establishment of tissue polarity during development. In yeast, DEAD-box helicases such as Prp8 [7] are components of the spliceosome and connect pre-mRNA splicing with the cell cycle. Disruption of the helC gene in D. discoideum led to developmental asynchrony, failure to differentiate and aberrant morphogenesis. We postulate that one reason for the existence of large families of homologous DEAD-box proteins in yeast, mammals and Dictyostelium could be that some DEAD-box proteins have developmentally specific roles regulating protein translation or mRNA stability. PMID:9601648

  11. 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

  12. 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

  13. A semidwarf phenotype of barley uzu results from a nucleotide substitution in the gene encoding a putative brassinosteroid receptor.

    PubMed

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

    2003-11-01

    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.

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

    PubMed Central

    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

  15. Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages.

    PubMed

    Hensel, M; Shea, J E; Waterman, S R; Mundy, R; Nikolaus, T; Banks, G; Vazquez-Torres, A; Gleeson, C; Fang, F C; Holden, D W

    1998-10-01

    The type III secretion system of Salmonella pathogenicity island 2 (SPI-2) is required for systemic infection of this pathogen in mice. Cloning and sequencing of a central region of SPI-2 revealed the presence of genes encoding putative chaperones and effector proteins of the secretion system. The predicted products of the sseB, sseC and sseD genes display weak but significant similarity to amino acid sequences of EspA, EspD and EspB, which are secreted by the type III secretion system encoded by the locus of enterocyte effacement of enteropathogenic Escherichia coli. The transcriptional activity of an sseA::luc fusion gene was shown to be dependent on ssrA, which is required for the expression of genes encoding components of the secretion system apparatus. Strains carrying nonpolar mutations in sseA, sseB or sseC were severely attenuated in virulence, strains carrying mutations in sseF or sseG were weakly attenuated, and a strain with a mutation in sseE had no detectable virulence defect. These phenotypes were reflected in the ability of mutant strains to grow within a variety of macrophage cell types: strains carrying mutations in sseA, sseB or sseC failed to accumulate, whereas the growth rates of strains carrying mutations in sseE, sseF or sseG were only modestly reduced. These data suggest that, in vivo, one of the functions of the SPI-2 secretion system is to enable intracellular bacterial proliferation.

  16. 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

  17. Overexpression of a cotton gene that encodes a putative transcription factor of AP2/EREBP family in Arabidopsis affects growth and development of transgenic plants.

    PubMed

    Zhou, Ying; Xia, Hui; Li, Xiao-Jie; Hu, Rong; Chen, Yun; Li, Xue-Bao

    2013-01-01

    In the study, a gene encoding a putative ethylene response factor of AP2/EREBP family was isolated from cotton (Gossypium hirsutum) and designated as GhERF12. Sequence alignment showed that GhERF12 protein contains a central AP2/ERF domain (58 amino acids) with two functional conserved amino acid residues (ala14 and asp19). Transactivation assay indicated that GhERF12 displayed strong transcription activation activity in yeast cells, suggesting that this protein may be a transcriptional activator in cotton. Quantitative RT-PCR analysis showed that GhERF12 expression in cotton was induced by ACC and IAA. Overexpression of GhERF12 in Arabidopsis affected seedling growth and development. The GhERF12 transgenic plants grew slowly, and displayed a dwarf phenotype. The mean bolting time of the transgenic plants was delayed for about 10 days, compared with that of wild type. Further study revealed that some ethylene-related and auxin-related genes were dramatically up-regulated in the transgenic plants, compared with those of wild type. Collectively, we speculated that GhERF12, as a transcription factor, may be involved in regulation of plant growth and development by activating the constitutive ethylene response likely related to auxin biosynthesis and/or signaling.

  18. The promoter of the barley aleurone-specific gene encoding a putative 7 kDa lipid transfer protein confers aleurone cell-specific expression in transgenic rice.

    PubMed

    Kalla, R; Shimamoto, K; Potter, R; Nielsen, P S; Linnestad, C; Olsen, O A

    1994-12-01

    This paper describes the aleurone-specific gene Ltp2 from barley, which encodes a putative 7 kDa non-specific lipid transfer protein. As shown by Northern and in situ hybridization analyses, the Ltp2 transcript is present in barley aleurone cells shortly after the initiation of aleurone cell differentiation. The expression of Ltp2 increases until grain mid-maturity, but the mRNA is absent from mature grains. The Ltp2 transcript is undetectable in the embryo and vegetative tissues, confirming the aleurone specificity of the Ltp2 gene. The ability of the isolated 801 bp Ltp2 promoter to direct aleurone-specific expression in immature barley grains is demonstrated by particle bombardment experiments. In these experiments, the activity of the Ltp2 promoter is 5% of the activity of the strong constitutive Actin1 promoter from rice, as quantified by GUS activity measurements. In stably transformed rice plants containing the Ltp2 promoter-Gus construct, the specificity of the Ltp2 promoter is confirmed in vivo by the presence of GUS activity exclusively in the aleurone layer. This study demonstrates the conserved nature of the regulatory signals involved in aleurone-specific gene transcription in cereal grains.

  19. Identification and characterization of a cDNA encoding a crustin-like, putative antibacterial protein from the American lobster Homarus americanus.

    PubMed

    Christie, Andrew E; Rus, Szymon; Goiney, Christopher C; Smith, Christine M; Towle, David W; Dickinson, Patsy S

    2007-07-01

    Pathogenic challenges in decapod crustaceans are combated by innate immune responses, including the production and secretion of soluble antibacterial proteins into the hemolymph. Among the antibacterials that have been identified in decapod species are the crustins, a group of four-disulfide core/whey-acidic-protein (WAP) domain-containing proteins, which target marine/salt tolerant Gram-positive bacteria. To begin to assess the possible role of crustins in combating bacterial invasion in the American lobster Homarus americanus, we identified and sequenced a 744 base pair cDNA that encodes a novel 96 amino acid crustin-like protein. Comparison of H. americanus crustin (Hoa-crustin) with crustins from other decapod species showed that it is most similar to an isoform predicted from the European lobster Homarus gammarus ( approximately 86% identity). With our identification of the Hoa-crustin cDNA, we are positioned to begin molecular and physiological investigations of the regulation and function of this putative antibacterial protein in H. americanus. PMID:17418897

  20. 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

  1. 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

  2. Putative multiadhesive protein from the marine sponge Geodia cydonium: cloning of the cDNA encoding a fibronectin-, an SRCR-, and a complement control protein module.

    PubMed

    Pahler, S; Blumbach, B; Müller, I; Müller, W E

    1998-10-15

    Sponges (Porifera) representing the simplest metazoan phylum so far have been thought to possess no basal lamina tissue structures. One major extracellular matrix protein that is also a constitutive glycoprotein of the basal lamina is fibronectin. It was the aim of the present study to identify the native protein from the marine sponge Geodia cydonium and to isolate the corresponding cDNA. In crude extracts from this sponge protein(s) of M(r) of approximately 230 and approximately 210 kDa could be visualized by Western-blotting using an anti-fibronectin [human] antibody. By PCR cloning from a cDNA library of G. cydonium we isolated a cDNA comprising one element of fibronectin, the type-III (FN3) module. The cDNA (2.3 kb long), encoding a 701 amino acid [aa] long putative "multiadhesive protein" termed MAP_GEOCY, was found to contain (i) a fibronectin-, (ii) a scavenger receptor cysteine-rich [SRCR]-, and (iii) a short consensus repeat [SCR] module. The 89 aa long fibronectin module comprises the characteristic topology and conserved aa found in fibronectin type-III (FN3) elements. The SRCR module (101 aa) features the characteristics of group B SRCR molecules. The predominant proteins belonging to this group are the mammalian WC1-, M130-, CD6- and CD5 antigens that probably are involved in immunological reactions. The SCR module (54 aa) shows the characteristics of type III SCR modules found in complement receptors. Phylogenetic analyses performed with all three building blocks of the "multiadhesive protein" showed that the respective sponge modules form independent, possibly basal, lineages in trees that include the corresponding modules from higher metazoan animals. In summary, these data demonstrate for the first time that the phylogenetically oldest Metazoa, the sponges, contain protein modules seen in higher animals in proteins of the extracellular matrix and in molecules involved in cell-mediated immune reactions in vertebrates. PMID:9755483

  3. [Changes in Cell Surface Properties and Biofilm Formation Efficiency in Azospirillum brasilense Sp245 Mutants in the Putative Genes of Lipid Metabolism mmsB1 and fabG1].

    PubMed

    Shumilova, E; Shelud'ko, A V; Filip'echeva, Yu A; Evstigneeva, S S; Ponomareva, E G; Petrova, L P; Katsy, E I

    2016-01-01

    The previously obtained insertion mutants ofAzospirillum brasilense Sp245 in the genes mmsBl and fabG1 (strains SK039 and Sp245.1610, respectively) were characterized by impaired flagellation and motility. The putative products of expression of these genes are 3-hydroxyisobutyrate dehydrogenase and 3-oxoacyl-[acyl-carrier protein] reductase, respectively. In the present work, A. brasilense- Sp245 strains SK039 and Sp245.1610 were found to have differences in the content of 3-hydroxyhexadecanoic, hexadecanoic, 3-hydroxytetradecanoic, hexadecenoic, octadecenoic, and nonadecanoic acids in their lipopolysaccharide prepa- rations, as well as in cell hydrophobicity and hemagglutination activity and dynamics of cell aggregation, in biomass amount, and in the relative content of lipopolysaccharide antigens in mature biofilms formed on hydrophilic or hydrophobic surfaces. PMID:27476204

  4. [Changes in Cell Surface Properties and Biofilm Formation Efficiency in Azospirillum brasilense Sp245 Mutants in the Putative Genes of Lipid Metabolism mmsB1 and fabG1].

    PubMed

    Shumilova, E; Shelud'ko, A V; Filip'echeva, Yu A; Evstigneeva, S S; Ponomareva, E G; Petrova, L P; Katsy, E I

    2016-01-01

    The previously obtained insertion mutants ofAzospirillum brasilense Sp245 in the genes mmsBl and fabG1 (strains SK039 and Sp245.1610, respectively) were characterized by impaired flagellation and motility. The putative products of expression of these genes are 3-hydroxyisobutyrate dehydrogenase and 3-oxoacyl-[acyl-carrier protein] reductase, respectively. In the present work, A. brasilense- Sp245 strains SK039 and Sp245.1610 were found to have differences in the content of 3-hydroxyhexadecanoic, hexadecanoic, 3-hydroxytetradecanoic, hexadecenoic, octadecenoic, and nonadecanoic acids in their lipopolysaccharide prepa- rations, as well as in cell hydrophobicity and hemagglutination activity and dynamics of cell aggregation, in biomass amount, and in the relative content of lipopolysaccharide antigens in mature biofilms formed on hydrophilic or hydrophobic surfaces.

  5. 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.

  6. Mycobacterium tuberculosis DosR Regulon Gene Rv0079 Encodes a Putative, ‘Dormancy Associated Translation Inhibitor (DATIN)’

    PubMed Central

    Kumar, Ashutosh; Majid, Mohammad; Kunisch, Ralph; Rani, Pittu Sandhya; Qureshi, Insaf A.; Lewin, Astrid; Hasnain, Seyed E.; Ahmed, Niyaz

    2012-01-01

    Mycobacterium tuberculosis is a major human pathogen that has evolved survival mechanisms to persist in an immune-competent host under a dormant condition. The regulation of M. tuberculosis metabolism during latent infection is not clearly known. The dormancy survival regulon (DosR regulon) is chiefly responsible for encoding dormancy related functions of M. tuberculosis. We describe functional characterization of an important gene of DosR regulon, Rv0079, which appears to be involved in the regulation of translation through the interaction of its product with bacterial ribosomal subunits. The protein encoded by Rv0079, possibly, has an inhibitory role with respect to protein synthesis, as revealed by our experiments. We performed computational modelling and docking simulation studies involving the protein encoded by Rv0079 followed by in vitro translation and growth curve analysis experiments, involving recombinant E. coli and Bacille Calmette Guérin (BCG) strains that overexpressed Rv0079. Our observations concerning the interaction of the protein with the ribosomes are supportive of its role in regulation/inhibition of translation. We propose that the protein encoded by locus Rv0079 is a ‘dormancy associated translation inhibitor’ or DATIN. PMID:22719925

  7. The yeast MOT2 gene encodes a putative zinc finger protein that serves as a global negative regulator affecting expression of several categories of genes, including mating-pheromone-responsive genes.

    PubMed

    Irie, K; Yamaguchi, K; Kawase, K; Matsumoto, K

    1994-05-01

    The STE4 gene encodes the beta subunit of a heterotrimeric G protein that is an essential component of the pheromone signal transduction pathway. To identify downstream component(s) of Ste4, we sought pseudo-revertants that restored mating competence to ste4 mutants. The suppressor mot2 was isolated as a recessive mutation that restored conjugational competence to a temperature-sensitive ste4 mutant and simultaneously conferred a temperature-sensitive growth phenotype. The MOT2 gene encodes a putative zinc finger protein, the deletion of which resulted in temperature-sensitive growth, increased expression of FUS1 in the absence of pheromones, and suppression of a deletion of the alpha-factor receptor. On the other hand, sterility resulting from deletion of STE4 was not suppressed by the mot2 deletion. These phenotypes are similar to those associated with temperature-sensitive mutations in CDC36 and CDC39, which are proposed to encode general negative regulators of transcription rather than factors involved in the pheromone response pathway. Deletion of MOT2 also caused increased transcription of unrelated genes such as GAL7 and PHO84. Overexpression of MOT2 suppresses the growth defect of temperature-sensitive mutations in CDC36 and CDC39. These observations suggest that Mot2 functions as a general negative regulator of transcription in the same processes as Cdc36 and Cdc39.

  8. Molecular characterization of the Jatropha curcas JcR1MYB1 gene encoding a putative R1-MYB transcription factor

    PubMed Central

    Li, Hui-Liang; Guo, Dong; Peng, Shi-Qing

    2014-01-01

    The cDNA encoding the R1-MYB transcription factor, designated as JcR1MYB1, was isolated from Jatropha curcas using rapid amplification of cDNA ends. JcR1MYB1 contains a 951 bp open reading frame that encodes 316 amino acids. The deduced JcR1MYB1 protein was predicted to possess the conserved, 56-amino acid-long DNA-binding domain, which consists of a single helix-turn-helix module and usually occurs in R1-MYBs. JcR1MYB1 is a member of the R1-MYB transcription factor subfamily. A subcellular localization study confirmed the nuclear localization of JcR1MYB1. Expression analysis showed that JcR1MYB1 transcripts accumulated in various examined tissues, with high expression levels in the root and low levels in the stem. JcR1MYB1 transcription was up-regulated by polyethylene glycol, NaCl, and cold treatments, as well as by abscisic acid, jasmonic acid, and ethylene treatment. Analysis of transgenic tobacco plants over-expressing JcR1MYB1 indicates an inportant function for this gene in salt stress. PMID:25249778

  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. Decreased cellulase and xylanase production in the fungus Talaromyces cellulolyticus by disruption of tacA and tctA genes, encoding putative zinc finger transcriptional factors.

    PubMed

    Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko

    2015-03-01

    Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) is one of the important strains for industrial cellulase production. An understanding of the control of cellulase gene expression in T. cellulolyticus is insufficient because only a few transcriptional factors related to cellulase gene expression have been identified. In the present study, we disrupted seven putative transcription regulator genes that showed similarity with cellulase or hemicellulase regulator genes in other filamentous fungi and investigated whether these genes are related to cellulase and xylanase production. Among the seven genes, five (tclA, tbgA, tlaA, tmcA, tclB2) had a smaller effect on cellulase and xylanase activities when culturing with cellulose. On the other hand, disruption of tacA and tctA, which are respectively homologues of ace1 (repressor of cellulase) and ctf1 (inducer of cutinase), led to a decrease in cellulase and hemicellulase production due to effects at both the enzymatic and transcriptional levels, indicating that tacA and tctA have positive roles in cellulase and xylanase production in T. cellulolyticus. These results suggest that cellulase and xylanase gene regulation in T. cellulolyticus differs from that in other filamentous fungi and imply that unknown transcriptional mechanisms function in T. cellulolyticus.

  11. Cell-Based Selection Expands the Utility of DNA-Encoded Small-Molecule Library Technology to Cell Surface Drug Targets: Identification of Novel Antagonists of the NK3 Tachykinin Receptor.

    PubMed

    Wu, Zining; Graybill, Todd L; Zeng, Xin; Platchek, Michael; Zhang, Jean; Bodmer, Vera Q; Wisnoski, David D; Deng, Jianghe; Coppo, Frank T; Yao, Gang; Tamburino, Alex; Scavello, Genaro; Franklin, G Joseph; Mataruse, Sibongile; Bedard, Katie L; Ding, Yun; Chai, Jing; Summerfield, Jennifer; Centrella, Paolo A; Messer, Jeffrey A; Pope, Andrew J; Israel, David I

    2015-12-14

    DNA-encoded small-molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, this technology has been used with soluble protein targets that are produced and used in a purified state. Here, we describe a cell-based method for identifying small-molecule ligands from DNA-encoded libraries against integral membrane protein targets. We use this method to identify novel, potent, and specific inhibitors of NK3, a member of the tachykinin family of G-protein coupled receptors (GPCRs). The method is simple and broadly applicable to other GPCRs and integral membrane proteins. We have extended the application of DNA-encoded library technology to membrane-associated targets and demonstrate the feasibility of selecting DNA-tagged, small-molecule ligands from complex combinatorial libraries against targets in a heterogeneous milieu, such as the surface of a cell.

  12. YtxR, a Conserved LysR-Like Regulator That Induces Expression of Genes Encoding a Putative ADP-Ribosyltransferase Toxin Homologue in Yersinia enterocolitica▿

    PubMed Central

    Axler-DiPerte, Grace L.; Miller, Virginia L.; Darwin, Andrew J.

    2006-01-01

    Yersinia enterocolitica causes human gastroenteritis, and many isolates have been classified as either “American” or “non-American” strains based on their geographic prevalence and virulence properties. In this study we describe identification of a transcriptional regulator that controls expression of the Y. enterocolitica ytxAB genes. The ytxAB genes have the potential to encode an ADP-ribosylating toxin with similarity to pertussis toxin. However, a ytxAB null mutation did not affect virulence in mice. Nevertheless, the ytxAB genes are conserved in many Y. enterocolitica strains. Interestingly, American and non-American strains have different ytxAB alleles encoding proteins that are only 50 to 60% identical. To obtain further insight into the ytxAB locus, we investigated whether it is regulated as part of a known or novel regulon. Transposon mutagenesis identified a LysR-like regulator, which we designated YtxR. Expression of ytxR from a nonnative promoter increased Φ(ytxA-lacZ) operon fusion expression up to 35-fold. YtxR also activated expression of its own promoter. DNase I footprinting showed that a His6-YtxR fusion protein directly interacted with the ytxA and ytxR control regions at similar distances upstream of their probable transcription initiation sites, identified by primer extension. Deletion analysis demonstrated that removal of the regions protected by His6-YtxR in vitro eliminated YtxR-dependent induction in vivo. The ytxAB locus is not present in most Yersinia species. In contrast, ytxR is conserved in multiple Yersinia species, as well as in the closely related organisms Photorhabdus luminescens and Photorhabdus asymbiotica. These observations suggest that YtxR may play a conserved role involving regulation of other genes besides ytxAB. PMID:16997967

  13. 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.

  14. 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

  15. ZmPIN1a and ZmPIN1b Encode Two Novel Putative Candidates for Polar Auxin Transport and Plant Architecture Determination of Maize1[W

    PubMed Central

    Carraro, Nicola; Forestan, Cristian; Canova, Sabrina; Traas, Jan; Varotto, Serena

    2006-01-01

    Shoot apical meristems produce organs in a highly stereotypic pattern that involves auxin. Auxin is supposed to be actively transported from cell to cell by influx (AUXIN/LIKE AUXIN proteins) and efflux (PIN-FORMED proteins) membrane carriers. Current hypotheses propose that, at the meristem surface, PIN proteins create patterns of auxin gradients that, in turn, create patterns of gene expression and morphogenesis. These hypotheses are entirely based on work in Arabidopsis (Arabidopsis thaliana). To verify whether these models also apply to other species, we studied the behavior of PIN proteins during maize (Zea mays) development. We identified two novel putative orthologs of AtPIN1 in maize and analyzed their expression pattern during development. The expression studies were complemented by immunolocalization studies using an anti-AtPIN1 antibody. Interestingly, the maize proteins visualized by this antibody are almost exclusively localized in subepidermal meristematic layers. Both tassel and ear were characterized by a compact group of cells, just below the surface, carrying PIN. In contrast to or to complement what was shown in Arabidopsis, these results point to the importance of internally localized cells in the patterning process. We chose the barren inflorescence2 (bif2) maize mutant to study the role of auxin polar fluxes in inflorescence development. In severe alleles of bif2, the tassel and the ear present altered ZmPIN1a and ZmPIN1b protein expression and localization patterns. In particular, the compact groups of cells in the tassel and ear of the mutant were missing. We conclude that BIF2 is important for PIN organization and could play a role in the establishment of polar auxin fluxes in maize inflorescence, indirectly modulating the process of axillary meristem formation and development. PMID:16844839

  16. Genome-wide identification, classification and expression analysis of genes encoding putative fasciclin-like arabinogalactan proteins in Chinese cabbage (Brassica rapa L.).

    PubMed

    Jun, Li; Xiaoming, Wu

    2012-12-01

    Fasciclin-like arabinogalactan proteins (FLAs), a subclass of arabinogalactan proteins (AGPs), have both predicted AGP-like glycosylated regions and putative fasciclin (FAS) domains, which may function in cell adhesion and communication. Previous studies have identified 21, 27, and 34 FLAs in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and wheat (Triticum aestivum), respectively. In this study, we identified 33 FLAs in the annotated genome of Chinese cabbage (Brassica rapa ssp. pekinensis line Chiifu-401-42). Sequence analysis indicated that FAS domains each contain two highly conserved regions, named H1 and H2, and that 17 FLAs from B. rapa (BrFLAs) possess both of these regions. Prediction of glycosylphosphatidylinositol (GPI) modification sites suggested that 15 BrFLAs were GPI-anchored to the plasma membrane. Additionally, 25 BrFLAs may have been duplicated during the processes that shaped the triplicated genome of the mesopolyploid B. rapa. Expression analyses indicated that BrFLA1, BrFLA11, BrFLA13, BrFLA28 and BrFLA32 were specifically expressed in inflorescence. Meanwhile, BrFLA9 (homologous to AtFLA12) is specifically expressed in stem, and BrFLA6/22 (homologous to AtFLA11) is also highly expressed in stem, suggesting BrFLA6/9/22 may have the same functions as AtFLA11/12 in A. thaliana. Taken together, the identification and bioinformatic analysis of FLAs in B. rapa will open the way for studying their biological functions in plant growth and development as well as evolutionary history of this gene family from A. thaliana to B. rapa. PMID:23053954

  17. Genome-wide identification, classification and expression analysis of genes encoding putative fasciclin-like arabinogalactan proteins in Chinese cabbage (Brassica rapa L.).

    PubMed

    Jun, Li; Xiaoming, Wu

    2012-12-01

    Fasciclin-like arabinogalactan proteins (FLAs), a subclass of arabinogalactan proteins (AGPs), have both predicted AGP-like glycosylated regions and putative fasciclin (FAS) domains, which may function in cell adhesion and communication. Previous studies have identified 21, 27, and 34 FLAs in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and wheat (Triticum aestivum), respectively. In this study, we identified 33 FLAs in the annotated genome of Chinese cabbage (Brassica rapa ssp. pekinensis line Chiifu-401-42). Sequence analysis indicated that FAS domains each contain two highly conserved regions, named H1 and H2, and that 17 FLAs from B. rapa (BrFLAs) possess both of these regions. Prediction of glycosylphosphatidylinositol (GPI) modification sites suggested that 15 BrFLAs were GPI-anchored to the plasma membrane. Additionally, 25 BrFLAs may have been duplicated during the processes that shaped the triplicated genome of the mesopolyploid B. rapa. Expression analyses indicated that BrFLA1, BrFLA11, BrFLA13, BrFLA28 and BrFLA32 were specifically expressed in inflorescence. Meanwhile, BrFLA9 (homologous to AtFLA12) is specifically expressed in stem, and BrFLA6/22 (homologous to AtFLA11) is also highly expressed in stem, suggesting BrFLA6/9/22 may have the same functions as AtFLA11/12 in A. thaliana. Taken together, the identification and bioinformatic analysis of FLAs in B. rapa will open the way for studying their biological functions in plant growth and development as well as evolutionary history of this gene family from A. thaliana to B. rapa.

  18. 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.

  19. 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 Central

    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

  20. 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.

  1. The Colletotrichum acutatum gene encoding a putative pH-responsive transcription regulator is a key virulence determinant during fungal pathogenesis on citrus.

    PubMed

    You, Bang-Jau; Choquer, Mathias; Chung, Kuang-Ren

    2007-09-01

    Postbloom fruit drop of citrus and Key lime anthracnose (KLA) are caused by different pathotypes of Colletotrichum acutatum. Both pathotypes are pathogenic to citrus flowers, resulting in blossom blight and induction of young fruit abscission. Two fungal mutants defective in pathogenicity were recovered from a KLA pathotype after Agrobacterium-mediated mutagenesis. A PacC(KLAP2) gene encoding a polypeptide that resembles many pH-responsive PacC/ Rim101 transcription regulators in fungi was identified from one of the mutants, and functionally characterized to play a crucial role in pathogenesis to both Key lime leaves and citrus flowers. Gene disruption at the Pac(KLAP2) locus created fungal mutants that were hypersensitive to alkaline pH, altered in conidium and appressorium production and germination, and concomitant with reduced virulence to both tissues. The pacC(KLAP2) null mutants had lower alkaline phosphatase and protease activities, but increased pectolytic and lipolytic activities. The mutants initiated penetration and incited lesion formation on Key lime, indistinguishable from the wild type, when a functional copy of PacC(KLAP2) was reintroduced or the leaves were wounded prior to inoculation. The null mutants were blocked at the penetration stage and, thus, failed to initiate the necrotrophic phase. The PacC(KLAP2) transcript was barely detectable when the fungus was grown on medium buffered to pH 3 or 4, yet accumulated to high levels at a pH between 5 and 7. The Pac(KLAP2) transcript was detected 2 days postinoculation on Key lime leaves, correlating with the time of lesion formation. We conclude that PacC(KLAP2) is essential for C. acutatum pathogenesis by regulating multiple physiological and developmental processes. PMID:17849717

  2. 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

    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

  3. 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

  4. Three cDNAs encoding vitellogenin homologs from Antarctic copepod, Tigriopus kingsejongensis: Cloning and transcriptional analysis in different maturation stages, temperatures, and putative reproductive hormones.

    PubMed

    Lee, Soo Rin; Lee, Ji-Hyun; Kim, Ah Ran; Kim, Sanghee; Park, Hyun; Baek, Hea Ja; Kim, Hyun-Woo

    2016-02-01

    Three full-length cDNAs encoding lipoprotein homologs were identified in Tigriopus kingsejongensis, a newly identified copepod from Antarctica. Structural and transcriptional analyses revealed homology with two vitellogenin-like proteins, Tik-Vg1 and Tik-Vg2, which were 1855 and 1795 amino acids in length, respectively, along with a third protein, Tik-MEP, which produced a 1517-residue protein with similarity to a melanin engaging protein (MEP) in insects Phylogenetic analysis showed that Vgs in Maxillopods including two Tik-Vgs belong to the arthropod vitellogenin-like clade, which includes clottable proteins (CPs) in decapod crustaceans and vitellogenins in insects. Tik-MEP clustered together with insect MEPs, which appear to have evolved before the apoB-like and arthropod Vg-like clades. Interestingly, no genes orthologous to those found in the apoB clade were identified in Maxillopoda, suggesting that functions of large lipid transfer proteins (LLTPs) in reproduction and lipid metabolism may be different from those in insect and decapod crustaceans. As suggested by phylogenetic analyses, the two Tik-Vgs belonging to the arthropod Vg-like clade appear to play major roles in oocyte maturation, while Vgs belonging to the apoB clade function primarily in the reproduction of decapod crustaceans. Transcriptional analysis of Tik-Vg expression revealed a 24-fold increase in mature and ovigerous females compared with immature female, whereas expression of Tik-MEP remained low through all reproductive stages. Acute temperature changes did not affect the transcription of Tik-Vg genes, whereas Tik-MEP appeared to be affected by temperature change. Among the three hormones thought to be involved in molting and reproduction in arthropods, only farnesoic acid (FA) induced transcription of the two Tik-Vg genes. Regardless of developmental stage and hormone treatment, Tik-Vg1 and Tik-Vg2 exhibited a strong positive correlation in expression, suggesting that expression of these

  5. Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

    PubMed Central

    Emond, Michelle R.; Jontes, James D.

    2014-01-01

    Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days. PMID:25350770

  6. Isolation of a fission yeast mutant that is sensitive to valproic acid and defective in the gene encoding Ric1, a putative component of Ypt/Rab-specific GEF for Ryh1 GTPase.

    PubMed

    Ma, Yan; Sugiura, Reiko; Zhang, Lili; Zhou, Xin; Takeuchi, Mai; He, Yi; Kuno, Takayoshi

    2010-09-01

    Valproic acid (VPA) causes various therapeutic and biological effects, but the exact mechanisms underlying these effects, however, remain elusive. To gain insights into the molecular mechanisms of VPA action, we performed in fission yeast a genetic screen for mutants that show VPA hypersensitivity and have identified several membrane-trafficking mutants including vas1-1/vps45 and vas2-1/aps1. Here, we describe the isolation and characterization of vas3-1/ric1-v3, a mutant allele of the ric1 (+) gene encoding a fission yeast homolog of the budding yeast Ric1p, a component of Ypt/Rab-specific guanyl-nucleotide exchange factor (GEF). The Rab GTPase Ryh1 knockout (Deltaryh1) cells and Deltaric1 cells exhibited similar phenotypes. The double knockout Deltaric1Deltaryh1 cells did not display synthetic growth defects. These results are consistent with the notion that Ric1 may be a component of the GEF complex for Ryh1. Overexpression of wild-type Ryh1 and the constitutively active Ryh1Q70L only partially suppressed the phenotypes of ric1-v3 and Deltaric1 cells, and they failed to localize to the Golgi/endosomes in ric1-v3 and Deltaric1 cells. Furthermore, we isolated vps15 (+) gene, encoding a serine/threonine protein kinase, as a dosage-dependent suppressor of the temperature-sensitive phenotype of ric1-v3 mutant, but not that of Deltaric1 cells. Our results showed that the ric1-v3 mutant allele has some residual functional activity and suggest that Vps15 plays a role in the regulation of Ric1 function. In conclusion, Ric1 is a putative component of GEF for Ryh1 and might be regulated by Vps15. Further studies are needed to reveal the mechanism underlying the regulation.

  7. 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

  8. 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.

  9. Structure of the cell surface.

    PubMed

    Singer, S J

    1982-01-01

    The cell surface is the locus for many important biochemical functions of cells and for the interactions of cells with one another and with their environment. The structure of the cell surface may be thought of as three-layered, with a central plasma membrane to which certain macromolecular components are attached on the outer face (the exoskeleton) and other components on the inner face (the membrane cytoskeleton). In the last decade, the basic molecular structure of the plasma membrane has been elucidated and can be represented by the fluid mosaic model as a first approximation. The binding of specific integral proteins of the membrane to individual peripheral proteins outside or inside the cell is most likely the basis for the three-layered structure of the cell surface. Studies of the last several years on the molecular structures of these three-layered cell surfaces of cultured normal fibroblasts and of fibroblasts transformed by oncogenic viruses are beginning to shed light on the molecular mechanisms responsible for changes in cell shape, adhesiveness, and in contact inhibition of motility associated with neoplastic transformation.

  10. Identification of Candida albicans ALS2 and ALS4 and Localization of Als Proteins to the Fungal Cell Surface

    PubMed Central

    Hoyer, L. L.; Payne, T. L.; Hecht, J. E.

    1998-01-01

    Additional genes in the growing ALS family of Candida albicans were isolated by PCR screening of a genomic fosmid library with primers designed from the consensus tandem-repeat sequence of ALS1. This procedure yielded fosmids encoding ALS2 and ALS4. ALS2 and ALS4 conformed to the three-domain structure of ALS genes, which consists of a central domain of tandemly repeated copies of a 108-bp motif, an upstream domain of highly conserved sequences, and a domain of divergent sequences 3′ of the tandem repeats. Alignment of five predicted Als protein sequences indicated conservation of N- and C-terminal hydrophobic regions which have the hallmarks of secretory signal sequences and glycosylphosphatidylinositol addition sites, respectively. Heterologous expression of an N-terminal fragment of Als1p in Saccharomyces cerevisiae demonstrated function of the putative signal sequence with cleavage following Ala17. This signal sequence cleavage site was conserved in the four other Als proteins analyzed, suggesting identical processing of each protein. Primary-structure features of the five Als proteins suggested a cell-surface localization, which was confirmed by indirect immunofluorescence with an anti-Als antiserum. Staining was observed on mother yeasts and germ tubes, although the intensity of staining on the mother yeast decreased with elongation of the germ tube. Similar to other ALS genes, ALS2 and ALS4 were differentially regulated. ALS4 expression was correlated with the growth phase of the culture; ALS2 expression was not observed under many different in vitro growth conditions. The data presented here demonstrate that ALS genes encode cell-surface proteins and support the conclusion that the size and number of Als proteins on the C. albicans cell surface vary with strain and growth conditions. PMID:9765564

  11. 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.

  12. Autosomal recessive hypercholesterolemia caused by mutations in a putative LDL receptor adaptor protein.

    PubMed

    Garcia, C K; Wilund, K; Arca, M; Zuliani, G; Fellin, R; Maioli, M; Calandra, S; Bertolini, S; Cossu, F; Grishin, N; Barnes, R; Cohen, J C; Hobbs, H H

    2001-05-18

    Atherogenic low density lipoproteins are cleared from the circulation by hepatic low density lipoprotein receptors (LDLR). Two inherited forms of hypercholesterolemia result from loss of LDLR activity: autosomal dominant familial hypercholesterolemia (FH), caused by mutations in the LDLR gene, and autosomal recessive hypercholesterolemia (ARH), of unknown etiology. Here we map the ARH locus to an approximately 1-centimorgan interval on chromosome 1p35 and identify six mutations in a gene encoding a putative adaptor protein (ARH). ARH contains a phosphotyrosine binding (PTB) domain, which in other proteins binds NPXY motifs in the cytoplasmic tails of cell-surface receptors, including the LDLR. ARH appears to have a tissue-specific role in LDLR function, as it is required in liver but not in fibroblasts. PMID:11326085

  13. Molecular cloning and tissue expression of FAT, the human homologue of the Drosophila fat gene that is located on chromosome 4q34-q35 and encodes a putative adhesion molecule

    SciTech Connect

    Dunne, P. Owen, M.J.; Hanby, A.M.; Poulsom, R.

    1995-11-20

    FAT, a new member of the human cadherin super-family, has been isolated from the T-leukemia cell line J6. The predicted protein closely resembles the Drosophila tumor suppressor fat, which is essential for controlling cell proliferation during Drosophila development. The gene has the potential to encode a large transmembrane protein of nearly 4600 residues with 34 tandem cadherin repeats, five EGF-like repeats, and a laminin A-G domain. The cytoplasmic sequence contains two domains with distant homology to the cadherin catenin-binding region. Northern blotting analysis of J6 mRNA demonstrated full-length, approximately 15-kb, FAT message in addition to several 5{prime}-truncated transcripts. In addition to its presence in J6 cells, in situ hybridization revealed FAT mRNA expression in epithelia and in some mesenchymal compartments. Furthermore, higher levels of expression were observed in fetal, as opposed to adult, tissue, suggesting that its expression may be developmentally regulated in these tissues. FAT shows homologies with a number of proteins important in developmental decisions and cell:cell communication and is the first fat-like protein reported in vertebrates. The gene encoding FAT was located by in situ hybridization on chromosome 4q34-q35. We propose that this family of molecules is likely to be important in mammalian developmental processes and cell communication. 80 refs., 10 figs., 1 tab.

  14. 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

  15. 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.

  16. 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.

  17. Introducing point mutations into the ATGs of the putative open reading frames of the HSV-1 gene encoding the latency associated transcript (LAT) reduces its anti-apoptosis activity.

    PubMed

    Carpenter, Dale; Henderson, Gail; Hsiang, Chinhui; Osorio, Nelson; BenMohamed, Lbachir; Jones, Clinton; Wechsler, Steven L

    2008-02-01

    The herpes simplex virus type 1 (HSV-1) latency associated transcript (LAT) gene has anti-apoptosis activity that directly or indirectly enhances the virus's reactivation phenotype in small animal models. The first 1.5 kb of the primary 8.3 kb LAT is sufficient and some or all of it is necessary for LAT's anti-apoptosis in transient transfection assays and for LAT's ability to enhance the reactivation phenotype. Based on LAT's genomic sequence, the first 1.5 kb contains eight potential open reading frames (ORFs) defined as an ATG followed by an in frame termination codon. In this study, point mutations were introduced into the ATGs of ORFs present in the 1.5 kb fragment of LAT. Mutagenesis of all eight ATGs in LAT ORFs consistently reduced the anti-apoptotic activity of LAT in transiently transfected mouse neuroblastoma cells regardless of whether apoptosis was induced by caspase 8 or caspase 9. Mutation of the six ATGs located in the stable intron sequences within the 1.5 kb LAT had a dramatic effect on caspase 9, but not caspase 8, induced apoptosis. For both caspase 8 and caspase 9 induced apoptosis, mutating the two ATGs in the exon of the LAT 1.5 kb fragment reduced, but did not eliminate the anti-apoptotic activity of LAT. These studies suggest that altering the fine structure of regulatory RNA or expression of a putative LAT ORF regulates the anti-apoptosis activity of LAT. These studies also indicate that more than one function is present in the 1.5 kb LAT fragment.

  18. Profiling of the cell surface proteome.

    PubMed

    Jang, Jun Ho; Hanash, Samir

    2003-10-01

    The in depth-mining of the proteome necessitates the comprehensive analysis of proteins in individual subcellular compartments to uncover interesting patterns of protein expression that include assessment of protein location, trafficking and of post-translational modifications that are location specific. One of the compartments of substantial interest from a diagnostic and therapeutic point of view is the plasma membrane which contains intrinsic membrane proteins and other proteins expressed on the cell surface. Technologies are currently available for the comprehensive profiling of the cell surface proteome that rely on protein tagging of intact cells. Studies are emerging that point to unexpected patterns of expression of specific proteins on the cell surface, with a common occurrence of proteins previously considered to occur predominantly in other compartments, notably the endoplasmic reticulum. The profiling of the cell surface and plasma membrane proteomes will likely provide novel insights and uncover disease related alterations. PMID:14625857

  19. Involvement of Golgi-associated retrograde protein complex in the recycling of the putative Dnf aminophospholipid flippases in yeast.

    PubMed

    Takagi, Keiko; Iwamoto, Kunihiko; Kobayashi, Shingo; Horiuchi, Hiroyuki; Fukuda, Ryouichi; Ohta, Akinori

    2012-01-01

    It is widely accepted that phosphatidylethanolamine (PE) is enriched in the cytosolic leaflet of the eukaryotic plasma membranes. To identify genes involved in the establishment and regulation of the asymmetric distribution of PE on the plasma membrane, we screened the deletion strain collection of the yeast Saccharomyces cerevisiae for hypersensitive mutants to the lantibiotic peptide Ro09-0198 (Ro) that specifically binds to PE on the cell surface and inhibits cellular growth. Deletion mutants of VPS51, VPS52, VPS53, and VPS54 encoding the components of Golgi-associated retrograde protein (GARP) complex, YPT6 encoding a Rab family small GTPase that functions with GARP complex, RIC1 and RGP1 encoding its guanine nucleotide exchange factor (GEF), and TLG2 encoding t-SNARE exhibited hypersensitivity to Ro. The mutants deleted for VPS51, VPS52, VPS53, and VPS54 were impaired in the uptake of fluorescently labeled PE. In addition, aberrant intracellular localization of the EGFP-tagged Dnf2p, the putative inward-directed phospholipid translocase (flippase) of the plasma membrane, was observed in the mutant defective in the GARP complex, Ypt6p, its GEF proteins, or Tlg2p. Our results suggest that the GARP complex is involved in the recycling of Dnf flippases.

  20. Cell-surface remodelling during mammalian erythropoiesis.

    PubMed

    Wraith, D C; Chesterton, C J

    1982-10-15

    Current evidence suggests that the major cell-surface modification occurring during mammalian erythropoiesis could be generated by two separate mechanisms: either selective loss of membrane proteins during enucleation or endocytosis at the subsequent reticulocyte and erythrocyte stages. The former idea was tested by collecting developing rabbit erythroid cells before and after the enucleation step and comparing their cell-surface protein composition via radiolabelling and electrophoresis. Few changes were observed. Our data thus lend support to the endocytosis mechanism.

  1. Enzyme Evolution by Yeast Cell Surface Engineering.

    PubMed

    Miura, Natsuko; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2015-01-01

    Artificial evolution of proteins with the aim of acquiring novel or improved functionality is important for practical applications of the proteins. We have developed yeast cell surface engineering methods (or arming technology) for evolving enzymes. Here, we have described yeast cell surface engineering coupled with in vivo homologous recombination and library screening as a method for the artificial evolution of enzymes such as firefly luciferases. Using this method, novel luciferases with improved substrate specificity and substrate reactivity were engineered. PMID:26060078

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

    PubMed

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

    2016-03-01

    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.

  3. Reduced expression of kan-1 (encoding putative bile acid-CoA-amino acid N-acyltransferase) mRNA in livers of rats after partial hepatectomy and during sepsis.

    PubMed Central

    Furutani, M; Arii, S; Higashitsuji, H; Mise, M; Fukumoto, M; Takano, S; Nakayama, H; Imamura, M; Fujita, J

    1995-01-01

    We isolated a cDNA clone, kan-1, from a rat liver cDNA library using a reverse transcriptase PCR cloning method. The kan-1 cDNA encoded a polypeptide of 420 amino acids, and was 70 and 69% identical in nucleotide and amino acid sequences respectively with human liver bile acid-CoA-amino acid N-acyltransferase (BAT). Thus Kan-1 is probably a rat homologue of human BAT (rBAT). Kan-1/rBAT mRNA was mainly expressed in the livers of adult rats and rats immediately after, but not before, birth. It was expressed in the hepatocytes, the sinusoidal endothelial cells and the Kupffer cells of the liver. An anti-Kan-1/rBAT polyclonal antibody detected a protein of molecular mass 46 kDa in the liver. After partial hepatectomy, the levels of Kan-1/rBAT mRNA decreased at 6 and 12 h in the regenerating liver. In a sepsis model, hepatic expression of Kan-1/rBAT mRNA decreased at 6 and 12 h after caecal ligation and puncture. The kinetics of Kan-1/rBAT mRNA expression suggests that it may play a role in acute-phase reactions. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7575455

  4. bldA-dependent expression of the Streptomyces exfoliatus M11 lipase gene (lipA) is mediated by the product of a contiguous gene, lipR, encoding a putative transcriptional activator.

    PubMed Central

    Servín-González, L; Castro, C; Pérez, C; Rubio, M; Valdez, F

    1997-01-01

    Extracellular lipase synthesis by Streptomyces lividans 66 carrying the cloned lipase gene (lipA) from Streptomyces exfoliatus M11 was found to be growth phase dependent, since lipase was secreted into the medium mainly during the stationary phase; S1 nuclease protection experiments revealed abundant lipA transcripts in RNA preparations obtained during the stationary phase but not in those obtained during exponential growth. Transcription from the lipA promoter was dependent on the presence of lipR, a contiguous downstream gene with a very high guanine-plus-cytosine content (80.2%). The deduced lipR product consists of a protein of 934 amino acids that shows similarity to known transcriptional activators and has a strong helix-turn-helix motif at its C terminus; this motif is part of a domain homologous to DNA-binding domains of bacterial regulators of the UhpA/LuxR superfamily. The lipR sequence revealed the presence of a leucine residue, encoded by the rare TTA codon, which caused bldA dependence of lipA transcription in Streptomyces coelicolor A3(2); replacement of the TTA codon by the alternate CTC leucine codon alleviated bidA dependence but not the apparent growth phase-dependent regulation of lipA transcription. When lipR expression was induced in a controlled fashion during the exponential growth phase, by placing it under the inducible tipA promoter, lipase synthesis was shifted to the exponential growth phase, indicating that the timing of lipR expression, and not its bldA dependence, is the main cause for stationary-phase transcription of lipA. PMID:9401043

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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)

  11. Reactivation of cell surface transport in Reticulomyxa.

    PubMed

    Orokos, D D; Bowser, S S; Travis, J L

    1997-01-01

    Granuloreticulosean protists transport particles (e.g., bacteria, algae, and sand grains) along the outer surfaces of their pseudopodia. This cell surface transport plays a vital role in feeding, reproduction, shell construction, and locomotion and can be visualized by the movements of extracellularly adherent polystyrene microspheres (i.e., latex beads). Our videomicroscopic analyses of transport associated with the pseudopodia of Reticulomyxa filosa revealed two distinct types of both intracellular and cell surface transport: (1) saltatory, bidirectional transport of individual or clustered organelles and/or surface-attached particles, and (2) continuous, unidirectional bulk or "resolute" motion of aggregated organelles and/or surface-bound particles. Organelles and surface-attached polystyrene microspheres remained firmly attached to the microtubule cytoskeletons of detergent-extracted pseudopodia. Both saltatory and resolute organelle and surface transport reactivated upon the addition of 0.01-1.0 mM ATP. At 1 mM ATP, the velocities of reactivated saltatory transport were indistinguishable from those observed in vivo. The reactivated transport was microtubule-dependent and was not inhibited by incubation with Ca(2+)-gelsolin under conditions that abolish rhodamine-phalloidin detection of actin filaments. These findings provide further support that both intracellular organelle and membrane surface transport are mediated by a common mechanism, and establish Reticulomyxa as a unique model system to further study the mechanochemistry of cell surface transport in vitro.

  12. Cell surface activation of the erythropoietin receptor by Friend spleen focus-forming virus gp55.

    PubMed Central

    Li, J P; Hu, H O; Niu, Q T; Fang, C

    1995-01-01

    The leukemogenic membrane glycoprotein gp55, encoded by Friend spleen focus-forming virus (SFFV), induces erythroid cell proliferation through its interaction with the erythropoietin receptor (EPO-R). There are two forms of gp55 in SFFV-infected cells: an intracellular form (more than 95% of the total protein), which is localized within the endoplasmic reticulum (ER) membranes, and a cell surface form (about 3 to 5%). Because both forms of the viral proteins bind to EPO-R, it is not clear whether the viral protein induces mitogenesis intracellularly or at the cell surface. To address this question, we constructed an EPO-R mutant that contained a 6-amino-acid (DEKKMP) C-terminus ER retention signal. Biochemical and functional analyses with this mutant indicated that it was completely retained in the ER and not expressed at the cell surface. Further analysis showed that the mutant, like the wild-type EPO-R, interacted with SFFV gp55. However, this apparent intracellular interaction between the two proteins failed to induce growth factor-independent proliferation of Ba/F3 cells. Furthermore, spontaneous variants of the ER-retained EPO-R selected on the basis of their ability to induce cell proliferation when coexpressed with gp55 were exclusively expressed at the cell surface. Thus, our results support the hypothesis that the mitogenic activation of the EPO-R by gp55 requires the interaction of the two proteins at the cell surface. PMID:7853508

  13. Five novel cell surface antigens of CNS neoplasms.

    PubMed

    Jennings, M T; Jennings, V D; Asadourian, L L; Rosenblum, M; Albino, A P; Cairncross, J G; Old, L J

    1989-01-01

    Optimal monoclonal antibody-mediated immunotherapy requires the identification of tumor-restricted cell surface antigens. We have identified and partially characterized 5 new monoclonal antibodies generated against malignant astrocytoma, medulloblastoma, neuroblastoma and melanoma which were used to define 5 neuroectodermal tumor antigenic systems. CNT/1 identifies a 57-kDa, heat-stable, trypsin-sensitive neuroblastoma surface antigen, which is expressed intracellularly in many malignant gliomas, medulloblastomas, ependymomas, breast and ovarian carcinomas. CNT/2 reacts with a 130-kDa, heat-labile, trypsin- and neuraminidase-resistant antigen restricted to low-grade astrocytomas and malignant gliomas. CNT/11 reacts with a 70-kDa, heat-labile, trypsin-sensitive antigen coded for by a gene on chromosome 12, and is restricted to astrocytomas, neuroblastomas and sarcomas. CNT/8 identifies a heat-labile, trypsin-sensitive antigen whose gene has been localized to chromosome 15 and is expressed by neuroectodermal and mesodermally derived tumors and few epithelial cancers. The B2.6 antigen is identified only in terms of serologic reactivity with a subset of cultured astrocytomas and melanomas. Neuroectodermal tumor-associated antigens may be categorized as lineage-consistent, lineage-independent and putatively tumor-restricted in their expression. These restricted antibodies may be potentially useful reagents to consider for monoclonal antibody-mediated immunotherapy of CNS neoplasms.

  14. 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.

  15. 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.

  16. Architecture of the Bacteroides cellulosolvens Cellulosome: Description of a Cell Surface-Anchoring Scaffoldin and a Family 48 Cellulase

    PubMed Central

    Xu, Qi; Bayer, Edward A.; Goldman, Milana; Kenig, Rina; Shoham, Yuval; Lamed, Raphael

    2004-01-01

    A large gene downstream of the primary Bacteroides cellulosolvens cellulosomal scaffoldin (cipBc, now renamed scaA) was sequenced. The gene, termed scaB, contained an N-terminal leader peptide followed by 10 type I cohesins, an “X” domain of unknown structure and function, and a C-terminal S-layer homology (SLH) surface-anchoring module. In addition, a previously identified gene in a different part of the genome, encoding for a dockerin-borne family 48 cellulosomal glycoside hydrolase (Cel48), was sequenced completely, and a putative cellulosome-related family 9 glycosyl hydrolase was detected. Recombinant fusion proteins, comprising dockerins derived from either the ScaA scaffoldin or Cel48, were overexpressed. Their interaction with ScaA and ScaB cohesins was examined by immunoassay. The results indicated that the ScaB type I cohesin of the new anchoring protein binds selectively to the ScaA dockerin, whereas the Cel48 dockerin binds specifically to the type II ScaA cohesin 5. Thus, by virtue of the 11 type II ScaA cohesins and the 10 type I ScaB cohesins, the relatively simple two-component cellulosome-integrating complex would potentially incorporate 110 enzyme molecules onto the cell surface via the ScaB SLH module. Compared to previously described cellulosome systems, the apparent roles of the B. cellulosolvens cohesins are reversed, in that the type II cohesins are located on the enzyme-binding primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldin. The results underscore the extensive diversity in the supramolecular architecture of cellulosome systems in nature. PMID:14761991

  17. 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.

  18. Cell-surface carbohydrates of Entamoeba invadens.

    PubMed

    Ribeiro, S; Soares, R M; Alviano, C S; Da Silva, E F; De Souza, W; Angluster, J

    1997-01-01

    Cell-surface carbohydrates of Entamoeba invadens trophozoites were analyzed using (a) a panel of highly purified lectins specific for molecules containing N-acetylglucosamine or sialic acid, N-acetylgalactosamine, galactose, mannose-like residues, and fucose; (b) Escherichia coli K-12 with mannose-sensitive fimbria; (c) enzymatic digestion; and (d) scanning electron microscopy. The presence of galactose (D-Gal) and N-acetylgalactosamine (D-GalNAc) was detected in the amoeba. Previous trypsinization induced the appearance of Glycine max (SBA, specific for D-GalNAc residues)-binding sites, whereas such treatment completely abolished the ability of Ricinus communis (RCAI) and Axinalla polypoides (APP, specific for D-Gal) lectins and partially abolished that of Euonymus europaeus (EEL, specific for D-Gal) lectins to agglutinate the trophozoites. The agglutinating activity of E. coli K-12 adheans with the amoeba was markedly increased after trypsin digestion, indicating that mannose units become exposed after enzyme treatment. These findings were essentially confirmed by scanning electron microscopy. After neuraminidase treatment the parasites became strongly agglutinated with SBA and Arachis hypogaea (PNA, specific for D-Gal) and the cell interaction with Wisteria floribunda (WFH, specific for D-GalNAc) was markedly increased. These results suggest that in E. invadens trophozoites, sialic acid residues are linked to D-Gal and D-GalNAc. PMID:9342747

  19. 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

  20. Signaling activities of the Drosophila wingless gene are separately mutable and appear to be transduced at the cell surface

    SciTech Connect

    Bejsovec, A.; Wieschaus, E.

    1995-01-01

    The Drosophila segment polarity gene wingless encodes an intercellular signaling molecule that transmits positional information during development of the embryonic epidermis. We have explored the mechanism of wg signal transduction by perturbing cellular processes genetically and by performing structure/function analysis of the Wg protein. We present evidence that Wingless protein may transduce signal at the cell surface and that Wg may bind to its cell surface receptor without necessarily activating it. We demonstrate that two specific signaling activities of the Wg molecule can be disrupted independently by mutation. Sequence analysis indicates that these different signaling activities are not promoted by discrete functional domains, but rather that the overall conformation of the molecule may control distinct signaling functions. We conclude that wg signaling may involve complex interactions between the Wg ligand and its cell surface receptor molecule(s) and that some of this complexity resides within the Wg ligand itself. 48 refs., 6 figs.

  1. Signaling Activities of the Drosophila Wingless Gene Are Separately Mutable and Appear to Be Transduced at the Cell Surface

    PubMed Central

    Bejsovec, A.; Wieschaus, E.

    1995-01-01

    The Drosophila segment polarity gene wingless encodes an intercellular signaling molecule that transmits positional information during development of the embryonic epidermis. We have explored the mechanism of wg signal transduction by perturbing cellular processes genetically and by performing structure/function analysis of the Wg protein. We present evidence that Wingless protein may transduce signal at the cell surface and that Wg may bind to its cell surface receptor without necessarily activating it. We demonstrate that two specific signaling activities of the Wg molecule can be disrupted independently by mutation. Sequence analysis indicates that these different signaling activities are not promoted by discrete functional domains, but rather that the overall conformation of the molecule may control distinct signaling functions. We conclude that wg signaling may involve complex interactions between the Wg ligand and its cell surface receptor molecule(s) and that some of this complexity resides within the Wg ligand itself. PMID:7705631

  2. 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

  3. 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

  4. Cell surface lectin-binding glycoconjugates on marine planktonic protists.

    PubMed

    Roberts, Emily C; Zubkov, Mikhail V; Martin-Cereceda, Mercedes; Novarino, Gianfranco; Wootton, Emma C

    2006-12-01

    Carbohydrate-protein interactions appear to play an important role in the phagocytosis of microbial prey by free-living protozoa. The present study utilizes FITC-labelled plant lectins to investigate the presence and localization of cell surface glycoconjugates on live and fixed planktonic protists (Dunaliella primolecta, Oxyrrhis marina, Goniomonas amphinema, Paraphysomonas vestita and Euplotes vannus). With live flagellate preparations, lectins primarily bound to external cell surfaces, with minimal internal staining observed. In contrast, cell fixation permeabilized cell membranes, allowing lectins to bind to internal structures, such as nuclear membranes and food vacuoles, interfering with the characterization of cell surface glycoconjugates. The method developed to label cell surface sugar moieties of live planktonic protists successfully overcomes the problems associated with fixation, and thus provides a useful protocol for future studies on protistan cell surface carbohydrate characterization.

  5. Cell surface fluctuations studied with defocusing microscopy

    NASA Astrophysics Data System (ADS)

    Agero, U.; Monken, C. H.; Ropert, C.; Gazzinelli, R. T.; Mesquita, O. N.

    2003-05-01

    cell surface, increases the relaxation time of cytoskeleton fluctuations, and increases the phagocytosis time. Our results suggest that the methods developed in this work can be of utility to assess the importance of cytoskeleton motility in the dynamics of cellular processes such as phagocytosis exhibited by macrophages.

  6. Nanoparticle energy transfer on the cell surface.

    PubMed

    Bene, László; Szentesi, Gergely; Mátyus, László; Gáspár, Rezso; Damjanovich, Sándor

    2005-01-01

    Membrane topology of receptors plays an important role in shaping transmembrane signalling of cells. Among the methods used for characterizing receptor clusters, fluorescence resonance energy transfer between a donor and acceptor fluorophore plays a unique role based on its capability of detecting molecular level (2-10 nm) proximities of receptors in physiological conditions. Recent development of biotechnology has made possible the usage of colloidal gold particles in a large size range for specific labelling of cells for the purposes of electron microscopy. However, by combining metal and fluorophore labelling of cells, the versatility of metal-fluorophore interactions opens the way for new applications by detecting the presence of the metal particles by the methods of fluorescence spectroscopy. An outstanding feature of the metal nanoparticle-fluorophore interaction is that the metal particle can enhance spontaneous emission of the fluorophore in a distance-dependent fashion, in an interaction range essentially determined by the size of the nanoparticle. In our work enhanced fluorescence of rhodamine and cyanine dyes was observed in the vicinity of immunogold nanoparticles on the surface of JY cells in a flow cytometer. The dyes and the immunogold were targetted to the cell surface receptors MHCI, MHCII, transferrin receptor and CD45 by monoclonal antibodies. The fluorescence enhancement was sensitive to the wavelength of the exciting light, the size and amount of surface bound gold beads, as well as the fluorophore-nanoparticle distance. The intensity of 90 degrees scattering of the incident light beam was enhanced by the immunogold in a concentration and size-dependent fashion. The 90 degrees light scattering varied with the wavelength of the incident light in a manner characteristic to gold nanoparticles of the applied sizes. A reduction in photobleaching time constant of the cyanine dye was observed in the vicinity of gold particles in a digital imaging

  7. 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

    2016-09-02

    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.

  8. 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

  9. Candida albicans CUG Mistranslation Is a Mechanism To Create Cell Surface Variation

    PubMed Central

    Miranda, Isabel; Silva-Dias, Ana; Rocha, Rita; Teixeira-Santos, Rita; Coelho, Carolina; Gonçalves, Teresa; Santos, Manuel A. S.; Pina-Vaz, Cidália; Solis, Norma V.; Filler, Scott G.; Rodrigues, Acácio G.

    2013-01-01

    ABSTRACT In the human fungal pathogen Candida albicans, the CUG codon is translated 97% of the time as serine and 3% of the time as leucine, which potentially originates an array of proteins resulting from the translation of a single gene. Genes encoding cell surface proteins are enriched in CUG codons; thus, CUG mistranslation may influence the interactions of the organism with the host. To investigate this, we compared a C. albicans strain that misincorporates 28% of leucine at CUGs with a wild-type parental strain. The first strain displayed increased adherence to inert and host molecules. In addition, it was less susceptible to phagocytosis by murine macrophages, probably due to reduced exposure of cell surface β-glucans. To prove that these phenotypes occurred due to serine/leucine exchange, the C. albicans adhesin and invasin ALS3 was expressed in Saccharomyces cerevisiae in its two natural isoforms (Als3p-Leu and Als3p-Ser). The cells with heterologous expression of Als3p-Leu showed increased adherence to host substrates and flocculation. We propose that CUG mistranslation has been maintained during the evolution of C. albicans due to its potential to generate cell surface variability, which significantly alters fungus-host interactions. PMID:23800396

  10. 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

  11. 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

  12. 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

  13. A mass spectrometric-derived cell surface protein atlas.

    PubMed

    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

  14. Crystal structure of a putative aspartic proteinase domain of the Mycobacterium tuberculosis cell surface antigen PE_PGRS16☆

    PubMed Central

    Barathy, Deivanayaga V.; Suguna, Kaza

    2013-01-01

    We report the crystal structure of the first prokaryotic aspartic proteinase-like domain identified in the genome of Mycobacterium tuberculosis. A search in the genomes of Mycobacterium species showed that the C-terminal domains of some of the PE family proteins contain two classic DT/SG motifs of aspartic proteinases with a low overall sequence similarity to HIV proteinase. The three-dimensional structure of one of them, Rv0977 (PE_PGRS16) of M. tuberculosis revealed the characteristic pepsin-fold and catalytic site architecture. However, the active site was completely blocked by the N-terminal His-tag. Surprisingly, the enzyme was found to be inactive even after the removal of the N-terminal His-tag. A comparison of the structure with pepsins showed significant differences in the critical substrate binding residues and in the flap tyrosine conformation that could contribute to the lack of proteolytic activity of Rv0977. PMID:23923105

  15. New Cell Surface Protein Involved in Biofilm Formation by Streptococcus parasanguinis ▿

    PubMed Central

    Liang, Xiaobo; Chen, Yi-Ywan M.; Ruiz, Teresa; Wu, Hui

    2011-01-01

    Dental biofilm formation is critical for maintaining the healthy microbial ecology of the oral cavity. Streptococci are predominant bacterial species in the oral cavity and play important roles in the initiation of plaque formation. In this study, we identified a new cell surface protein, BapA1, from Streptococcus parasanguinis FW213 and determined that BapA1 is critical for biofilm formation. Sequence analysis revealed that BapA1 possesses a typical cell wall-sorting signal for cell surface-anchored proteins from Gram-positive bacteria. No functional orthologue was reported in other streptococci. BapA1 possesses nine putative pilin isopeptide linker domains which are crucial for pilus assembly in a number of Gram-positive bacteria. Deletion of the 3′ portion of bapA1 generated a mutant that lacks surface-anchored BapA1 and abolishes formation of short fibrils on the cell surface. The mutant failed to form biofilms and exhibited reduced adherence to an in vitro tooth model. The BapA1 deficiency also inhibited bacterial autoaggregation. The N-terminal muramidase-released-protein-like domain mediated BapA1-BapA1 interactions, suggesting that BapA1-mediated cell-cell interactions are important for bacterial autoaggregation and biofilm formation. Furthermore, the BapA1-mediated bacterial adhesion and biofilm formation are independent of a fimbria-associated serine-rich repeat adhesin, Fap1, demonstrating that BapA1 is a new streptococcal adhesin. PMID:21576336

  16. Cell surface proteoglycan associates with the cytoskeleton at the basolateral cell surface of mouse mammary epithelial cells

    PubMed Central

    1986-01-01

    The cell surface proteoglycan on normal murine mammary gland mouse mammary epithelial cells consists of an ectodomain bearing heparan and chondroitin sulfate chains and a lipophilic domain that is presumed to be intercalated into the plasma membrane. Because the ectodomain binds to matrix components produced by stromal cells with specificity and high affinity, we have proposed that the cell surface proteoglycan is a matrix receptor that binds epithelial cells to their underlying basement membrane. We now show that the proteoglycan surrounds cells grown in subconfluent or newly confluent monolayers, but becomes restricted to the basolateral surface of cells that have been confluent for a week or more; Triton X-100 extraction distinguishes three fractions of cell surface proteoglycan: a fraction released by detergent and presumed to be free in the membrane, a fraction bound via a salt-labile linkage, and a nonextractable fraction; the latter two fractions co-localize with actin filament bundles at the basal cell surface; and when proteoglycans at the apical cell surface are cross- linked by antibodies, they initially assimilate into detergent- resistant, immobile clusters that are subsequently aggregated by the cytoskeleton. These findings suggest that the proteoglycan, initially present on the entire surface and free in the plane of the membrane, becomes sequestered at the basolateral cell surface and bound to the actin-rich cytoskeleton as the cells become polarized in vitro. Binding of matrix components may cross-link proteoglycans at the basal cell surface and cause them to associate with the actin cytoskeleton, providing a mechanism by which the cell surface proteoglycan acts as a matrix receptor to stabilize the morphology of epithelial sheets. PMID:3025223

  17. Synthesis of putative uniflorine A.

    PubMed

    Davis, Andrew S; Pyne, Stephen G; Skelton, Brian W; White, Allan H

    2004-04-30

    A diastereoselective synthesis of the putative structure of the natural product uniflorine A has been achieved by using the Petasis borono-Mannich reaction and ring-closing metathesis as key steps. The NMR data of the synthetic material did not match that reported for the natural product. The structure of the final synthetic product was unequivocally determined by single-crystal X-ray study of its pentaacetate derivative. Thus it was concluded that the proposed structure of uniflorine A is incorrect.

  18. Streptomycin favors biofilm formation by altering cell surface properties.

    PubMed

    Kumar, Amit; Ting, Yen-Peng

    2016-10-01

    Studies have shown that external stress induces biofilm formation, but the underlying details are not clearly understood. This study investigates the changes in cell surface properties leading to increase in biofilm formation by Staphylococcus aureus and Pseudomonas aeruginosa in the presence of streptomycin. Bacterial attachment in the presence and absence of streptomycin was quantified by fluorescence spectroscopy. In addition, cell surface charge and contact angle were measured and the free energy barrier for attachment was modeled using extended Derjaguin-Landau-Verwey-Overbeek (xDLVO) theory. Peptides from bacterial cell surface were shaved by protease treatment and identified with ultra-performance liquid chromatography (UPLC)-QTOF and a homology search program SPIDER. Biofilm formation increased significantly in the presence of streptomycin (10 mg/L) in the culture. Bacterial cell surface charge reduced, and hydrophobicity increased leading to a net decrease in the free energy barrier for attachment. Extracellular matrix-binding protein was positively regulated in S. aureus under stress, indicating stronger interaction between bacterial cells and solid surface. In addition, several other proteins including biofilm regulatory proteins, multidrug efflux pumps, transporters, signaling proteins, and virulence factors were differentially expressed on bacterial cell surface, which is indicative of a strong stress response by bacteria to streptomycin treatment. PMID:27568380

  19. Advances in cell surface glycoengineering reveal biological function.

    PubMed

    Nischan, Nicole; Kohler, Jennifer J

    2016-08-01

    Cell surface glycans are critical mediators of cell-cell, cell-ligand, and cell-pathogen interactions. By controlling the set of glycans displayed on the surface of a cell, it is possible to gain insight into the biological functions of glycans. Moreover, control of glycan expression can be used to direct cellular behavior. While genetic approaches to manipulate glycosyltransferase gene expression are available, their utility in glycan engineering has limitations due to the combinatorial nature of glycan biosynthesis and the functional redundancy of glycosyltransferase genes. Biochemical and chemical strategies offer valuable complements to these genetic approaches, notably by enabling introduction of unnatural functionalities, such as fluorophores, into cell surface glycans. Here, we describe some of the most recent developments in glycoengineering of cell surfaces, with an emphasis on strategies that employ novel chemical reagents. We highlight key examples of how these advances in cell surface glycan engineering enable study of cell surface glycans and their function. Exciting new technologies include synthetic lipid-glycans, new chemical reporters for metabolic oligosaccharide engineering to allow tandem and in vivo labeling of glycans, improved chemical and enzymatic methods for glycoproteomics, and metabolic glycosyltransferase inhibitors. Many chemical and biochemical reagents for glycan engineering are commercially available, facilitating their adoption by the biological community.

  20. 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.

  1. 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

  2. Enhancement of Biological Reactions on Cell Surfaces via Macromolecular Crowding

    PubMed Central

    Chapanian, Rafi; Kwan, David H.; Constantinescu, Iren; Shaikh, Fathima A.; Rossi, Nicholas A.A.; Withers, Stephen G.; Kizhakkedathu, Jayachandran N.

    2016-01-01

    The reaction of macromolecules such as enzymes and antibodies with cell surfaces is often an inefficient process, requiring large amounts of expensive reagent. Here we report a general method based on macromolecular crowding with a range of neutral polymers to enhance such reactions, using red blood cells (RBCs) as a model system. Rates of conversion of Type A and B red blood cells to universal O type by removal of antigenic carbohydrates with selective glycosidases are increased up to 400-fold in the presence of crowders. Similar enhancements are seen for antibody binding. We further explore the factors underlying these enhancements using confocal microscopy and fluorescent recovery after bleaching (FRAP) techniques with various fluorescent protein fusion partners. Increased cell-surface concentration due to volume exclusion, along with two-dimensionally confined diffusion of enzymes close to the cell surface, appear to be the major contributing factors. PMID:25140641

  3. Cell surface engineering of industrial microorganisms for biorefining applications.

    PubMed

    Tanaka, Tsutomu; Kondo, Akihiko

    2015-11-15

    In order to decrease carbon emissions and negative environmental impacts of various pollutants, biofuel/biochemical production should be promoted for replacing fossil-based industrial processes. Utilization of abundant lignocellulosic biomass as a feedstock has recently become an attractive option. In this review, we focus on recent efforts of cell surface display using industrial microorganisms such as Escherichia coli and yeast. Cell surface display is used primarily for endowing cellulolytic activity on the host cells, and enables direct fermentation to generate useful fuels and chemicals from lignocellulosic biomass. Cell surface display systems are systematically summarized, and the drawbacks/perspectives as well as successful application of surface display for industrial biotechnology are discussed.

  4. 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

  5. Cell surface engineering of industrial microorganisms for biorefining applications.

    PubMed

    Tanaka, Tsutomu; Kondo, Akihiko

    2015-11-15

    In order to decrease carbon emissions and negative environmental impacts of various pollutants, biofuel/biochemical production should be promoted for replacing fossil-based industrial processes. Utilization of abundant lignocellulosic biomass as a feedstock has recently become an attractive option. In this review, we focus on recent efforts of cell surface display using industrial microorganisms such as Escherichia coli and yeast. Cell surface display is used primarily for endowing cellulolytic activity on the host cells, and enables direct fermentation to generate useful fuels and chemicals from lignocellulosic biomass. Cell surface display systems are systematically summarized, and the drawbacks/perspectives as well as successful application of surface display for industrial biotechnology are discussed. PMID:26070720

  6. 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

  7. Analysis of cell surface properties using derivatized agarose beads.

    PubMed

    Salbilla, B A; Vaghefi, H; Chhabra, P; Hall, G; Brown, D; Sadoughi, F; Francisco, E; Attas, L; Walker, S L; Nguyen, B N; Oppenheimer, S B

    1999-07-01

    An assay has been developed to analyse cell surface properties using agarose beads derivatized with amino acids, sugars, proteins, and other molecules. The assay is simple and rapid and is useful to identify new cell surface markers. Various species and strains of yeast, paramecium, and Euglena were tested for their ability to bind to over 100 types of derivatized beads. A variety of specificity studies were performed in order to understand the nature of cell-bead binding. Our results indicate that cell-bead binding is often specific enough to distinguish between configurational isomers and spacer sizes and can be blocked by addition of specific molecules to the incubation medium. In some cases, different species or strains differed only by their binding to a single bead type. This simple and rapid assay may help to uncover new cell surface receptors and may lead to the development of clinically useful compounds for therapeutic applications.

  8. 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

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

    PubMed Central

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

    2013-01-01

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

  10. 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.

  11. 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

  12. 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...

  13. 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

  14. The Cell Surface Proteome of Human Mesenchymal Stromal Cells

    PubMed Central

    Pursche, Theresia; Bornhäuser, Martin; Corbeil, Denis; Hoflack, Bernard

    2011-01-01

    Background Multipotent human mesenchymal stromal cells (hMSCs) are considered as promising biological tools for regenerative medicine. Their antibody-based isolation relies on the identification of reliable cell surface markers. Methodology/Principal Findings To obtain a comprehensive view of the cell surface proteome of bone marrow-derived hMSCs, we have developed an analytical pipeline relying on cell surface biotinylation of intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin to enrich the plasma membrane proteins and mass spectrometry for identification with extremely high confidence. Among the 888 proteins identified, we found ≈200 bona fide plasma membrane proteins including 33 cell adhesion molecules and 26 signaling receptors. In total 41 CD markers including 5 novel ones (CD97, CD112, CD239, CD276, and CD316) were identified. The CD markers are distributed homogenously within plastic-adherent hMSC populations and their expression is modulated during the process of adipogenesis or osteogenesis. Moreover, our in silico analysis revealed a significant difference between the cell surface proteome of hMSCs and that of human embryonic stem cells reported previously. Conclusions/Significance Collectively, our analytical methods not only provide a basis for further studies of mechanisms maintaining the multipotency of hMSCs within their niches and triggering their differentiation after signaling, but also a toolbox for a refined antibody-based identification of hMSC populations from different tissues and their isolation for therapeutic intervention. PMID:21637820

  15. 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

  16. Sequence and tissue-specific expression of a putative peroxidase gene from wheat (Triticum aestivum L.).

    PubMed

    Hertig, C; Rebmann, G; Bull, J; Mauch, F; Dudler, R

    1991-01-01

    We have used a cDNA clone encoding a pathogen-induced putative wheat peroxidase to screen a genomic library of wheat (Triticum aestivum L. cv. Cheyenne) and isolated one positive clone, lambda POX1. Sequence analysis revealed that this clone contains a gene encoding a putative peroxidase with a calculated pI of 8.1 which exhibits 58% and 83% sequence identity to the amino acid sequence of the turnip (Brassica rapa) peroxidase and a pathogen-induced putative wheat peroxidase, respectively. The two introns in the wheat gene are at the same positions as introns in the peroxidase genes of tomato and horseradish. Results of S1-mapping experiments suggest that this gene is neither pathogen- nor wound-induced in leaves but is constitutively expressed in roots.

  17. Sequence and tissue-specific expression of a putative peroxidase gene from wheat (Triticum aestivum L.).

    PubMed

    Hertig, C; Rebmann, G; Bull, J; Mauch, F; Dudler, R

    1991-01-01

    We have used a cDNA clone encoding a pathogen-induced putative wheat peroxidase to screen a genomic library of wheat (Triticum aestivum L. cv. Cheyenne) and isolated one positive clone, lambda POX1. Sequence analysis revealed that this clone contains a gene encoding a putative peroxidase with a calculated pI of 8.1 which exhibits 58% and 83% sequence identity to the amino acid sequence of the turnip (Brassica rapa) peroxidase and a pathogen-induced putative wheat peroxidase, respectively. The two introns in the wheat gene are at the same positions as introns in the peroxidase genes of tomato and horseradish. Results of S1-mapping experiments suggest that this gene is neither pathogen- nor wound-induced in leaves but is constitutively expressed in roots. PMID:1653627

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. A Collagen-Binding Adhesin, Acb, and Ten Other Putative MSCRAMM and Pilus Family Proteins of Streptococcus gallolyticus subsp. gallolyticus (Streptococcus bovis Group, Biotype I)▿ §

    PubMed Central

    Sillanpää, Jouko; Nallapareddy, Sreedhar R.; Qin, Xiang; Singh, Kavindra V.; Muzny, Donna M.; Kovar, Christie L.; Nazareth, Lynne V.; Gibbs, Richard A.; Ferraro, Mary J.; Steckelberg, James M.; Weinstock, George M.; Murray, Barbara E.

    2009-01-01

    Members of the Streptococcus bovis group are important causes of endocarditis. However, factors associated with their pathogenicity, such as adhesins, remain uncharacterized. We recently demonstrated that endocarditis-derived Streptococcus gallolyticus subsp. gallolyticus isolates frequently adhere to extracellular matrix (ECM) proteins. Here, we generated a draft genome sequence of an ECM protein-adherent S. gallolyticus subsp. gallolyticus strain and found, by genome-wide analyses, 11 predicted LPXTG-type cell wall-anchored proteins with characteristics of MSCRAMMs, including a modular architecture of domains predicted to adopt immunoglobulin (Ig)-like folding. A recombinant segment of one of these, Acb, showed high-affinity binding to immobilized collagen, and cell surface expression of Acb correlated with the presence of acb and collagen adherence of isolates. Three of the 11 proteins have similarities to major pilus subunits and are organized in separate clusters, each including a second Ig-fold-containing MSCRAMM and a class C sortase, suggesting that the sequenced strain encodes three distinct types of pili. Reverse transcription-PCR demonstrated that all three genes of one cluster, acb-sbs7-srtC1, are cotranscribed, consistent with pilus operons of other gram-positive bacteria. Further analysis detected expression of all 11 genes in cells grown to mid to late exponential growth phases. Wide distribution of 9 of the 11 genes was observed among S. gallolyticus subsp. gallolyticus isolates with fewer genes present in other S. bovis group species/subspecies. The high prevalence of genes encoding putative MSCRAMMs and pili, including a collagen-binding MSCRAMM, among S. gallolyticus subsp. gallolyticus isolates may play an important role in the predominance of this subspecies in S. bovis endocarditis. PMID:19717590

  13. 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.

  14. Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells.

    PubMed

    Katz, Adam J; Tholpady, Ashok; Tholpady, Sunil S; Shang, Hulan; Ogle, Roy C

    2005-03-01

    Adult human subcutaneous adipose tissue contains cells with intriguing multilineage developmental plasticity, much like marrow-derived mesenchymal stem cells. Putative stem or progenitor cells from fat have been given many different names in the literature, reflecting an early and evolving consensus regarding their phenotypic characterization. The study reported here used microarrays to evaluate over 170 genes relating to angiogenesis and extracellular matrix in undifferentiated, early-passage human adipose-derived adherent stromal (hADAS) cells isolated from three separate donors. The hADAS populations unanimously transcribed 66% of the screened genes, and 83% were transcribed by at least two of the three populations. The most highly transcribed genes relate to functional groupings such as cell adhesion, matrix proteins, growth factors and receptors, and proteases. The transcriptome of hADAS cells demonstrated by this work reveals many similarities to published profiles of bone marrow mesenchymal stem cells (MSCs). In addition, flow analysis of over 24 hADAS cell surface proteins (n = 7 donors) both confirms and expands on the existing literature and reveals strong intergroup correlation, despite an inconsistent nomenclature and the lack of standardized protocols for cell isolation and culture. Finally, based on flow analysis and reverse transcription polymerase chain reaction studies, our results suggest that hADAS cells do not express several proteins that are implicated as markers of "stemness" in other stem cell populations, including telomerase, CD133, and the membrane transporter ABCG2.

  15. 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

  16. Externalization and binding of galectin-1 on cell surface of K562 cells upon erythroid differentiation.

    PubMed

    Lutomski, D; Fouillit, M; Bourin, P; Mellottée, D; Denize, N; Pontet, M; Bladier, D; Caron, M; Joubert-Caron, R

    1997-12-01

    Galectin 1 (GAL1) is a beta-galactoside-binding lectin involved in cell cycle progression. GAL1 overexpression is associated with neoplastic transformation and loss of differentiation. The gene encoding for human GAL1 resides on chromosome 22(q12; q13), and its expression is developmentally regulated. Although devoid of signal peptide GAL1 can be externalized from cells by a mechanism independent of the normal secretory process. We report here on a study of the effects of erythroid differentiation of the human leukemia cell line K562 on GAL1 protein expression. In undifferentiated K562 cells, GAL1 was expressed into the cytosol. However, the amount of GAL1 was surprisingly weaker in K562 cells than in other leukemia cell lines such as TF-1 or KG1a. Treatment of K562 cells with erythropoietin (EPO) or with aphidicolin (APH), an inhibitor for DNA polymerase alpha, induced an erythroid phenotype and led to the externalization of cytosolic GAL1 which was then bound to ligands on cell surface in a galactoside-inhibitable fashion. Our results demonstrate that acquisition of an erythroid phenotype is associated with an externalization of GAL1. The autocrine binding of GAL1 to cell surface ligands of non adherent cells such as K562 suggest that GAL1 is implicated rather in signal transduction than in cell-cell or cell-matrix interaction. Moreover, the reciprocal translocation involving chromosomes 9 and 22 t(9;22) present in K562 cells might explain the weak expression of GAL1 in K562 leukemia cells.

  17. Saccharomyces cerevisiae mutant displaying beta-glucans on cell surface.

    PubMed

    Sakai, Yumiko; Azuma, Masayuki; Takada, Yuki; Umeyama, Takashi; Kaneko, Aki; Fujita, Tsuyoshi; Igarashi, Koichi; Ooshima, Hiroshi

    2007-02-01

    The deletion of MCD4 leads to an increase in beta-1,6-glucan level and a decrease in glycosylphosphatidylinositol-anchored protein and mannan levels in the cell wall of Saccharomyces cerevisiae, suggesting that mcd4 deletion mutant (mcd4Delta) displays beta-glucans on the cell surface without a mannan cover. An observation of the cell surface of mcd4Delta cells and an examination of the effect of contact between mcd4Delta cells and mouse macrophages indicated that macrophages were activated by contact with mcd4Delta cells displaying beta-glucans on the cell surface. We further examined the effect of intraperitoneal ethanol-fixed mcd4Delta cells on the survival period of mice infected with Candida albicans. mcd4Delta cells prolonged the survival period, implying that mcd4Delta cells may enhance the immune function of mice via macrophage activation. Moreover, we examined the structures of beta-glucans (i.e., alkali- and acetic acid-insoluble beta-glucans) extracted from mcd4Delta with (13)C-NMR and the effect of extracted beta-glucans on TNF-alpha secretion from macrophages. The structures of the beta-glucans from mcd4Delta differed from those of wild type (WT); however, there was no difference in tumor necrosis factor-alpha (TNF-alpha) secretion level between beta-glucans from mcd4Delta and those from WT. The yield of purified beta-glucans obtained from dry cells of mcd4Delta was higher than that obtained from dry cells of WT. mcd4Delta may be a superior strain for the preparation of beta-glucans. PMID:17368399

  18. 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

  19. Polymer-supported membranes as models of the cell surface

    NASA Astrophysics Data System (ADS)

    Tanaka, Motomu; Sackmann, Erich

    2005-09-01

    Lipid-bilayer membranes supported on solid substrates are widely used as cell-surface models that connect biological and artificial materials. They can be placed either directly on solids or on ultrathin polymer supports that mimic the generic role of the extracellular matrix. The tools of modern genetic engineering and bioorganic chemistry make it possible to couple many types of biomolecule to supported membranes. This results in sophisticated interfaces that can be used to control, organize and study the properties and function of membranes and membrane-associated proteins. Particularly exciting opportunities arise when these systems are coupled with advanced semiconductor technology.

  20. Photodynamic induction of a bacterial cell surface polypeptide.

    PubMed Central

    Hoober, J K

    1977-01-01

    The photodynamic action of several dyes on cells of a bacterium, tentatively identified as a species of Arthrobacter, resulted in remarkable stimulation of synthesis of a polypeptide 21,000 daltons in mass. This polypeptide resides on the cell surface and can be solubilized by sodium dodecyl sulfate without lysis of the cells. Chlorophyllin and rose bengal are effective in inducing synthesis of the polypeptide in proportion to their ability to sensitize the photooxidation of histidine. Etiolated cells of the alga Chlamydomonas reinhardtii y-1 excrete a substance into the medium that also sensitized the photoinduction of the polypeptide. Images PMID:885841

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. Spatial patterns and cell surface clusters in perineuronal nets.

    PubMed

    Arnst, Nikita; Kuznetsova, Svetlana; Lipachev, Nikita; Shaikhutdinov, Nurislam; Melnikova, Anastasiya; Mavlikeev, Mikhail; Uvarov, Pavel; Baltina, Tatyana V; Rauvala, Heikki; Osin, Yuriy N; Kiyasov, Andrey P; Paveliev, Mikhail

    2016-10-01

    Perineuronal nets (PNN) ensheath GABAergic and glutamatergic synapses on neuronal cell surface in the central nervous system (CNS), have neuroprotective effect in animal models of Alzheimer disease and regulate synaptic plasticity during development and regeneration. Crucial insights were obtained recently concerning molecular composition and physiological importance of PNN but the microstructure of the network remains largely unstudied. Here we used histochemistry, fluorescent microscopy and quantitative image analysis to study the PNN structure in adult mouse and rat neurons from layers IV and VI of the somatosensory cortex. Vast majority of meshes have quadrangle, pentagon or hexagon shape with mean mesh area of 1.29µm(2) in mouse and 1.44µm(2) in rat neurons. We demonstrate two distinct patterns of chondroitin sulfate distribution within a single mesh - with uniform (nonpolar) and node-enriched (polar) distribution of the Wisteria floribunda agglutinin-positive signal. Vertices of the node-enriched pattern match better with local maxima of chondroitin sulfate density as compared to the uniform pattern. PNN is organized into clusters of meshes with distinct morphologies on the neuronal cell surface. Our findings suggest the role for the PNN microstructure in the synaptic transduction and plasticity.

  7. Intracellular transport and cell surface delivery of the neural cell adhesion molecule (NCAM).

    PubMed

    Leshchyns'ka, Iryna; Sytnyk, Vladimir

    2015-01-01

    The neural cell adhesion molecule (NCAM) regulates differentiation and functioning of neurons by accumulating at the cell surface where it mediates the interactions of neurons with the extracellular environment. NCAM also induces a number of intracellular signaling cascades, which coordinate interactions at the cell surface with intracellular processes including changes in gene expression, transport and cytoskeleton remodeling. Since NCAM functions at the cell surface, its transport and delivery to the cell surface play a critical role. Here, we review recent advances in our understanding of the molecular mechanisms of the intracellular transport and cell surface delivery of NCAM. We also discuss the data suggesting a possibility of cross talk between activation of NCAM at the cell surface and the intracellular transport and cell surface delivery of NCAM.

  8. 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

  9. 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

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

    PubMed Central

    1989-01-01

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

  11. Modulation of cell-surface antigens of a murine neuroblastoma.

    PubMed

    Akeson, R; Herschman, H R

    1974-01-01

    Antisera were produced in rabbits to morphologically differentiated cells from the C1300 murine neuroblastoma (i.e., cells in which process formation was induced by maintenance on serum-free medium for 5 days). These antisera reacted more strongly in the complement fixation reaction with such "differentiated" cells than with "undifferentiated" (nonprocess-bearing) neuroblastoma cells. Adsorption of the antisera with undifferentiated cells removed the reactivity to cells without processes, while the reactivity with serum-free cells which possess processes was retained. Indirect immunofluorescence studies confirmed the results obtained by complement fixation and demonstrated that antibodies to the surface antigens of process-bearing cells could be adsorbed by particulate preparations from brain but not liver, spleen, or kidney. This is the first description of neural-associated cell-surface changes that correlate with the morphological differentiation in culture of neuroblastoma cells.

  12. Only scratching the cell surface: extracellular signals in cerebrum development.

    PubMed

    Hébert, Jean M

    2013-08-01

    Numerous roles have been identified for extracellular signals such as Fibroblast Growth Factors (FGFs), Transforming Growth Factor-βs (TGFβs), Wingless-Int proteins (WNTs), and Sonic Hedgehog (SHH) in assigning fates to cells during development of the cerebrum. However, several fundamental questions remain largely unexplored. First, how does the same extracellular signal instruct precursor cells in different locations or at different stages to adopt distinct fates? And second, how does a precursor cell integrate multiple signals to adopt a specific fate? Answers to these questions require knowing the mechanisms that underlie each cell type's competence to respond to certain extracellular signals. This brief review provides illustrative examples of potential mechanisms that begin to bridge the gap between cell surface and cell fate during cerebrum development.

  13. Mechanotransduction Across the Cell Surface and Through the Cytoskeleton

    NASA Astrophysics Data System (ADS)

    Wang, Ning; Butler, James P.; Ingber, Donald E.

    1993-05-01

    Mechanical stresses were applied directly to cell surface receptors with a magnetic twisting device. The extracellular matrix receptor, integrin β_1, induced focal adhesion formation and supported a force-dependent stiffening response, whereas nonadhesion receptors did not. The cytoskeletal stiffness (ratio of stress to strain) increased in direct proportion to the applied stress and required intact microtubules and intermediate filaments as well as microfilaments. Tensegrity models that incorporate mechanically interdependent struts and strings that reorient globally in response to a localized stress mimicked this response. These results suggest that integrins act as mechanoreceptors and transmit mechanical signals to the cytoskeleton. Mechanotransduction, in turn, may be mediated simultaneously at multiple locations inside the cell through force-induced rearrangements within a tensionally integrated cytoskeleton.

  14. Mechanotransduction across the cell surface and through the cytoskeleton

    NASA Technical Reports Server (NTRS)

    Wang, N.; Butler, J. P.; Ingber, D. E.

    1993-01-01

    Mechanical stresses were applied directly to cell surface receptors with a magnetic twisting device. The extracellular matrix receptor, integrin beta 1, induced focal adhesion formation and supported a force-dependent stiffening response, whereas nonadhesion receptors did not. The cytoskeletal stiffness (ratio of stress to strain) increased in direct proportion to the applied stress and required intact microtubules and intermediate filaments as well as microfilaments. Tensegrity models that incorporate mechanically interdependent struts and strings that reorient globally in response to a localized stress mimicked this response. These results suggest that integrins act as mechanoreceptors and transmit mechanical signals to the cytoskeleton. Mechanotransduction, in turn, may be mediated simultaneously at multiple locations inside the cell through force-induced rearrangements within a tensionally integrated cytoskeleton.

  15. Advances in targeting cell surface signalling molecules for immune modulation

    PubMed Central

    Yao, Sheng; Zhu, Yuwen; Chen, Lieping

    2013-01-01

    The past decade has witnessed a surge in the development of immunomodulatory approaches to combat a broad range of human diseases, including cancer, viral infections, autoimmunity and inflammation as well as in the prevention of transplant rejection. Immunomodulatory approaches mostly involve the use of monoclonal antibodies or recombinant fusion proteins that target cell surface signalling molecules on immune cells to drive immune responses towards the desired direction. Advances in our understanding of the human immune system, along with valuable lessons learned from the first generation of therapeutic biologics, are aiding the design of the next generation of immunomodulatory biologics with better therapeutic efficacy, minimized adverse effects and long-lasting clinical benefit. The recent encouraging results from antibodies targeting programmed cell death protein 1 (PD1) and B7 homolog 1 (B7H1; also known as PDL1) for the treatment of various advanced human cancers show that immunomodulatory therapy has come of age. PMID:23370250

  16. Autonomous molecular cascades for evaluation of cell surfaces

    NASA Astrophysics Data System (ADS)

    Rudchenko, Maria; Taylor, Steven; Pallavi, Payal; Dechkovskaia, Alesia; Khan, Safana; Butler, Vincent P., Jr.; 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.

  17. Biological Functionalization of Carbon Nanotubes Using Cell Surface Mucin Mimics

    NASA Astrophysics Data System (ADS)

    Chen, Xing; Lee, Goo Soo; Zettl, Alex; Bertozzi, Carolyn

    2004-03-01

    Carbon Nanotubes (CNTs) are molecular wires with remarkable structural, electrical, and mechanical properties. Their potential applications in biology include sensing, imaging, and scaffolding for cell growth, but are presently limited by chemical incompatibility of the CNT surface with biological components and their aqueous milieu. Here we describe a biomimetic surface modification of CNTs using glycosylated polymers designed to mimic natural cell surface mucins. The polymers were end-functionalized with lipid tails for self-assembly on the CNT surface through hydrophobic interactions. Mucin mimic-coated CNTs were soluble in water, resisted non-specific protein binding and bound specifically to biomolecules via receptor-ligand interactions. This strategy for biomimetic surface engineering provides a means to bridge nanomaterials and biological systems.

  18. 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

  19. Substrate recognition by the cell surface palmitoyl transferase DHHC5.

    PubMed

    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

    2014-12-01

    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.

  20. Novel eukaryotic enzymes modifying cell-surface biopolymers

    PubMed Central

    2010-01-01

    Background Eukaryotic extracellular matrices such as proteoglycans, sclerotinized structures, mucus, external tests, capsules, cell walls and waxes contain highly modified proteins, glycans and other composite biopolymers. Using comparative genomics and sequence profile analysis we identify several novel enzymes that could be potentially involved in the modification of cell-surface glycans or glycoproteins. Results Using sequence analysis and conservation we define the acyltransferase domain prototyped by the fungal Cas1p proteins, identify its active site residues and unify them to the superfamily of classical 10TM acyltransferases (e.g. oatA). We also identify a novel family of esterases (prototyped by the previously uncharacterized N-terminal domain of Cas1p) that have a similar fold as the SGNH/GDSL esterases but differ from them in their conservation pattern. Conclusions We posit that the combined action of the acyltransferase and esterase domain plays an important role in controlling the acylation levels of glycans and thereby regulates their physico-chemical properties such as hygroscopicity, resistance to enzymatic hydrolysis and physical strength. We present evidence that the action of these novel enzymes on glycans might play an important role in host-pathogen interaction of plants, fungi and metazoans. We present evidence that in plants (e.g. PMR5 and ESK1) the regulation of carbohydrate acylation by these acylesterases might also play an important role in regulation of transpiration and stress resistance. We also identify a subfamily of these esterases in metazoans (e.g. C7orf58), which are fused to an ATP-grasp amino acid ligase domain that is predicted to catalyze, in certain animals, modification of cell surface polymers by amino acid or peptides. Reviewers This article was reviewed by Gaspar Jekely and Frank Eisenhaber PMID:20056006

  1. Thrombomucin, a Novel Cell Surface Protein that Defines Thrombocytes and Multipotent Hematopoietic Progenitors

    PubMed Central

    McNagny, Kelly M.; Pettersson, Inger; Rossi, Fabio; Flamme, Ingo; Shevchenko, Andrej; Mann, Matthias; Graf, Thomas

    1997-01-01

    MEP21 is an avian antigen specifically expressed on the surface of Myb-Ets–transformed multipotent hematopoietic precursors (MEPs) and of normal thrombocytes. Using nanoelectrospray tandem mass spectrometry, we have sequenced and subsequently cloned the MEP21 cDNA and named the gene thrombomucin as it encodes a 571–amino acid protein with an extracellular domain typical of the mucin family of proteoglycans. Thrombomucin is distantly related to CD34, the best characterized and most used human hematopoietic stem cell marker. It is also highly homologous in its transmembrane/intracellular domain to podocalyxinlike protein–1, a rabbit cell surface glycoprotein of kidney podocytes. Single cell analysis of yolk sac cells from 3-d-old chick embryos revealed that thrombomucin is expressed on the surface of both lineage-restricted and multipotent progenitors. In the bone marrow, thrombomucin is also expressed on mono- and multipotent progenitors, showing an overlapping but distinct expression pattern from that of the receptor-type stem cell marker c-kit. These observations strengthen the notion that the Myb-Ets oncoprotein can induce the proliferation of thrombomucin-positive hematopoietic progenitors that have retained the capacity to differentiate along multiple lineages. They also suggest that thrombomucin and CD34 form a family of stem cell–specific proteins with possibly overlapping functions in early hematopoietic progenitors. PMID:9298993

  2. 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

  3. Heterogeneous distribution of Candida albicans cell-surface antigens demonstrated with an Als1-specific monoclonal antibody

    PubMed Central

    Coleman, David A.; Oh, Soon-Hwan; Zhao, Xiaomin; Hoyer, Lois L.

    2010-01-01

    Despite an abundance of data describing expression of genes in the Candida albicans ALS (agglutinin-like sequence) gene family, little is known about the production of Als proteins on individual cells, their spatial localization or stability. Als proteins are most commonly discussed with respect to function in adhesion of C. albicans to host and abiotic surfaces. Development of a mAb specific for Als1, one of the eight large glycoproteins encoded by the ALS family, provided the opportunity to detect Als1 during growth of yeast and hyphae, both in vitro and in vivo, and to demonstrate the utility of the mAb in blocking C. albicans adhesion to host cells. Although most C. albicans yeast cells in a saturated culture are Als1-negative by indirect immunofluorescence, Als1 is detected on the surface of nearly all cells shortly after transfer into fresh growth medium. Als1 covers the yeast cell surface, with the exception of bud scars. Daughters of the inoculum cells, and sometimes granddaughters, also have detectable Als1, but Als1 is not detectable on cells from subsequent generations. On germ tubes and hyphae, most Als1 is localized proximal to the mother yeast. Once deposited on yeasts or hyphae, Als1 persists long after the culture has reached saturation. Growth stage-dependent production of Als1, coupled with its persistence on the cell surface, results in a heterogeneous population of cells within a C. albicans culture. Anti-Als1 immunolabelling patterns vary depending on the source of the C. albicans cells, with obvious differences between cells recovered from culture and those from a murine model of disseminated candidiasis. Results from this work highlight the temporal parallels for ALS1 expression and Als1 production in yeasts and germ tubes, the specialized spatial localization and persistence of Als1 on the C. albicans cell surface, and the differences in Als1 localization that occur in vitro and in vivo. PMID:20705663

  4. Complete Genome Sequence of the Filamentous Fungus Aspergillus westerdijkiae Reveals the Putative Biosynthetic Gene Cluster of Ochratoxin A

    PubMed Central

    Chakrabortti, Alolika; Li, Jinming

    2016-01-01

    Ochratoxin A (OTA) is a common mycotoxin that contaminates food and agricultural products. Sequencing of the complete genome of Aspergillus westerdijkiae, a major producer of OTA, reveals more than 50 biosynthetic gene clusters, including a putative OTA biosynthetic gene cluster that encodes a dozen of enzymes, transporters, and regulatory proteins. PMID:27635003

  5. Complete Genome Sequence of the Filamentous Fungus Aspergillus westerdijkiae Reveals the Putative Biosynthetic Gene Cluster of Ochratoxin A.

    PubMed

    Chakrabortti, Alolika; Li, Jinming; Liang, Zhao-Xun

    2016-01-01

    Ochratoxin A (OTA) is a common mycotoxin that contaminates food and agricultural products. Sequencing of the complete genome of Aspergillus westerdijkiae, a major producer of OTA, reveals more than 50 biosynthetic gene clusters, including a putative OTA biosynthetic gene cluster that encodes a dozen of enzymes, transporters, and regulatory proteins. PMID:27635003

  6. 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

  7. 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.

  8. Evaluation of the effects of Streptococcus mutans chaperones and protein secretion machinery components on cell surface protein biogenesis, competence, and mutacin production.

    PubMed

    Crowley, P J; Brady, L J

    2016-02-01

    The respective contributions of components of the protein translocation/maturation machinery to cell surface biogenesis in Streptococcus mutans are not fully understood. Here we used a genetic approach to characterize the effects of deletion of genes encoding the ribosome-associated chaperone RopA (Trigger Factor), the surface-localized foldase PrsA, and the membrane-localized chaperone insertases YidC1 and YidC2, both singly and in combination, on bacterial growth, chain length, self-aggregation, cell surface hydrophobicity, autolysis, and antigenicity of surface proteins P1 (AgI/II, PAc), WapA, GbpC, and GtfD. The single and double deletion mutants, as well as additional mutant strains lacking components of the signal recognition particle pathway, were also evaluated for their effects on mutacin production and genetic competence. PMID:26386361

  9. Somatic cell genetic analysis of human cell surface antigens 5.1H11 and F35/9 (gp45).

    PubMed

    Rettig, W J; Triche, T J; Bander, N H

    1990-01-01

    Serologic analysis of rodent-human somatic cell hybrids has permitted the assignment of loci coding for cell surface differentiation antigens 5.1H11 (gene symbol MSK39) and F35/9 (MSK40) to human chromosomes 11q13-qter and 22, respectively. Both antigens are expressed in hybrids constructed with antigen-positive human cells and certain hybrids constructed with antigen-negative human cells, indicating that the coding genes are not irreversibly silenced in human nonexpressor cells. Antigens 5.1H11 and F35/9, and at least six additional cell surface antigens encoded by chromosomes 11 and 22, are expressed on human Ewing sarcoma and peripheral neuroepithelioma cells, providing selectable markers for isolating and characterizing the specific t(11;22)(q24;q12) marker chromosomes of these tumors in interspecies hybrids.

  10. Miniaturised optical encoder

    NASA Astrophysics Data System (ADS)

    Carr, John; Desmulliez, Marc P. Y.; Weston, Nick; McKendrick, David; Cunningham, Graeme; McFarland, Geoff; Meredith, Wyn; McKee, Andrew; Langton, Conrad; Eddie, Iain

    2008-08-01

    Optical encoders are pervasive in many sectors of industry including metrology, motion systems, electronics, medical, scanning/ printing, scientific instruments, space research and specialist machine tools. The precision of automated manufacture and assembly has been revolutionised by the adoption of optical diffractive measurement methods. Today's optical encoders comprise discrete components: light source(s), reference and analyser gratings, and a photodiode array that utilise diffractive optic methods to achieve high resolution. However the critical alignment requirements between the optical gratings and to the photodiode array, the bulky nature of the encoder devices and subsequent packaging mean that optical encoders can be prohibitively expensive for many applications and unsuitable for others. We report here on the design, manufacture and test of a miniaturised optical encoder to be used in precision measurement systems. Microsystems manufacturing techniques facilitate the monolithic integration of the traditional encoder components onto a single compound semiconductor chip, radically reducing the size, cost and set-up time. Fabrication of the gratings at the wafer level, by standard photo-lithography, allows for the simultaneous alignment of many devices in a single process step. This development coupled with a unique photodiode configuration not only provides increased performance but also significantly improves the alignment tolerances in both manufacture and set-up. A National Research and Development Corporation type optical encoder chip has been successfully demonstrated under test conditions on both amplitude and phase scales with pitches of 20 micron, 8 micron and 4 micron, showing significantly relaxed alignment tolerances with signal-to-noise ratios greater than 60:1. Various reference mark schemes have also been investigated. Results are presented here.

  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. Structure and transforming potential of the human cot oncogene encoding a putative protein kinase.

    PubMed Central

    Miyoshi, J; Higashi, T; Mukai, H; Ohuchi, T; Kakunaga, T

    1991-01-01

    A new transforming gene has been molecularly cloned from hamster SHOK cells transformed with DNA extracted from a human thyroid carcinoma cell line and named the cot (cancer Osaka thyroid) oncogene. cDNA sequencing disclosed that this oncogene codes for a protein with 415 amino acid residues, and computer matching showed 42 to 48% similarity matches with serine protein kinases. Its gene product was identified as a 52-kDa protein by transcription and translation in vitro. Expression of cot cDNA under transcriptional control by a retroviral long terminal repeat induced morphological transformation of NIH 3T3 cells as well as SHOK cells. Protein kinase activity associated with constructed p60gag-cot was detected by immune complex kinase assay with anti-gag antiserum. The cot oncogene was overexpressed in transformed SHOK cells and found to have a rearranged 3' end in the last coding exon, which probably resulted in a deletion and an altered C' terminus in the transforming protein. This DNA rearrangement appeared to have occurred during transfection of the tumor DNA into hamster SHOK cells and not in the original thyroid tumor. Images PMID:2072910

  13. 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

  14. 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

  15. 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.

  16. CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

    PubMed Central

    Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Nuñez-Prado, Natalia; Diaz, Rosa M; Vile, Richard; Alvarez-Vallina, Luis

    2013-01-01

    A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells. PMID:23695536

  17. 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

  18. 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

  19. Molecular and Genetic Analyses of the Putative Proteus O Antigen Gene Locus▿ †

    PubMed Central

    Wang, Quan; Torzewska, Agnieszka; Ruan, Xiaojuan; Wang, Xiaoting; Rozalski, Antoni; Shao, Zhujun; Guo, Xi; Zhou, Haijian; Feng, Lu; Wang, Lei

    2010-01-01

    Proteus species are well-characterized opportunistic pathogens primarily associated with urinary tract infections (UTI) of humans. The Proteus O antigen is one of the most variable constituents of the cell surface, and O antigen heterogeneity is used for serological classification of Proteus isolates. Even though most Proteus O antigen structures have been identified, the O antigen locus has not been well characterized. In this study, we identified the putative Proteus O antigen locus and demonstrated this region's high degree of heterogeneity by comparing sequences of 40 Proteus isolates using PCR-restriction fragment length polymorphism (RFLP). This analysis identified five putative Proteus O antigen gene clusters, and the probable functions of these O antigen-related genes were proposed, based on their similarity to genes in the available databases. Finally, Proteus-specific genes from these five serogroups were identified by screening 79 strains belonging to the 68 Proteus O antigen serogroups. To our knowledge, this is the first molecular characterization of the putative Proteus O antigen locus, and we describe a novel molecular classification method for the identification of different Proteus serogroups. PMID:20581173

  20. ABC transporters involved in export of cell surface glycoconjugates.

    PubMed

    Cuthbertson, Leslie; Kos, Veronica; Whitfield, Chris

    2010-09-01

    Complex glycoconjugates play critical roles in the biology of microorganisms. Despite the remarkable diversity in glycan structures and the bacteria that produce them, conserved themes are evident in the biosynthesis-export pathways. One of the primary pathways involves representatives of the ATP-binding cassette (ABC) transporter superfamily. These proteins are responsible for the export of a wide variety of cell surface oligo- and polysaccharides in both Gram-positive and Gram-negative bacteria. Recent investigations of the structure and function of ABC transporters involved in the export of lipopolysaccharide O antigens have revealed two fundamentally different strategies for coupling glycan polymerization to export. These mechanisms are distinguished by the presence (or absence) of characteristic nonreducing terminal modifications on the export substrates, which serve as chain termination and/or export signals, and by the presence (or absence) of a discrete substrate-binding domain in the nucleotide-binding domain polypeptide of the ABC transporter. A bioinformatic survey examining ABC exporters from known oligo- and polysaccharide biosynthesis loci identifies conserved nucleotide-binding domain protein families that correlate well with themes in the structures and assembly of glycans. The familial relationships among the ABC exporters generate hypotheses concerning the biosynthesis of structurally diverse oligo- and polysaccharides, which play important roles in the biology of bacteria with different lifestyles.

  1. Encephalitis and antibodies to synaptic and neuronal cell surface proteins

    PubMed Central

    Lancaster, Eric; Martinez-Hernandez, Eugenia

    2011-01-01

    The identification of encephalitis associated with antibodies against cell surface and synaptic proteins, although recent, has already had a substantial impact in clinical neurology and neuroscience. The target antigens are receptors and proteins that have critical roles in synaptic transmission and plasticity, including the NMDA receptor, the AMPA receptor, the GABAB receptor, and the glycine receptor. Other autoantigens, such as leucine-rich glioma-inactivated 1 and contactin-associated protein-like 2, form part of trans-synaptic complexes and neuronal cell adhesion molecules involved in fine-tuning synaptic transmission and nerve excitability. Syndromes resulting from these immune responses resemble those of pharmacologic or genetic models in which the antigens are disrupted. For some immune responses, there is evidence that the antibodies alter the structure and function of the antigen, suggesting a direct pathogenic effect. These disorders are important because they can affect children and young adults, are severe and protracted, occur with or without tumor association, and respond to treatment but may relapse. This review provides an update on these syndromes and autoantigens with special emphasis on clinical diagnosis and treatment. PMID:21747075

  2. Characterizing cell surface of blooming Microcystis in Lake Taihu, China.

    PubMed

    Liu, Lizhen; Huang, Qi; Qin, Boqiang; Zhu, Guangwei; Wu, Pan; Wu, Yongming

    2016-01-01

    Microcystis occurs as colonies in the natural environment but disaggregates into single cells in laboratory cultures. In order to explore the mechanism of how Microcystis forms colonies, the zeta potentials of Microcystis cells from the laboratory and the field were studied, and the hydrophobicity of Microcystis colonies in different sizes was investigated in Lake Taihu. The incubation experiment indicated that the zeta potentials of Microcystis cells were affected by growth phase and species. The absolute values in exponential phase were lower than those in stationary phase, suggesting that the cells with rapid growth easily formed colonies due to more instability on the cell surface. The values of Microcystis aeruginosa were higher than those of Microcystis flos-aquae, which confirmed that M. aeruginosa prevailed in waters for a longer time and at a larger size compared with M. flos-aquae. In another aspect, the absolute zeta potentials of Microcystis spp. at pH 7.0 decreased from spring to autumn in the field; the values in spring were higher than those in summer, suggesting that a large-sized Microcystis colony would more easily form in summer. Additionally, differences in hydrophobicity exist among Microcystis colonies of various sizes. The surface hydrophobicity of colonies in the <20 μm size class was higher than that of larger colonies. This characteristic allowed small colonies to easily form large colonies to survive better. These results would be helpful to understand the mechanism of the bloom formation, especially the colony formation, in Microcystis. PMID:27232410

  3. CELL SURFACE ANTIGENS OF A MOUSE TESTICULAR TERATOMA

    PubMed Central

    Gooding, Linda R.; Edidin, Michael

    1974-01-01

    Rabbit antisera to a mouse testicular teratoma, absorbed with normal mouse tissues, react by immunofluorescence with plasma membrane antigens of a variety of transplantable mouse tumor cells and transformed fibroblast cell lines including Clone 1D, SV-40-3T3, and 3T12. Trypsin treatment of cells of "normal" lines, 3T3 and FR-SV-3T3, uncovers reactivity on these as well. Early passage mouse embryo fibroblast cell cultures do not react even after trypsinization. By cross-absorbtion studies, the anti-teratoma serum appears to react with an antigen common to most tumor cells investigated thus far. When this antigen on Clone 1D cells is "capped," H-2 antigens collect with the teratoma antigens in the cap indicating a physical association between the molecules. Molecules specified by both the H-2D and H-2K regions are bound to the teratoma antigens in the Clone 1D plasma membrane. This antigen is also found in soluble tumor cell fractions where it is believed to be free of H-2. A second cell surface antigen defined by anti-teratoma serum is expressed only by hepatoma and teratoma itself. This second antigen is apparently a secretory product of teratoma cells. A third surface antigen defined by anti-teratoma serum appears to be specific for the teratoma. PMID:4365513

  4. 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

  5. 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.

  6. Characterization and use of crystalline bacterial cell surface layers

    NASA Astrophysics Data System (ADS)

    Sleytr, Uwe B.; Sára, Margit; Pum, Dietmar; Schuster, Bernhard

    2001-10-01

    Crystalline bacterial cell surface layers (S-layers) are one of the most common outermost cell envelope components of prokaryotic organisms (archaea and bacteria). S-layers are monomolecular arrays composed of a single protein or glycoprotein species and represent the simplest biological membranes developed during evolution. S-layers as the most abundant of prokaryotic cellular proteins are appealing model systems for studying the structure, synthesis, genetics, assembly and function of proteinaceous supramolecular structures. The wealth of information existing on the general principle of S-layers have revealed a broad application potential. The most relevant features exploited in applied S-layer research are: (i) pores passing through S-layers show identical size and morphology and are in the range of ultrafiltration membranes; (ii) functional groups on the surface and in the pores are aligned in well-defined positions and orientations and accessible for chemical modifications and binding functional molecules in very precise fashion; (iii) isolated S-layer subunits from a variety of organisms are capable of recrystallizing as closed monolayers onto solid supports (e.g., metals, polymers, silicon wafers) at the air-water interface, on lipid films or onto the surface of liposomes; (iv) functional domains can be incorporated in S-layer proteins by genetic engineering. Thus, S-layer technologies particularly provide new approaches for biotechnology, biomimetics, molecular nanotechnology, nanopatterning of surfaces and formation of ordered arrays of metal clusters or nanoparticles as required for nanoelectronics.

  7. Dual-laser homo-FRET on the cell surface.

    PubMed

    Bene, László; Ungvári, Tamás; Fedor, Roland; Nagy, István; Damjanovich, László

    2015-05-01

    Inhomogeneous broadening and red-edge effects have been detected on a highly mobile system of fluorescently conjugated mAbs targeted to cell surface receptors. By exploiting site-selective spectroscopy and the characteristic loss of homo-FRET on increasing excitation and decreasing emission wavelengths, contributions of physical rotation and homo-FRET to the depolarization of fluorescence anisotropy have been separated. Absolute homo-FRET efficiency has been determined by ratioing two anisotropies: a homo-FRET-sensitive one, which is excited at the absorption main band and detected at the long wavelength region of emission, and a homo-FRET-insensitive one, which is excited at the long wavelength region of absorption and detected at the short wavelength region of emission. Because the anisotropies are simultaneously detected in a unified detection scheme of a dual T-format arrangement, the method is applicable for the real-time tracking of dynamical changes of physical rotations and proximities. The utility of the method is demonstrated in the context of the MHCII molecule and the heavy and light chains of the MHCI molecule, a system of three receptors with well-characterized close mutual proximities. Although the method is presented for a flow cytometer, it can also be realized in a fluorescence microscope capable for dual-laser excitation and dual-anisotropy detection.

  8. Analysis of cell surface antigens by Surface Plasmon Resonance imaging.

    PubMed

    Stojanović, Ivan; Schasfoort, Richard B M; Terstappen, Leon W M M

    2014-02-15

    Surface Plasmon Resonance (SPR) is most commonly used to measure bio-molecular interactions. SPR is used significantly less frequent for measuring whole cell interactions. Here we introduce a method to measure whole cells label free using the specific binding of cell surface antigens expressed on the surface of cancer cells and specific ligands deposited on sensor chips using an IBIS MX96 SPR imager (SPRi). As a model system, cells from the breast cancer cell line HS578T, SKBR3 and MCF7 were used. SPRi responses to Epithelial Cell Adhesion Molecule (EpCAM) antibody and other ligands coated on the sensor chips were measured. SPR curves show a response attributable to the sedimentation of the cells and a specific binding response on top of the initial response, the magnitude of which is dependent on the ligand density and the cell type used. Comparison of SPRi with flow cytometry showed similar EpCAM expression on MCF7, SKBR3 and HS578T cells.

  9. 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.

  10. Cell surface proteome of the marine planctomycete Rhodopirellula baltica.

    PubMed

    Voigt, Birgit; Hieu, Cao Xuan; Hempel, Kristina; Becher, Dörte; Schlüter, Rabea; Teeling, Hanno; Glöckner, Frank Oliver; Amann, Rudolf; Hecker, Michael; Schweder, Thomas

    2012-06-01

    The surface proteome (surfaceome) of the marine planctomycete Rhodopirellula baltica SH1(T) was studied using a biotinylation and a proteinase K approach combined with SDS-PAGE and mass spectrometry. 52 of the proteins identified in both approaches could be assigned to the group of potential surface proteins. Among them are some high molecular weight proteins, potentially involved in cell-cell attachment, that contain domains shown before to be typical for surface proteins like cadherin/dockerin domains, a bacterial adhesion domain or the fasciclin domain. The identification of proteins with enzymatic functions in the R. baltica surfaceome provides further clues for the suggestion that some degradative enzymes may be anchored onto the cell surface. YTV proteins, which have been earlier supposed to be components of the proteinaceous cell wall of R. baltica, were detected in the surface proteome. Additionally, 8 proteins with a novel protein structure combining a conserved type IV pilin/N-methylation domain and a planctomycete-typical DUF1559 domain were identified. PMID:22623273

  11. ABC Transporters Involved in Export of Cell Surface Glycoconjugates

    PubMed Central

    Cuthbertson, Leslie; Kos, Veronica; Whitfield, Chris

    2010-01-01

    Summary: Complex glycoconjugates play critical roles in the biology of microorganisms. Despite the remarkable diversity in glycan structures and the bacteria that produce them, conserved themes are evident in the biosynthesis-export pathways. One of the primary pathways involves representatives of the ATP-binding cassette (ABC) transporter superfamily. These proteins are responsible for the export of a wide variety of cell surface oligo- and polysaccharides in both Gram-positive and Gram-negative bacteria. Recent investigations of the structure and function of ABC transporters involved in the export of lipopolysaccharide O antigens have revealed two fundamentally different strategies for coupling glycan polymerization to export. These mechanisms are distinguished by the presence (or absence) of characteristic nonreducing terminal modifications on the export substrates, which serve as chain termination and/or export signals, and by the presence (or absence) of a discrete substrate-binding domain in the nucleotide-binding domain polypeptide of the ABC transporter. A bioinformatic survey examining ABC exporters from known oligo- and polysaccharide biosynthesis loci identifies conserved nucleotide-binding domain protein families that correlate well with themes in the structures and assembly of glycans. The familial relationships among the ABC exporters generate hypotheses concerning the biosynthesis of structurally diverse oligo- and polysaccharides, which play important roles in the biology of bacteria with different lifestyles. PMID:20805402

  12. Labile disulfide bonds are common at the leucocyte cell surface

    PubMed Central

    Metcalfe, Clive; Cresswell, Peter; Ciaccia, Laura; Thomas, Benjamin; Barclay, A. Neil

    2011-01-01

    Redox conditions change in events such as immune and platelet activation, and during viral infection, but the biochemical consequences are not well characterized. There is evidence that some disulfide bonds in membrane proteins are labile while others that are probably structurally important are not exposed at the protein surface. We have developed a proteomic/mass spectrometry method to screen for and identify non-structural, redox-labile disulfide bonds in leucocyte cell-surface proteins. These labile disulfide bonds are common, with several classes of proteins being identified and around 30 membrane proteins regularly identified under different reducing conditions including using enzymes such as thioredoxin. The proteins identified include integrins, receptors, transporters and cell–cell recognition proteins. In many cases, at least one cysteine residue was identified by mass spectrometry as being modified by the reduction process. In some cases, functional changes are predicted (e.g. in integrins and cytokine receptors) but the scale of molecular changes in membrane proteins observed suggests that widespread effects are likely on many different types of proteins including enzymes, adhesion proteins and transporters. The results imply that membrane protein activity is being modulated by a ‘redox regulator’ mechanism. PMID:22645650

  13. 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.

  14. 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

  15. 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

  16. A third secreted protein that is encoded by the enteropathogenic Escherichia coli pathogenicity island is required for transduction of signals and for attaching and effacing activities in host cells.

    PubMed Central

    Lai, L C; Wainwright, L A; Stone, K D; Donnenberg, M S

    1997-01-01

    Enteropathogenic Escherichia coli strains are able to signal host cells, cause dramatic cytoskeletal rearrangements, and adhere intimately to the cell surface in a process known as the attaching and effacing effect. A pathogenicity island of 35 kb known as the locus of enterocyte effacement (LEE) is necessary and sufficient for this effect. The LEE encodes an outer membrane adhesin called intimin, a type III secretion apparatus, and the EspA and EspB secreted proteins. The DNA sequence of the region between espA and espB revealed a new gene, espD. The product of espD was demonstrated by using a T7 expression system. We constructed a nonpolar mutation in espD and found that the mutant is incapable of the signal transduction events that lead to activation of the putative intimin receptor in host cells and that the mutant fails to induce the attaching and effacing effect. These phenotypes were restored to the mutant by complementation with a plasmid containing the cloned espD locus. We demonstrated by immunoblotting and microsequencing that the EspD protein is secreted via the type III apparatus. Thus, we describe a novel locus encoding a secreted protein that is required for attaching and effacing activity. PMID:9169753

  17. 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…

  18. 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

  19. 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

  20. 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.

  1. Fractal Dimension Analysis of Putative Martian Coastlines

    NASA Astrophysics Data System (ADS)

    Gianelli, G. A.

    2005-08-01

    Prior research is equivocal on the existence and location of Martian coastlines. This study proposes a novel method of analyzing putative coastlines; fractal dimensions provide a quantitative measurement of the complexity and nature of a fractal. Geological evidence points to a coastline at the elevation of -3790 meters, called the Deuteronilus contact. It is hypothesized that the fractal dimensions of this putative Martian coastline will be comparable to those of Earth shorelines. A topographic map with a contour line at -3790 meters was obtained from the U. S. Geological Survey, reflecting the most recent Mars Orbiter Laser Altimeter data. The map was cropped into sixty and twenty degree segments, and the putative coastline was isolated from extraneous features. A program which used the box-counting method calculated the fractal dimensions of the putative shorelines. The 22 results were tabulated and compared to 17 fractal dimensions of Earth shorelines, collected from published articles. Ranges were 1.07 to 1.66 for Earth and 1.141 to 1.436 for Mars. The mean was 1.28 for the Mars data and 1.22 for the Earth data, a slight difference that asteroid craters could account for. An unpaired t-test could not prove that the two data sets were significantly different. Although the past existence of a coastline at the Deuteronilus contact cannot be definitively proven without on site investigations, the hypothesis that the fractal dimensions of the putative Martian coastline would be comparable to those of Earth's was accepted, thereby substantiating the claims for the existence of a large northern ocean.

  2. Actin polymerization and intracellular solvent flow in cell surface blebbing

    PubMed Central

    1995-01-01

    The cortical actin gel of eukaryotic cells is postulated to control cell surface activity. One type of protrusion that may offer clues to this regulation are the spherical aneurysms of the surface membrane known as blebs. Blebs occur normally in cells during spreading and alternate with other protrusions, such as ruffles, suggesting similar protrusive machinery is involved. We recently reported that human melanoma cell lines deficient in the actin filament cross-linking protein, ABP-280, show prolonged blebbing, thus allowing close study of blebs and their dynamics. Blebs expand at different rates of volume increase that directly predict the final size achieved by each bleb. These rates decrease as the F-actin concentration of the cells increase over time after plating on a surface, but do so at lower concentrations in ABP-280 expressing cells. Fluorescently labeled actin and phalloidin injections of blebbing cells indicate that a polymerized actin structure is not present initially, but appears later and is responsible for stopping further bleb expansion. Therefore, it is postulated that blebs occur when the fluid-driven expansion of the cell membrane is sufficiently rapid to initially outpace the local rate of actin polymerization. In this model, the rate of intracellular solvent flow driving this expansion decreases as cortical gelation is achieved, whether by factors such as ABP-280, or by concentrated actin polymers alone, thereby leading to decreased size and occurrence of blebs. Since the forces driving bleb extension would always be present in a cell, this process may influence other cell protrusions as well. PMID:7790356

  3. 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.

  4. 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

  5. Video time encoding machines.

    PubMed

    Lazar, Aurel A; Pnevmatikakis, Eftychios A

    2011-03-01

    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.

  6. 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.

  7. Identification and Validation of Human Papillomavirus Encoded microRNAs

    PubMed Central

    Rönty, Mikko; Michon, Frederic; Frilander, Mikko J.; Ritari, Jarmo; Tarkkanen, Jussi; Paulín, Lars; Auvinen, Petri; Auvinen, Eeva

    2013-01-01

    We report here identification and validation of the first papillomavirus encoded microRNAs expressed in human cervical lesions and cell lines. We established small RNA libraries from ten human papillomavirus associated cervical lesions including cancer and two human papillomavirus harboring cell lines. These libraries were sequenced using SOLiD 4 technology. We used the sequencing data to predict putative viral microRNAs and discovered nine putative papillomavirus encoded microRNAs. Validation was performed for five candidates, four of which were successfully validated by qPCR from cervical tissue samples and cell lines: two were encoded by HPV 16, one by HPV 38 and one by HPV 68. The expression of HPV 16 microRNAs was further confirmed by in situ hybridization, and colocalization with p16INK4A was established. Prediction of cellular target genes of HPV 16 encoded microRNAs suggests that they may play a role in cell cycle, immune functions, cell adhesion and migration, development, and cancer. Two putative viral target sites for the two validated HPV 16 miRNAs were mapped to the E5 gene, one in the E1 gene, two in the L1 gene and one in the LCR region. This is the first report to show that papillomaviruses encode their own microRNA species. Importantly, microRNAs were found in libraries established from human cervical disease and carcinoma cell lines, and their expression was confirmed in additional tissue samples. To our knowledge, this is also the first paper to use in situ hybridization to show the expression of a viral microRNA in human tissue. PMID:23936163

  8. Identification and validation of human papillomavirus encoded microRNAs.

    PubMed

    Qian, Kui; Pietilä, Tuuli; Rönty, Mikko; Michon, Frederic; Frilander, Mikko J; Ritari, Jarmo; Tarkkanen, Jussi; Paulín, Lars; Auvinen, Petri; Auvinen, Eeva

    2013-01-01

    We report here identification and validation of the first papillomavirus encoded microRNAs expressed in human cervical lesions and cell lines. We established small RNA libraries from ten human papillomavirus associated cervical lesions including cancer and two human papillomavirus harboring cell lines. These libraries were sequenced using SOLiD 4 technology. We used the sequencing data to predict putative viral microRNAs and discovered nine putative papillomavirus encoded microRNAs. Validation was performed for five candidates, four of which were successfully validated by qPCR from cervical tissue samples and cell lines: two were encoded by HPV 16, one by HPV 38 and one by HPV 68. The expression of HPV 16 microRNAs was further confirmed by in situ hybridization, and colocalization with p16INK4A was established. Prediction of cellular target genes of HPV 16 encoded microRNAs suggests that they may play a role in cell cycle, immune functions, cell adhesion and migration, development, and cancer. Two putative viral target sites for the two validated HPV 16 miRNAs were mapped to the E5 gene, one in the E1 gene, two in the L1 gene and one in the LCR region. This is the first report to show that papillomaviruses encode their own microRNA species. Importantly, microRNAs were found in libraries established from human cervical disease and carcinoma cell lines, and their expression was confirmed in additional tissue samples. To our knowledge, this is also the first paper to use in situ hybridization to show the expression of a viral microRNA in human tissue.

  9. 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

  10. Coupling Binding to Catalysis: Using Yeast Cell Surface Display to Select Enzymatic Activities.

    PubMed

    Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

    2015-01-01

    We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence-activated cell sorting. PMID:26060080

  11. 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

  12. Coupling Binding to Catalysis: Using Yeast Cell Surface Display to Select Enzymatic Activities.

    PubMed

    Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

    2015-01-01

    We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence-activated cell sorting.

  13. 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

  14. Cell Surface Receptors for Signal Transduction and Ligand Transport: A Design Principles Study

    PubMed Central

    Shankaran, Harish; Resat, Haluk; Wiley, H. Steven

    2007-01-01

    Receptors constitute the interface of cells to their external environment. These molecules bind specific ligands involved in multiple processes, such as signal transduction and nutrient transport. Although a variety of cell surface receptors undergo endocytosis, the systems-level design principles that govern the evolution of receptor trafficking dynamics are far from fully understood. We have constructed a generalized mathematical model of receptor–ligand binding and internalization to understand how receptor internalization dynamics encodes receptor function and regulation. A given signaling or transport receptor system represents a particular implementation of this module with a specific set of kinetic parameters. Parametric analysis of the response of receptor systems to ligand inputs reveals that receptor systems can be characterized as being: i) avidity-controlled where the response control depends primarily on the extracellular ligand capture efficiency, ii) consumption-controlled where the ability to internalize surface-bound ligand is the primary control parameter, and iii) dual-sensitivity where both the avidity and consumption parameters are important. We show that the transferrin and low-density lipoprotein receptors are avidity-controlled, the vitellogenin receptor is consumption-controlled, and the epidermal growth factor receptor is a dual-sensitivity receptor. Significantly, we show that ligand-induced endocytosis is a mechanism to enhance the accuracy of signaling receptors rather than merely serving to attenuate signaling. Our analysis reveals that the location of a receptor system in the avidity-consumption parameter space can be used to understand both its function and its regulation. PMID:17542642

  15. Novel Properties for Endoglucanase Acquired by Cell-Surface Display Technique.

    PubMed

    Shi, Baosheng; Ke, Xiaojing; Yu, Hongwei; Xie, Jing; Jia, Yingmin; Guo, Runfang

    2015-11-01

    In order to improve the stability of endoglucanase under thermal and acidic conditions, the endoglucanase gene was fused to the N-terminus of the Saccharomyces cerevisiae pir gene, encoding the cell wall protein PIR. The fusion gene was transformed into Pichia pastoris GS115 for expression. A resulting strain with high expression and high activity was identified by examining resistance to Geneticin 418, Congo red staining, and quantitative analysis of enzyme activity. SDS-PAGE analysis revealed that the endoglucanase was successfully displayed on the yeast cell surface. The displayed endoglucanase (DEG) showed maximum activity towards sodium carboxyl methyl cellulose at approximately 275 IU/g cell dry weight. DEG exhibited greater than 60% residual activity in the pH range 2.5-8.5, higher than free endoglucanase (FEG), which had 40% residual activity at the same pH range. The highest tolerated temperature for DEG was 70°C, much higher than that of FEG, which was approximately 50°C. Moreover, DEG showed 91.1% activity at 65°C for 120 min, while FEG only kept 77.8% residual activity over the same period. The half-life of DEG was 270 min at 65°C, compared with only 150 min for FEG. DEG could be used repeatedly at least three times. These results suggest that the DEG has broad applications as a yeast whole-cell biocatalyst, due to its novel properties of high catalytic efficiency, acid-thermal stabilities, and reusability. PMID:26198121

  16. 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

  17. 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.

  18. Novel Properties for Endoglucanase Acquired by Cell-Surface Display Technique.

    PubMed

    Shi, Baosheng; Ke, Xiaojing; Yu, Hongwei; Xie, Jing; Jia, Yingmin; Guo, Runfang

    2015-11-01

    In order to improve the stability of endoglucanase under thermal and acidic conditions, the endoglucanase gene was fused to the N-terminus of the Saccharomyces cerevisiae pir gene, encoding the cell wall protein PIR. The fusion gene was transformed into Pichia pastoris GS115 for expression. A resulting strain with high expression and high activity was identified by examining resistance to Geneticin 418, Congo red staining, and quantitative analysis of enzyme activity. SDS-PAGE analysis revealed that the endoglucanase was successfully displayed on the yeast cell surface. The displayed endoglucanase (DEG) showed maximum activity towards sodium carboxyl methyl cellulose at approximately 275 IU/g cell dry weight. DEG exhibited greater than 60% residual activity in the pH range 2.5-8.5, higher than free endoglucanase (FEG), which had 40% residual activity at the same pH range. The highest tolerated temperature for DEG was 70°C, much higher than that of FEG, which was approximately 50°C. Moreover, DEG showed 91.1% activity at 65°C for 120 min, while FEG only kept 77.8% residual activity over the same period. The half-life of DEG was 270 min at 65°C, compared with only 150 min for FEG. DEG could be used repeatedly at least three times. These results suggest that the DEG has broad applications as a yeast whole-cell biocatalyst, due to its novel properties of high catalytic efficiency, acid-thermal stabilities, and reusability.

  19. MCD4 Encodes a Conserved Endoplasmic Reticulum Membrane Protein Essential for Glycosylphosphatidylinositol Anchor Synthesis in Yeast

    PubMed Central

    Gaynor, Erin C.; Mondésert, Guillaume; Grimme, Stephen J.; Reed, Steve I.; Orlean, Peter; Emr, Scott D.

    1999-01-01

    Glycosylphosphatidylinositol (GPI)-anchored proteins are cell surface-localized proteins that serve many important cellular functions. The pathway mediating synthesis and attachment of the GPI anchor to these proteins in eukaryotic cells is complex, highly conserved, and plays a critical role in the proper targeting, transport, and function of all GPI-anchored protein family members. In this article, we demonstrate that MCD4, an essential gene that was initially identified in a genetic screen to isolate Saccharomyces cerevisiae mutants defective for bud emergence, encodes a previously unidentified component of the GPI anchor synthesis pathway. Mcd4p is a multimembrane-spanning protein that localizes to the endoplasmic reticulum (ER) and contains a large NH2-terminal ER lumenal domain. We have also cloned the human MCD4 gene and found that Mcd4p is both highly conserved throughout eukaryotes and has two yeast homologues. Mcd4p’s lumenal domain contains three conserved motifs found in mammalian phosphodiesterases and nucleotide pyrophosphases; notably, the temperature-conditional MCD4 allele used for our studies (mcd4–174) harbors a single amino acid change in motif 2. The mcd4–174 mutant (1) is defective in ER-to-Golgi transport of GPI-anchored proteins (i.e., Gas1p) while other proteins (i.e., CPY) are unaffected; (2) secretes and releases (potentially up-regulated cell wall) proteins into the medium, suggesting a defect in cell wall integrity; and (3) exhibits marked morphological defects, most notably the accumulation of distorted, ER- and vesicle-like membranes. mcd4–174 cells synthesize all classes of inositolphosphoceramides, indicating that the GPI protein transport block is not due to deficient ceramide synthesis. However, mcd4–174 cells have a severe defect in incorporation of [3H]inositol into proteins and accumulate several previously uncharacterized [3H]inositol-labeled lipids whose properties are consistent with their being GPI precursors

  20. 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?

  1. 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.

  2. 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

  3. Characterization of three putative xylulose 5-phosphate/fructose 6-phosphate phosphoketolases in the cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    Moriyama, Takashi; Tajima, Naoyuki; Sekine, Kohsuke; Sato, Naoki

    2015-01-01

    Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) is a key enzyme in the central carbohydrate metabolism in heterofermentative bacteria, in which enzymatic property of Xfps is well characterized. This is not the case in other microbes. The cyanobacterium Anabaena sp. PCC 7120 possesses three putative genes encoding Xfp, all1483, all2567, and alr1850. We purified three putative Xfps as recombinant proteins. The results of gel filtration indicated that these proteins form homomultimer complex. All1483 and All2567 showed phosphoketolase activity, whereas Alr1850 did not show the activity. Kinetic analyses demonstrated that substrates, fructose 6-phosphate and inorganic phosphate, are cooperatively bound to enzymes positively and negatively, respectively.

  4. Cell surface markers for T and B lymphocytes activation and adhesion as putative prognostic biomarkers for head and neck squamous cell carcinoma.

    PubMed

    Andrade, Mariléia Chaves; Ferreira, Shirlene Barbosa Pimentel; Gonçalves, Luciana C; De-Paula, Alfredo Maurício Batista; de Faria, Elaine Speziali; Teixeira-Carvalho, Andréa; Martins-Filho, Olindo Assis

    2013-12-01

    The study population comprised HNSCC patients, risk-positive controls (tabagism and alcoholism habits), and risk-negative controls (without risk factors). Significant increases in the activation status of CD4(+)and CD8(+) T-cells, and higher migration potentials of lymphocytes were observed in HNSCC patients compared with control groups. Although decreased frequency of CD19(+)-B lymphocytes was observed in HSNCC patients, a higher percentage of HLA-DR(+)CD19(+)-B lymphocytes was detected in these individuals as compared with other evaluated groups. Metastasis and tumor grading were the major pathological parameters associated with significant alterations in the expression of activation molecules on circulating CD4(+) and CD8(+) T-cells. A reduced frequency of CD38-expressing CD8(+) T-cells was the most relevant biomarker associated with HNSCC aggressiveness. Performance analysis suggested a cut-off point for the CD8(+)CD38(+)/CD8(+) T-cell ratio of 7.0 for segregating patients according to tumor grading. In contrast, a higher proportion of CD8(+)CD54(+)/CD8(+) T-cells could represent a relevant biomarker associated with metastasis in HNSCC patients, and performance analysis suggested a cut-off point for the CD8(+)CD54(+)/CD8(+) T-cell ratio of 30 for segregating patients according to absence or presence of metastasis. The results obtained can increment immunological aspects of HNSCC and provide tools for the determination of cut-off scores of clinically relevant immunophenotypic prognostic biomarkers. PMID:23994583

  5. 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

  6. 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.

  7. 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…

  8. [Development and application of Saccharomyces cerevisiae cell-surface display for bioethanol production].

    PubMed

    Yang, Fei; Cao, Meng; Jin, Yi; Yang, Xiushan; Tian, Shen

    2012-08-01

    Saccharomyces cerevisiae is useful as a host for genetic engineering, since it allows the folding and glycosylation of expressed heterologous eukaryotic proteins and can be subjected to many genetic manipulations. 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. We reviewed the basic principle and progress of S. cerevisiae cell-surface engineering and gave an insight into the recent technological developments in the production of bioethanol using surface engineered yeast. PMID:23185890

  9. Putative impact of RNA editing on drug discovery.

    PubMed

    Decher, Niels; Netter, Michael F; Streit, Anne K

    2013-01-01

    Virtually all organisms use RNA editing as a powerful post-transcriptional mechanism to recode genomic information and to increase functional protein diversity. The enzymatic editing of pre-mRNA by ADARs and CDARs is known to change the functional properties of neuronal receptors and ion channels regulating cellular excitability. However, RNA editing is also an important mechanism for genes expressed outside the brain. The fact that RNA editing breaks the 'one gene encodes one protein' hypothesis is daunting for scientists and a probable drawback for drug development, as scientists might search for drugs targeting the 'wrong' protein. This possible difficulty for drug discovery and development became more evident from recent publications, describing that RNA editing events have profound impact on the pharmacology of some common drug targets. These recent studies highlight that RNA editing can cause massive discrepancies between the in vitro and in vivo pharmacology. Here, we review the putative impact of RNA editing on drug discovery, as RNA editing has to be considered before using high-throughput screens, rational drug design or choosing the right model organism for target validation.

  10. Large-Scale Identification of Putative Exported Proteins in Candida albicans by Genetic Selection

    PubMed Central

    Monteoliva, L.; López Matas, M.; Gil, C.; Nombela, C.; Pla, J.

    2002-01-01

    In all living organisms, secreted proteins play essential roles in different processes. Of special interest is the construction of the fungal cell wall, since this structure is absent from mammalian cells. The identification of the proteins involved in its biogenesis is therefore a primary goal in antifungal research. To perform a systematic identification of such proteins in Candida albicans, we carried out a genetic screening in which in-frame fusions with an intracellular allele of invertase gene SUC2 of Saccharomyces cerevisiae can be used to select and identify putatively exported proteins in the heterologous host S. cerevisiae. Eighty-three clones were selected, including 11 previously identified genes from C. albicans as well as 41 C. albicans genes that encode proteins homologous to already described proteins from related organisms. They include enzymes involved in cell wall synthesis and protein secretion. We also found membrane receptors and transporters presumably related to the interaction of C. albicans with the environment as well as extracellular enzymes and proteins involved in different morphological transitions. In addition, 11 C. albicans open reading frames (ORFs) identified in this screening encode proteins homologous to unknown or putative proteins, while 5 ORFs encode novel secreted proteins without known homologues in other organisms. This screening procedure therefore not only identifies a set of targets of interest in antifungal research but also provides new clues for understanding the topological locations of many proteins involved in processes relevant to the pathogenicity of this microorganism. PMID:12456000

  11. 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

  12. Disorders of phonological encoding.

    PubMed

    Butterworth, B

    1992-03-01

    Studies of phonological disturbances in aphasic speech are reviewed. It is argued that failure to test for error consistency in individual patients makes it generally improper to draw inferences about specific disorders of phonological encoding. A minimalist interpretation of available data on phonological errors is therefore proposed that involves variable loss of information in transmission between processing subsystems. Proposals for systematic loss or corruption of phonological information in lexical representations or in translation subsystems is shown to be inadequately grounded. The review concludes with some simple methodological prescriptions for future research.

  13. 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

  14. The influence of cell surface properties of thermophilic streptococci on attachment to stainless steel.

    PubMed

    Flint, S H; Brooks, J D; Bremer, P J

    1997-10-01

    The quality of milk products is threatened by the formation of biofilms of thermophilic streptococci on the internal surfaces of plate heat exchangers used in milk processing. Although attachment to stainless steel surfaces is one of the first stages in the development of a biofilm, the mechanisms involved in attachment have not been reported. The cell surface properties of 12 strains of thermophilic streptococci were examined to determine their importance in attachment to stainless steel surfaces. Hydrophobicity, extracellular polysaccharide production and cell surface charge varied between the different strains but could not be related to numbers attaching. Treating the cells with sodium metaperiodate, lysozyme or trichloroacetic acid to disrupt cell surface polysaccharide had no effect on attachment. Treatment with trypsin or sodium dodecyl sulphate to remove cell surface proteins resulted in a 100-fold reduction in the number of bacteria attaching. This result suggests that the surface proteins of the thermophilic streptococci are important in their attachment to stainless steel. PMID:9351231

  15. 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...

  16. Cell-Surface Phenol Soluble Modulins Regulate Staphylococcus aureus Colony Spreading

    PubMed Central

    Kizaki, Hayato; Omae, Yosuke; Tabuchi, Fumiaki; Saito, Yuki; Sekimizu, Kazuhisa

    2016-01-01

    Staphylococcus aureus produces phenol-soluble modulins (PSMs), which are amphipathic small peptides with lytic activity against mammalian cells. We previously reported that PSMα1–4 stimulate S. aureus colony spreading, the phenomenon of S. aureus colony expansion on the surface of soft agar plates, whereas δ-toxin (Hld, PSMγ) inhibits colony-spreading activity. In this study, we revealed the underlying mechanism of the opposing effects of PSMα1–4 and δ-toxin in S. aureus colony spreading. PSMα1–4 and δ-toxin are abundant on the S. aureus cell surface, and account for 18% and 8.5% of the total amount of PSMα1–4 and δ-toxin, respectively, in S. aureus overnight cultures. Knockout of PSMα1–4 did not affect the amount of cell surface δ-toxin. In contrast, knockout of δ-toxin increased the amount of cell surface PSMα1–4, and decreased the amount of culture supernatant PSMα1–4. The δ-toxin inhibited PSMα3 and PSMα2 binding to the S. aureus cell surface in vitro. A double knockout strain of PSMα1–4 and δ-toxin exhibited decreased colony spreading compared with the parent strain. Expression of cell surface PSMα1–4, but not culture supernatant PSMα1–4, restored the colony-spreading activity of the PSMα1-4/δ-toxin double knockout strain. Expression of δ-toxin on the cell surface or in the culture supernatant did not restore the colony-spreading activity of the PSMα1-4/δ-toxin double knockout strain. These findings suggest that cell surface PSMα1–4 promote S. aureus colony spreading, whereas δ-toxin suppresses colony-spreading activity by inhibiting PSMα1–4 binding to the S. aureus cell surface. PMID:27723838

  17. Structure, functions, and biosynthesis of glycoconjugates of Leishmania spp. cell surface.

    PubMed

    Novozhilova, N M; Bovin, N V

    2010-06-01

    Cell surface of leishmaniasis causal agent, a parasitic member of Protozoa of Leishmania genus, is covered by thick glycocalix consisting of various phosphatidylinositol-anchored molecules. This review deals with the structure and biosynthesis of the main phosphoglycans and glycoproteins of Leishmania cell surface, many of which incorporate the rare natural D-arabinopyranose, and the problem concerning the involvement of these molecules in support of Leishmania survival during their intricate life cycle is discussed.

  18. 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

  19. 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...

  20. Angiopoietin-3 is tethered on the cell surface via heparan sulfate proteoglycans.

    PubMed

    Xu, Yin; Liu, Yao-Juan; Yu, Qin

    2004-09-24

    Angiopoietins are a family of factors that play important roles in angiogenesis, and their receptor, Tie-2 receptor tyrosine kinase, is expressed primarily by endothelial cells. Three angiopoietins have been identified so far, angiopoietin-1 (Ang-1), angiopietin-2 (Ang-2), and angiopoietin-3 (Ang-3). It has been established that Ang-1 and Tie-2 play essential roles in embryonic angiogenesis. We have demonstrated recently that, unlike Ang-2, Ang-1 binds to the extracellular matrix, which regulates the availability and activity of Ang-1 (Xu, Y., and Yu, Q. (2001) J. Biol. Chem. 276, 34990-34998). However, the role and biochemical characteristics of Ang-3 are unknown. In our current study, we demonstrated that, unlike Ang-1 and Ang-2, Ang-3 is tethered on cell surface via heparan sulfate proteoglycans (HSPGs), especially perlecan. The cell surface-bound Ang-3 is capable of binding to its receptor, Tie-2; suggesting HSPGs concentrate Ang-3 on the cell surface and present Ang-3 to its receptor to elicit specific local reaction. Mutagenesis experiment revealed that the coiled-coil domain of Ang-3 is responsible for its binding to the cell surface. In addition, we demonstrated that the cell surface-bound Ang-3 but not soluble Ang-3 induces retraction and loss of integrity of endothelial monolayer, indicating the binding of Ang-3 to the cell surface via HSPGs is required for this bioactivity of Ang-3. PMID:15280392

  1. 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

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

    PubMed

    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

    2015-01-13

    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.

  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. 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.

  5. 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.

  6. Putative respiratory chain of Porphyromonas gingivalis.

    PubMed

    Meuric, Vincent; Rouillon, Astrid; Chandad, Fatiha; Bonnaure-Mallet, Martine

    2010-05-01

    The electron transfer chain in Porphyromonas gingivalis, or periodontopathogens, has not yet been characterized. P. gingivalis, a strict anaerobic bacteria and the second colonizer of the oral cavity, is considered to be a major causal agent involved in periodontal diseases. Primary colonizers create a favorable environment for P. gingivalis growth by decreasing oxygen pressure. Oxygen does not appear to be the final electron acceptor of the respiratory chain. Fumarate and cytochrome b have been implicated as major components of the respiratory activity. However, the P. gingivalis genome shows many other enzymes that could be implicated in aerobic or nitrite respiration. Using bioinformatic tools and literature studies of respiratory pathways, the ATP synthesis mechanism from the sodium cycle and nutrients metabolism, the putative respirasome of P. gingivalis has been proposed.

  7. Putative neuroprotective agents in neuropsychiatric disorders.

    PubMed

    Dodd, Seetal; Maes, Michael; Anderson, George; Dean, Olivia M; Moylan, Steven; Berk, Michael

    2013-04-01

    In many individuals with major neuropsychiatric disorders including depression, bipolar disorder and schizophrenia, their disease characteristics are consistent with a neuroprogressive illness. This includes progressive structural brain changes, cognitive and functional decline, poorer treatment response and an increasing vulnerability to relapse with chronicity. The underlying molecular mechanisms of neuroprogression are thought to include neurotrophins and regulation of neurogenesis and apoptosis, neurotransmitters, inflammatory, oxidative and nitrosative stress, mitochondrial dysfunction, cortisol and the hypothalamic-pituitary-adrenal axis, and epigenetic influences. Knowledge of the involvement of each of these pathways implies that specific agents that act on some or multiple of these pathways may thus block this cascade and have neuroprotective properties. This paper reviews the potential of the most promising of these agents, including lithium and other known psychotropics, aspirin, minocycline, statins, N-acetylcysteine, leptin and melatonin. These agents are putative neuroprotective agents for schizophrenia and mood disorders.

  8. 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.

  9. 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).

  10. 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.

  11. Identification and Analysis of Putative Homologues of Mechanosensitive Channels in Pathogenic Protozoa

    PubMed Central

    Prole, David L.; Taylor, Colin W.

    2013-01-01

    Mechanosensitive channels play important roles in the physiology of many organisms, and their dysfunction can affect cell survival. This suggests that they might be therapeutic targets in pathogenic organisms. Pathogenic protozoa lead to diseases such as malaria, dysentery, leishmaniasis and trypanosomiasis that are responsible for millions of deaths each year worldwide. We analyzed the genomes of pathogenic protozoa and show the existence within them of genes encoding putative homologues of mechanosensitive channels. Entamoeba histolytica, Leishmania spp., Trypanosoma cruzi and Trichomonas vaginalis have genes encoding homologues of Piezo channels, while most pathogenic protozoa have genes encoding homologues of mechanosensitive small-conductance (MscS) and K+-dependent (MscK) channels. In contrast, all parasites examined lack genes encoding mechanosensitive large-conductance (MscL), mini-conductance (MscM) and degenerin/epithelial Na+ (DEG/ENaC) channels. Multiple sequence alignments of evolutionarily distant protozoan, amoeban, plant, insect and vertebrate Piezo channel subunits define an absolutely conserved motif that may be involved in channel conductance or gating. MscS channels are not present in humans, and the sequences of protozoan and human homologues of Piezo channels differ substantially. This suggests the possibility for specific targeting of mechanosensitive channels of pathogens by therapeutic drugs. PMID:23785469

  12. 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

  13. 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

  14. 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.

  15. Expression of heat shock protein 90 at the cell surface in human neuroblastoma cells

    PubMed Central

    Cid, Cristina; Regidor, Ignacio; Poveda, Pedro D.

    2008-01-01

    In addition to the activity of heat shock protein 90 (Hsp90/HSPC) as a chaperone, some recent studies have reported expression of Hsp90 at the cell surface in certain types of cancer and nervous system cells. We study the expression of Hsp90 at the cell surface in human neuroblastoma (NB69) cells. Immunofluorescence experiments labeling with anti-Hsp90 antibodies on both nonpermeabilized cells and live cells detected Hsp90 at the cell surface. Hsp90 was also identified in a membrane fraction from subcellular fractionation. Cell-surface Hsp90 was significantly more expressed in undifferentiated proliferative spherical neuroblastoma cells than in differentiated flattened cells. In addition, spherical cells were significantly more sensitive to Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin compared to flattened cells. This paper describes the first evidence of cell-surface Hsp90 expression in a cancer cell line from nervous tissue and may indicate a novel target for anti-tumoral agents. PMID:18800240

  16. 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

  17. 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.

  18. 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.

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

    PubMed Central

    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

  20. 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

  1. 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

  2. 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.

  3. Cleavage of a putative metal permease in Chlamydia trachomatis yields an iron-dependent transcriptional repressor.

    PubMed

    Thompson, Christopher C; Nicod, Sophie S; Malcolm, Denise S; Grieshaber, Scott S; Carabeo, Rey A

    2012-06-26

    The regulation of iron homeostasis is essential for most organisms, because iron is required for a variety of conserved biochemical processes, yet can be toxic at high concentrations. Upon experiencing iron starvation in vitro, the obligate intracellular human pathogen Chlamydia trachomatis exhibits elevated expression of a putative iron-transport system encoded by the ytg operon. The third component of the ytg operon, CT069 (YtgCR), encodes a protein with two distinct domains: a membrane-anchored metal ion permease and a diphtheria toxin repressor (DtxR)-like transcriptional repressor. In this report, we demonstrate that the C-terminal domain of CT069 (YtgR) serves as an iron-dependent autorepressor of the ytg operon. Moreover, the nascent full-length metal permease-transcriptional repressor protein was processed during the course of infection, and heterologously when expressed in Escherichia coli. The products produced by heterologous cleavage in E. coli were functional in the repression of a reporter gene downstream of a putative YtgR operator. We report a bona fide mechanism of iron-dependent regulation of transcription in Chlamydia. Moreover, the unusual membrane permease-DNA-binding polypeptide fusion configuration was found in several bacteria. Therefore, the DNA-binding capability and liberation of the YtgR domain from a membrane-anchored permease in C. trachomatis could represent a previously uncharacterized mechanism for prokaryotic regulation of iron-homeostasis.

  4. 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

  5. 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

  6. Atomic force microscopy – looking at mechanosensors on the cell surface

    PubMed Central

    Heinisch, Jürgen J.; Lipke, Peter N.; Beaussart, Audrey; El Kirat Chatel, Sofiane; Dupres, Vincent; Alsteens, David; Dufrêne, Yves F.

    2012-01-01

    Summary Living cells use cell surface proteins, such as mechanosensors, to constantly sense and respond to their environment. However, the way in which these proteins respond to mechanical stimuli and assemble into large complexes remains poorly understood at the molecular level. In the past years, atomic force microscopy (AFM) has revolutionized the way in which biologists analyze cell surface proteins to molecular resolution. In this Commentary, we discuss how the powerful set of advanced AFM techniques (e.g. live-cell imaging and single-molecule manipulation) can be integrated with the modern tools of molecular genetics (i.e. protein design) to study the localization and molecular elasticity of individual mechanosensors on the surface of living cells. Although we emphasize recent studies on cell surface proteins from yeasts, the techniques described are applicable to surface proteins from virtually all organisms, from bacteria to human cells. PMID:23077172

  7. 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.

  8. 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

  9. Chemical remodeling of cell-surface sialic acids through a palladium-triggered bioorthogonal elimination reaction.

    PubMed

    Wang, Jie; Cheng, Bo; Li, Jie; Zhang, Zhaoyue; Hong, Weiyao; Chen, Xing; Chen, Peng R

    2015-04-27

    We herein report a chemical decaging strategy for the in situ generation of neuramic acid (Neu), a unique type of sialic acid, on live cells by the use of a palladium-mediated bioorthogonal elimination reaction. Palladium nanoparticles (Pd NPs) were found to be a highly efficient and biocompatible depropargylation catalyst for the direct conversion of metabolically incorporated N-(propargyloxycarbonyl)neuramic acid (Neu5Proc) into Neu on cell-surface glycans. This conversion chemically mimics the enzymatic de-N-acetylation of N-acetylneuramic acid (Neu5Ac), a proposed mechanism for the natural occurrence of Neu on cell-surface glycans. The bioorthogonal elimination was also exploited for the manipulation of cell-surface charge by unmasking the free amine at C5 to neutralize the negatively charged carboxyl group at C1 of sialic acids.

  10. 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.

  11. Cell surface differences of Naegleria fowleri and Naegleria lovaniensis exposed with surface markers.

    PubMed

    González-Robles, Arturo; Castañón, Guadalupe; Cristóbal-Ramos, Ana Ruth; Hernández-Ramírez, Verónica Ivonne; Omaña-Molina, Maritza; Martínez-Palomo, Adolfo

    2007-12-01

    Differences in the distribution of diverse cell surface coat markers were found between Naegleria fowleri and Naegleria lovaniensis. The presence of carbohydrate-containing components in the cell coat of the two species was detected by selective staining with ruthenium red and alcian blue. Using both markers, N. fowleri presented a thicker deposit than N. lovaniensis. The existence of exposed mannose or glucose residues was revealed by discriminatory agglutination with the plant lectin Concanavalin A. These sugar residues were also visualized at the cell surface of these parasites either by transmission electron microscopy or by fluorescein-tagged Concanavalin A. Using this lectin cap formation was induced only in N. fowleri. The anionic sites on the cell surface detected by means of cationized ferritin were more apparent in N. fowleri. Biotinylation assays confirmed that even though the two amoebae species have some analogous plasma membrane proteins, there is a clear difference in their composition.

  12. 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.

  13. 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

  14. Isolation and characterization of two mitoviruses and a putative alphapartitivirus from Fusarium spp.

    PubMed

    Osaki, Hideki; Sasaki, Atsuko; Nomiyama, Koji; Sekiguchi, Hiroyuki; Tomioka, Keisuke; Takehara, Toshiaki

    2015-06-01

    The filamentous fungus Fusarium spp. includes several important plant pathogens. We attempted to reveal presence of double-stranded (ds) RNAs in the genus. Thirty-seven Fusarium spp. at the MAFF collection were analyzed. In the strains of Fusarium coeruleum, Fusarium globosum and Fusarium solani f. sp. pisi, single dsRNA bands were detected. The strains of F. coeruleum and F. solani f. sp. pisi cause potato dry rot and mulberry twig blight, respectively. Sequence analyses revealed that dsRNAs in F. coeruleum and F. globosum consisted of 2423 and 2414 bp, respectively. Using the fungal mitochondrial translation table, the positive strands of these cDNAs were found to contain single open reading frames with the potential to encode a protein of putative 757 and 717 amino acids (molecular mass 88.5 and 84.0 kDa, respectively), similar to RNA-dependent RNA polymerases of members of the genus Mitovirus. These dsRNAs in F. coeruleum and F. globosum were assigned to the genus Mitovirus (family Narnaviridae), and these two mitoviruses were designated as Fusarium coeruleum mitovirus 1 and Fusarium globosum mitovirus 1. On the other hand, a positive strand of cDNA (1950 bp) from dsRNA in F. solani f. sp. pisi contained an ORF potentially encoding a putative RdRp of 608 amino acids (72.0 kDa). The putative RdRp was shown to be related to those of members of the genus of Alphapartitivirus (family Partitiviridae). We coined the name Fusarium solani partitivirus 2 for dsRNA in F. solani f. sp. pisi.

  15. 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…

  16. Physical Features of Intracellular Proteins that Moonlight on the Cell Surface

    PubMed Central

    Amblee, Vaishak; Jeffery, Constance J.

    2015-01-01

    Moonlighting proteins comprise a subset of multifunctional proteins that perform two or more biochemical functions that are not due to gene fusions, multiple splice variants, proteolytic fragments, or promiscuous enzyme activities. The project described herein focuses on a sub-set of moonlighting proteins that have a canonical biochemical function inside the cell and perform a second biochemical function on the cell surface in at least one species. The goal of this project is to consider the biophysical features of these moonlighting proteins to determine whether they have shared characteristics or defining features that might suggest why these particular proteins were adopted for a second function on the cell surface, or if these proteins resemble typical intracellular proteins. The latter might suggest that many other normally intracellular proteins found on the cell surface might also be moonlighting in this fashion. We have identified 30 types of proteins that have different functions inside the cell and on the cell surface. Some of these proteins are found to moonlight on the surface of multiple species, sometimes with different extracellular functions in different species, so there are a total of 98 proteins in the study set. Although a variety of intracellular proteins (enzymes, chaperones, etc.) are observed to be re-used on the cell surface, for the most part, these proteins were found to have physical characteristics typical of intracellular proteins. Many other intracellular proteins have also been found on the surface of bacterial pathogens and other organisms in proteomics experiments. It is quite possible that many of those proteins also have a moonlighting function on the cell surface. The increasing number and variety of known moonlighting proteins suggest that there may be more moonlighting proteins than previously thought, and moonlighting might be a common feature of many more proteins. PMID:26110848

  17. Nucleolin on the cell surface as a new molecular target for gastric cancer treatment.

    PubMed

    Watanabe, Tatsuro; Hirano, Kazuya; Takahashi, Atsushi; Yamaguchi, Kensei; Beppu, Masatoshi; Fujiki, Hirota; Suganuma, Masami

    2010-01-01

    Nucleolin is an abundant non-ribosomal protein found in nucleolus and a major component of silver-stained nucleolar organizer region (AgNOR), a histopathological marker of cancer which is highly elevated in cancer cells. We recently reported that nucleolin on the cell surface of mouse gastric cancer cells acts as a receptor for tumor necrosis factor-alpha-inducing protein (Tipalpha), a new carcinogenic factor of Helicobacter pylori. In this study, we first examined the localization of nucleolin on cell surface of five gastric cancer cell lines by cell fractionation and flow cytometry: We found that large amounts of nucleolin were present on surface of MKN-45, KATOIII, MKN-74, and AGS cells, with smaller amounts on surface of MKN-1 cells. The membrane fraction of normal epithelial cells of mouse glandular stomach did not contain much nucleolin, suggesting that translocation of nucleolin to the cell surface occurs during carcinogenesis, making for easier binding with Tipalpha. AS1411, a nucleolin targeted DNA aptamer, inhibited growth of gastric cancer cell lines in this order of potency: MKN-45>KATOIII>AGS>MKN-74=MKN-1, associated with induction of S-phase cell cycle arrest. Fluorescein isothiocyanate (FITC)-AS1411 was more rapidly incorporated into MKN-45 and AGS than into MKN-1 cells, based on varying amounts of cell surface nucleolin. We think that AS1411 first binds to nucleolin on the cell surface and that the binding complex is then incorporated into the cells. All results indicate that nucleolin on the cell surface is a new and promising therapeutic target for treatment of gastric cancer. PMID:20460757

  18. 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.

  19. Putative bronchopulmonary flagellated protozoa in immunosuppressed patients.

    PubMed

    Kilimcioglu, Ali Ahmet; Havlucu, Yavuz; Girginkardesler, Nogay; Celik, Pınar; Yereli, Kor; Özbilgin, Ahmet

    2014-01-01

    Flagellated protozoa that cause bronchopulmonary symptoms in humans are commonly neglected. These protozoal forms which were presumed to be "flagellated protozoa" have been previously identified in immunosuppressed patients in a number of studies, but have not been certainly classified so far. Since no human cases of bronchopulmonary flagellated protozoa were reported from Turkey, we aimed to investigate these putative protozoa in immunosuppressed patients who are particularly at risk of infectious diseases. Bronchoalveolar lavage fluid samples of 110 immunosuppressed adult patients who were admitted to the Department of Chest Diseases, Hafsa Sultan Hospital of Celal Bayar University, Manisa, Turkey, were examined in terms of parasites by light microscopy. Flagellated protozoal forms were detected in nine (8.2%) of 110 cases. Metronidazole (500 mg b.i.d. for 30 days) was given to all positive cases and a second bronchoscopy was performed at the end of the treatment, which revealed no parasites. In conclusion, immunosuppressed patients with bronchopulmonary symptoms should attentively be examined with regard to flagellated protozoa which can easily be misidentified as epithelial cells.

  20. Mechanosensory neurons, cutaneous mechanoreceptors, and putative mechanoproteins.

    PubMed

    Del Valle, M E; Cobo, T; Cobo, J L; Vega, J A

    2012-08-01

    The mammalian skin has developed sensory structures (mechanoreceptors) that are responsible for different modalities of mechanosensitivity like touch, vibration, and pressure sensation. These specialized sensory organs are anatomically and functionally connected to a special subset of sensory neurons called mechanosensory neurons, which electrophysiologically correspond with Aβ fibers. Although mechanosensory neurons and cutaneous mechanoreceptors are rather well known, the biology of the sense of touch still remains poorly understood. Basically, the process of mechanosensitivity requires the conversion of a mechanical stimulus into an electrical signal through the activation of ion channels that gate in response to mechanical stimuli. These ion channels belong primarily to the family of the degenerin/epithelium sodium channels, especially the subfamily acid-sensing ion channels, and to the family of transient receptor potential channels. This review compiles the current knowledge on the occurrence of putative mechanoproteins in mechanosensory neurons and mechanoreceptors, as well as the involvement of these proteins on the biology of touch. Furthermore, we include a section about what the knock-out mice for mechanoproteins are teaching us. Finally, the possibilities for mechanotransduction in mechanoreceptors, and the common involvement of the ion channels, extracellular membrane, and cytoskeleton, are revisited.

  1. 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

  2. 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

  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. Distinct Trafficking of Cell Surface and Endosomal TIM-1 to the Immune Synapse.

    PubMed

    Echbarthi, Meriem; Zonca, Manuela; Mellwig, Rachel; Schwab, Yannick; Kaplan, Gerardo; DeKruyff, Rosemarie H; Roda-Navarro, Pedro; Casasnovas, Jose M

    2015-11-01

    The T cell costimulatory molecule TIM-1 (T cell/transmembrane, mucin and immunoglobulin domain protein 1) sorts mainly to endosomes in lymphoid cells. At difference from the cell surface protein, endosomal TIM-1 translocates to the immune synapse (IS), where it can contribute to antigen-dependent T cell costimulation. TIM-1 ligands increase the amount of cell surface protein, preventing its traffic to the IS. The bipolar sorting of TIM-1 observed during IS formation is determined by differences in its subcellular location, and probably modulates antigen-driven immune responses.

  5. 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

  6. Labeling Cell Surface GPIs and GPI-Anchored Proteins through Metabolic Engineering with Artificial Inositol Derivatives.

    PubMed

    Lu, Lili; Gao, Jian; Guo, Zhongwu

    2015-08-10

    Glycosylphosphatidylinositol (GPI) anchoring of proteins to the cell surface is important for various biological processes, but GPI-anchored proteins are difficult to study. An effective strategy was developed for the metabolic engineering of cell-surface GPIs and GPI-anchored proteins by using inositol derivatives carrying an azido group. The azide-labeled GPIs and GPI-anchored proteins were then tagged with biotin on live cells through a click reaction, which allows further elaboration with streptavidin-conjugated dyes or other molecules. The strategy can be used to label GPI-anchored proteins with various tags for biological studies.

  7. Distinct Trafficking of Cell Surface and Endosomal TIM-1 to the Immune Synapse.

    PubMed

    Echbarthi, Meriem; Zonca, Manuela; Mellwig, Rachel; Schwab, Yannick; Kaplan, Gerardo; DeKruyff, Rosemarie H; Roda-Navarro, Pedro; Casasnovas, Jose M

    2015-11-01

    The T cell costimulatory molecule TIM-1 (T cell/transmembrane, mucin and immunoglobulin domain protein 1) sorts mainly to endosomes in lymphoid cells. At difference from the cell surface protein, endosomal TIM-1 translocates to the immune synapse (IS), where it can contribute to antigen-dependent T cell costimulation. TIM-1 ligands increase the amount of cell surface protein, preventing its traffic to the IS. The bipolar sorting of TIM-1 observed during IS formation is determined by differences in its subcellular location, and probably modulates antigen-driven immune responses. PMID:26332704

  8. Chemistry-enabled methods for the visualization of cell-surface glycoproteins in Metazoans.

    PubMed

    Chuh, Kelly N; Pratt, Matthew R

    2015-10-01

    The majority of cell-surface and secreted proteins are glycosylated, which can directly affect their macromolecular interactions, stability, and localization. Investigating these effects is critical to developing a complete understanding of the role of glycoproteins in fundamental biology and human disease. The development of selective and unique chemical reactions have revolutionized the visualization, identification, and characterization of glycoproteins. Here, we review the chemical methods that have been created to enable the visualization of the major types of cell-surface glycoproteins in living systems, from mammalian cells to whole animals.

  9. 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.

  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. Sorting Motifs in the Cytoplasmic Tail of the Immunomodulatory E3/49K Protein of Species D Adenoviruses Modulate Cell Surface Expression and Ectodomain Shedding.

    PubMed

    Windheim, Mark; Höning, Stefan; Leppard, Keith N; Butler, Larissa; Seed, Christina; Ponnambalam, Sreenivasan; Burgert, Hans-Gerhard

    2016-03-25

    The E3 transcription unit of human species C adenoviruses (Ads) encodes immunomodulatory proteins that mediate direct protection of infected cells. Recently, we described a novel immunomodulatory function for E3/49K, an E3 protein uniquely expressed by species D Ads. E3/49K of Ad19a/Ad64, a serotype that causes epidemic keratokonjunctivitis, is synthesized as a highly glycosylated type I transmembrane protein that is subsequently cleaved, resulting in secretion of its large ectodomain (sec49K). sec49K binds to CD45 on leukocytes, impairing activation and functions of natural killer cells and T cells. E3/49K is localized in the Golgi/trans-Golgi network (TGN), in the early endosomes, and on the plasma membrane, yet the cellular compartment where E3/49K is cleaved and the protease involved remained elusive. Here we show that TGN-localized E3/49K comprises both newly synthesized and recycled molecules. Full-length E3/49K was not detected in late endosomes/lysosomes, but the C-terminal fragment accumulated in this compartment at late times of infection. Inhibitor studies showed that cleavage occurs in a post-TGN compartment and that lysosomotropic agents enhance secretion. Interestingly, the cytoplasmic tail of E3/49K contains two potential sorting motifs, YXXΦ (where Φ represents a bulky hydrophobic amino acid) and LL, that are important for binding the clathrin adaptor proteins AP-1 and AP-2in vitro Surprisingly, mutating the LL motif, either alone or together with YXXΦ, did not prevent proteolytic processing but increased cell surface expression and secretion. Upon brefeldin A treatment, cell surface expression was rapidly lost, even for mutants lacking all known endocytosis motifs. Together with immunofluorescence data, we propose a model for intracellular E3/49K transport whereby cleavage takes place on the cell surface by matrix metalloproteases.

  13. Vasopressin-inducible ubiquitin-specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3.

    PubMed

    Boulkroun, Sheerazed; Ruffieux-Daidié, Dorothée; Vitagliano, Jean-Jacques; Poirot, Olivier; Charles, Roch-Philippe; Lagnaz, Dagmara; Firsov, Dmitri; Kellenberger, Stephan; Staub, Olivier

    2008-10-01

    Adjustment of Na+ balance in extracellular fluids is achieved by regulated Na+ transport involving the amiloride-sensitive epithelial Na+ channel (ENaC) in the distal nephron. In this context, ENaC is controlled by a number of hormones, including vasopressin, which promotes rapid translocation of water and Na+ channels to the plasma membrane and long-term effects on transcription of vasopressin-induced and -reduced transcripts. We have identified a mRNA encoding the deubiquitylating enzyme ubiquitin-specific protease 10 (Usp10), whose expression is increased by vasopressin at both the mRNA and the protein level. Coexpression of Usp10 in ENaC-transfected HEK-293 cells causes a more than fivefold increase in amiloride-sensitive Na+ currents, as measured by whole cell patch clamping. This is accompanied by a three- to fourfold increase in surface expression of alpha- and gamma-ENaC, as shown by cell surface biotinylation experiments. Although ENaC is well known to be regulated by its direct ubiquitylation, Usp10 does not affect the ubiquitylation level of ENaC, suggesting an indirect effect. A two-hybrid screen identified sorting nexin 3 (SNX3) as a novel substrate of Usp10. We show that it is a ubiquitylated protein that is degraded by the proteasome; interaction with Usp10 leads to its deubiquitylation and stabilization. When coexpressed with ENaC, SNX3 increases the channel's cell surface expression, similarly to Usp10. In mCCD(cl1) cells, vasopressin increases SNX3 protein but not mRNA, supporting the idea that the vasopressin-induced Usp10 deubiquitylates and stabilizes endogenous SNX3 and consequently promotes cell surface expression of ENaC.

  14. Sorting Motifs in the Cytoplasmic Tail of the Immunomodulatory E3/49K Protein of Species D Adenoviruses Modulate Cell Surface Expression and Ectodomain Shedding.

    PubMed

    Windheim, Mark; Höning, Stefan; Leppard, Keith N; Butler, Larissa; Seed, Christina; Ponnambalam, Sreenivasan; Burgert, Hans-Gerhard

    2016-03-25

    The E3 transcription unit of human species C adenoviruses (Ads) encodes immunomodulatory proteins that mediate direct protection of infected cells. Recently, we described a novel immunomodulatory function for E3/49K, an E3 protein uniquely expressed by species D Ads. E3/49K of Ad19a/Ad64, a serotype that causes epidemic keratokonjunctivitis, is synthesized as a highly glycosylated type I transmembrane protein that is subsequently cleaved, resulting in secretion of its large ectodomain (sec49K). sec49K binds to CD45 on leukocytes, impairing activation and functions of natural killer cells and T cells. E3/49K is localized in the Golgi/trans-Golgi network (TGN), in the early endosomes, and on the plasma membrane, yet the cellular compartment where E3/49K is cleaved and the protease involved remained elusive. Here we show that TGN-localized E3/49K comprises both newly synthesized and recycled molecules. Full-length E3/49K was not detected in late endosomes/lysosomes, but the C-terminal fragment accumulated in this compartment at late times of infection. Inhibitor studies showed that cleavage occurs in a post-TGN compartment and that lysosomotropic agents enhance secretion. Interestingly, the cytoplasmic tail of E3/49K contains two potential sorting motifs, YXXΦ (where Φ represents a bulky hydrophobic amino acid) and LL, that are important for binding the clathrin adaptor proteins AP-1 and AP-2in vitro Surprisingly, mutating the LL motif, either alone or together with YXXΦ, did not prevent proteolytic processing but increased cell surface expression and secretion. Upon brefeldin A treatment, cell surface expression was rapidly lost, even for mutants lacking all known endocytosis motifs. Together with immunofluorescence data, we propose a model for intracellular E3/49K transport whereby cleavage takes place on the cell surface by matrix metalloproteases. PMID:26841862

  15. Structure, regulation, and putative function of the arginine deiminase system of Streptococcus suis.

    PubMed

    Gruening, Petra; Fulde, Marcus; Valentin-Weigand, Peter; Goethe, Ralph

    2006-01-01

    Streptococcus suis is an important cause of infectious diseases in young pigs. Little is known about the virulence factors or protective antigens of S. suis. Recently, we have identified two proteins of the arginine deiminase system (ADS) of S. suis, which were temperature induced and expressed on the streptococcal surface (N. Winterhoff, R. Goethe, P. Gruening, M. Rohde, H. Kalisz, H. E. Smith, and P. Valentin-Weigand, J. Bacteriol. 184:6768-6776, 2002). In the present study, we analyzed the complete ADS of S. suis. Due to their homologies to the recently published S. gordonii ADS genes, the genes for arginine deiminase, ornithine carbamoyl-transferase, and carbamate kinase, which were previously designated adiS, octS, and ckS, respectively, were renamed arcA, arcB, and arcC, respectively. Our data revealed that arcA, arcB, and arcC of the S. suis ADS are transcribed from an operon (arcABC operon). Additionally, putative ADS-associated genes were cloned and sequenced which, however, did not belong to the arcABC operon. These were the flpS gene upstream of the arcABC operon with homology to the flp transcription regulator of S. gordonii and the arcD, arcT, arcH, and argR genes downstream of the arcABC operon with high homologies to a putative arginine-ornithine antiporter, a putative dipeptidase of S. gordonii, a putative beta-N-acetylhexosaminidase of S. pneumoniae, and a putative arginine repressor of S. gordonii, respectively. The transcriptional start point of the arcABC operon was determined, and promoter analysis provided evidence that multiple factors contribute to the regulation of the ADS. Thus, a putative binding site for a transcription regulator of the Crp/Fnr family, an ArgR-binding site, and two cis-acting catabolite response elements were identified in the promoter-operator region of the operon. Consistent with this, we could demonstrate that the ADS of S. suis is inducible by arginine and reduced O2 tension and subject to carbon catabolite

  16. Amino acid sequence of a putative sodium channel expressed in the giant axon of the squid Loligo opalescens.

    PubMed Central

    Rosenthal, J J; Gilly, W F

    1993-01-01

    A full-length cDNA encoding a putative Na+ channel (GFLN1) has been cloned from a library prepared from the stellate ganglion of Loligo opalescens. The cDNA encodes a predicted protein of 1784 amino acids. Regions of the GFLN1 protein with defined functional importance (membrane span S4, the SS1 and SS2 segments, and interdomain III-IV) are highly conserved among all vertebrate Na+ channel alpha-subunit structures. Northern blot hybridization and RNase protection assays verify that mRNA corresponding to GFLN1 is expressed in neurons of the giant fiber lobe that form the giant axon. We propose that GFLN1 encodes the Na+ channel that has been extensively studied in the squid axon. Images Fig. 3 PMID:8234251

  17. CbrA is a stationary-phase regulator of cell surface physiology and legume symbiosis in Sinorhizobium meliloti.

    PubMed

    Gibson, Katherine E; Campbell, Gordon R; Lloret, Javier; Walker, Graham C

    2006-06-01

    Sinorhizobium meliloti produces an exopolysaccharide called succinoglycan that plays a critical role in promoting symbiosis with its host legume, alfalfa (Medicago sativa). We performed a transposon mutagenesis and screened for mutants with altered succinoglycan production and a defect in symbiosis. In this way, we identified a putative two-component histidine kinase associated with a PAS sensory domain, now designated CbrA (calcofluor-bright regulator A). The cbrA::Tn5 mutation causes overproduction of succinoglycan and results in increased accumulation of low-molecular-weight forms of this exopolysaccharide. Our results suggest the cbrA::Tn5 allele leads to this succinoglycan phenotype through increased expression of exo genes required for succinoglycan biosynthesis and modification. Interestingly, CbrA-dependent regulation of exo and exs genes is observed almost exclusively during stationary-phase growth. The cbrA::Tn5 mutant also has an apparent cell envelope defect, based on increased sensitivity to a number of toxic compounds, including the bile salt deoxycholate and the hydrophobic dye crystal violet. Growth of the cbrA mutant is also slowed under oxidative-stress conditions. The CbrA-regulated genes exsA and exsE encode putative inner membrane ABC transporters with a high degree of similarity to lipid exporters. ExsA is homologous to the Escherichia coli MsbA protein, which is required for lipopolysaccharide transport, while ExsE is a member of the eukaryotic family of ABCD/hALD peroxisomal membrane proteins involved in transport of very long-chain fatty acids, which are a unique component of the lipopolysaccharides of alphaproteobacteria. Thus, CbrA could play a role in regulating the lipopolysaccharide or lipoprotein components of the cell envelope. PMID:16740957

  18. 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

  19. 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

  20. PNA-encoded chemical libraries.

    PubMed

    Zambaldo, Claudio; Barluenga, Sofia; Winssinger, Nicolas

    2015-06-01

    Peptide nucleic acid (PNA)-encoded chemical libraries along with DNA-encoded libraries have provided a powerful new paradigm for library synthesis and ligand discovery. PNA-encoding stands out for its compatibility with standard solid phase synthesis and the technology has been used to prepare libraries of peptides, heterocycles and glycoconjugates. Different screening formats have now been reported including selection-based and microarray-based methods that have yielded specific ligands against diverse target classes including membrane receptors, lectins and challenging targets such as Hsp70.

  1. 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.

  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. 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.

  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. 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

  6. 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

  7. 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.

  8. 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

  9. Monocyte cell surface glycosaminoglycans positively modulate IL-4-induced differentiation toward dendritic cells.

    PubMed

    den Dekker, Els; Grefte, Sander; Huijs, Tonnie; ten Dam, Gerdy B; Versteeg, Elly M M; van den Berk, Lieke C J; Bladergroen, Bellinda A; van Kuppevelt, Toin H; Figdor, Carl G; Torensma, Ruurd

    2008-03-15

    IL-4 induces the differentiation of monocytes toward dendritic cells (DCs). The activity of many cytokines is modulated by glycosaminoglycans (GAGs). In this study, we explored the effect of GAGs on the IL-4-induced differentiation of monocytes toward DCs. IL-4 dose-dependently up-regulated the expression of DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), CD80, CD206, and CD1a. Monocytes stained positive with Abs against heparan sulfate (HS) and chondroitin sulfate (CS) B (CSB; dermatan sulfate), but not with Abs that recognize CSA, CSC, and CSE. Inhibition of sulfation of monocyte/DC cell surface GAGs by sodium chlorate reduced the reactivity of sulfate-recognizing single-chain Abs. This correlated with hampered IL-4-induced DC differentiation as evidenced by lower expression of DC-SIGN and CD1a and a decreased DC-induced PBL proliferation, suggesting that sulfated monocyte cell surface GAGs support IL-4 activity. Furthermore, removal of cell surface chondroitin sulfates by chondroitinase ABC strongly impaired IL-4-induced STAT6 phosphorylation, whereas removal of HS by heparinase III had only a weak inhibitory effect. IL-4 bound to heparin and CSB, but not to HS, CSA, CSC, CSD, and CSE. Binding of IL-4 required iduronic acid, an N-sulfate group (heparin) and specific O sulfates (CSB and heparin). Together, these data demonstrate that monocyte cell surface chondroitin sulfates play an important role in the IL-4-driven differentiation of monocytes into DCs.

  10. 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

  11. Molecular regulation of lysophosphatidic acid receptor 1 trafficking to the cell surface.

    PubMed

    Zhao, Jing; Wei, Jianxin; Bowser, Rachel K; Dong, Su; Xiao, Shuqi; Zhao, Yutong

    2014-11-01

    The lysophosphatidic acid receptor 1 (LPA1), a G-protein coupled receptor, regulates cell proliferation, migration, and cytokine release. Here, we investigate the molecular signature of LPA1 trafficking to the cell surface. The overexpressed LPA1 with a C-terminal V5 tag (LPA1-V5) is majorly expressed on the cell surface, while two deletion mutants (C320 and ∆84-87) failed to be trafficked to the cell surface. Further, site-directed mutagenesis analysis of the LPA1 revealed that Ile325, Tyr85, and Leu87 within these two fragments regulate LPA1 maturation and trafficking to the cell surface. Over-expression of Sar1, a component of coat protein complex II (COPII), enhances glycosylation of LPA1 wild type, but not these mutants. The mutants of LPA1 are majorly localized in the endoplasmic reticulum (ER) and exhibit a higher binding affinity to heat shock protein 70 (Hsp70), when compared to the LPA1 wild type. Further, we found that all these mutants failed to increase phosphorylation of Erk, and the cytokine release in response to LPA treatment. These results suggest that Ile325, Tyr85, and Leu87 within LPA1 are essential for LPA1 protein properly folding in the ER.

  12. The transmembrane protein of HIV-1 primary isolates modulates cell surface expression of their envelope glycoproteins.

    PubMed

    Lebigot, S; Roingeard, P; Thibault, G; Lemiale, F; Verrier, B; Barin, F; Brand, D

    2001-11-10

    We have recently shown that the level of cell surface expression of envelope glycoproteins derived from various human immunodeficiency virus type 1 (HIV-1) primary isolates (PI) was lower than those of envelope glycoproteins derived from T-cell laboratory-adapted (TCLA) HIV-1 (D. Brand et al., 2000, Virology 271, 350-362). We investigated this phenomenon by comparing the cell surface expression of chimeric envelope glycoproteins constructed by swapping the gp120 surface and gp41 transmembrane glycoproteins of the TCLA HIV-1MN and the PI HIV-1(133), HIV-1G365, or HIV-1EFRA. We found that each chimeric envelope construct had a cell surface-specific pattern of expression similar to that of the parental envelope glycoproteins corresponding to the gp41. Thus, the difference in cell surface expression observed between TCLA viruses and various PI is probably due to a signal located in gp41. Identification of this signal may be important for the design of PI envelope-derived immunogens and may increase our understanding of the mechanisms by which HIV-1 escapes from the immune system.

  13. Increased cell surface hydrophobicity of a Serratia marcescens NS 38 mutant lacking wetting activity.

    PubMed

    Bar-Ness, R; Avrahamy, N; Matsuyama, T; Rosenberg, M

    1988-09-01

    The cell surface hydrophobicity of Serratia marcescens appears to be an important factor in its adhesion to and colonization of various interfaces. The cell surface components responsible for mediating the hydrophobicity of S. marcescens have not been completely elucidated, but may include prodigiosin and other factors. In the present report we have investigated the potential role of serratamolide, an amphipathic aminolipid present on the surfaces of certain S. marcescens strains, in modulating cell surface hydrophobicity. The hydrophobic properties of a serratamolide-producing strain (NS 38) were compared with those of a serratamolide-deficient mutant (NS 38-9) by monitoring the kinetics of adhesion to hexadecane. Serratamolide production was monitored by thin-layer chromatography and the wetting activity of washed-cell suspensions on polystyrene. Wild-type NS 38 cells were far less hydrophobic than the serratamolide-deficient mutant cells were; the removal coefficients were 48 min-1 for the mutant, as compared with only 18 min-1 for the wild type. The data suggest that the presence of serratamolide on S. marcescens cells results in a reduction in hydrophobicity, presumably by blocking hydrophobic sites on the cell surface.

  14. 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

  15. Escherichia coli K-12 YfgF is an anaerobic cyclic di-GMP phosphodiesterase with roles in cell surface remodelling and the oxidative stress response.

    PubMed

    Lacey, Melissa M; Partridge, Jonathan D; Green, Jeffrey

    2010-09-01

    The Escherichia coli K-12 yfgF gene encodes a protein with domains associated with cyclic di-GMP signalling: GGDEF (associated with diguanylate cyclase activity) and EAL (associated with cyclic di-GMP phosphodiesterase activity). Here, it is shown that yfgF is expressed under anaerobic conditions from a class II FNR (regulator of fumarate and nitrate reduction)-dependent promoter. Anaerobic expression of yfgF is greatest in stationary phase, and in cultures grown at 28 degrees C, suggesting that low growth rates promote yfgF expression. Mutation of yfgF resulted in altered cell surface properties and enhanced sensitivity when anaerobic cultures were exposed to peroxides. The purified YfgF GGDEF-EAL (YfgF(GE)) and EAL (YfgF(E)) domains possessed cyclic di-GMP-specific phosphodiesterase activity, but lacked diguanylate cyclase activity. However, the catalytically inactive GGDEF domain was required for YfgF(GE) dimerization and enhanced cyclic di-GMP phosphodiesterase activity in the presence of physiological concentrations of Mg(2+). The cyclic di-GMP phosphodiesterase activity of YfgF(GE) and YfgF(E) was inhibited by the product of the reaction, 5'-phosphoguanylyl-(3'-5')-guanosine (pGpG). Thus, it is shown that the yfgF gene encodes an anaerobic cyclic di-GMP phosphodiesterase that is involved in remodelling the cell surface of E. coli K-12 and in the response to peroxide shock, with implications for integrating three global regulatory networks, i.e. oxygen regulation, cyclic di-GMP signalling and the oxidative stress response.

  16. 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.

  17. Identification of Putative Receptors for the Novel Adipokine CTRP3 Using Ligand-Receptor Capture Technology

    PubMed Central

    Li, Ying; Ozment, Tammy; Wright, Gary L.

    2016-01-01

    C1q TNF Related Protein 3 (CTRP3) is a member of a family of secreted proteins that exert a multitude of biological effects. Our initial work identified CTRP3’s promise as an effective treatment for Nonalcoholic fatty liver disease (NAFLD). Specifically, we demonstrated that mice fed a high fat diet failed to develop NAFLD when treated with CTRP3. The purpose of this current project is to identify putative receptors which mediate the hepatic actions of CTRP3. Methods We used Ligand-receptor glycocapture technology with TriCEPS™-based ligand-receptor capture (LRC-TriCEPS; Dualsystems Biotech AG). The LRC-TriCEPS experiment with CTRP3-FLAG protein as ligand and insulin as a control ligand was performed on the H4IIE rat hepatoma cell line. Results Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis (FACS) demonstrated successful oxidation and crosslinking of CTRP3-TriCEPS and Insulin-TriCEPS complexes to cell surface glycans. Demonstrating the utility of TriCEPS under these conditions, the insulin receptor was identified in the control dataset. In the CTRP3 treated cells a total enrichment of 261 peptides was observed. From these experiments 5 putative receptors for CTRP3 were identified with two reaching statistically significance: Lysosomal-associated membrane protein 1 (LAMP-1) and Lysosome membrane protein 2 (LIMP II). Follow-up Co-immunoprecipitation analysis confirmed the association between LAMP1 and CTRP3 and further testing using a polyclonal antibody to block potential binding sites of LAMP1 prevented CTRP3 binding to the cells. Conclusion The LRC-TriCEPS methodology was successful in identifying potential novel receptors for CTRP3. Relevance The identification of the receptors for CTRP3 are important prerequisites for the development of small molecule drug candidates, of which none currently exist, for the treatment NAFLD. PMID:27727322

  18. A27L protein mediates vaccinia virus interaction with cell surface heparan sulfate.

    PubMed

    Chung, C S; Hsiao, J C; Chang, Y S; Chang, W

    1998-02-01

    Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.

  19. 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

  20. Selective labelling of cell-surface proteins using CyDye DIGE Fluor minimal dyes.

    PubMed

    Hagner-McWhirter, Asa; Winkvist, Maria; Bourin, Stephanie; Marouga, Rita

    2008-01-01

    Surface proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan DIGE technology and analysis of protein expression changes using DeCyder 2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

  1. [Immunoglobulin genes encoding antibodies directed to oncodevelopmental carbohydrate antigens].

    PubMed

    Zenita, K; Yago, K; Fujimoto, E; Kannagi, R

    1990-07-01

    We investigated the immunoglobulin genes which encode the variable region of the monoclonal antibodies directed to the onco-developmental carbohydrate antigens such SSEA-1, fucosyl SSEA-1, SSEA-3 and SSEA-4. The VH region of these antibodies was preferentially encoded by the gene members of the X24, VH7183 and Q52 families, the families which are known to be located at the 3'-end region of the murine germ line VH gene. This result is interesting particularly when considering that the members of the 3'-end VH families are known to be preferentially expressed in embryonic B lymphocytes by an intrinsic genetic program. The comparative study of the nucleic acid sequences of mRNAs encoding these antibodies and the sequences of the corresponding germ line VH genes disclosed that the sequences encoding the antibodies contain no mutation from the germ line VH genes, or contain only a few somatic mutations, which are thought to be insignificant for the reactivity of the antibodies to the nominal antigens. These results imply that some of the embryonic B lymphocytes that express the unmutated germ line VH genes of the 3'-end families can be reactive with embryonic carbohydrate antigens, albeit rearranged with appropriate D-JH gene segments, and coupled with proper light chains. The VH region of the syngenic monoclonal anti-idiotypic antibodies directed to these anti-carbohydrate antibodies were also encoded preferentially by the members of the 3'-end VH families. We propose here that a part of the virgin embryonic B lymphocytes, which express the antibody encoded by the gene members of the 3'-end VH families at the cell surface, will be stimulated by the embryonic carbohydrate antigens which are abundantly present in the internal milieu of the embryo. The clonally expanded B lymphocytes, in turn, will facilitate the proliferation of other populations of embryonic B lymphocytes expressing the corresponding anti-idiotypic antibodies, which are also encoded by the gene members

  2. Deubiquitinating activity of Sdu1, a putative member of the PPPDE peptidase family, in Schizosaccharomyces pombe.

    PubMed

    Kim, Yunsik; Jo, Hannah; Lim, Chang-Jin

    2013-12-01

    The Schizosaccharomyces pombe sdu⁺ gene encoding a putative member of the PPPDE (Permuted Papain fold Peptidases of DsRNA viruses and Eukaryotes) superfamily was cloned into an Escherichia coli - yeast shuttle vector pRS316, resulting in the recombinant plasmid pYSTP. The determined nucleotide sequence carries 1207 bp, which would encode a protein of 201 amino acid residues. The S. pombe cells harboring pYSTP contained higher sdu1⁺ mRNA and deubiquitinating activity levels than the vector control cells, indicating that the sdu1⁺ gene is functioning. They exhibited a better growth in normal rich medium than the vector control cells. When shifted into the fresh medium containing hydrogen peroxide, menadione, or sodium nitroprusside, the S. pombe cells harboring pYSTP were able to grow reasonably well, while the growth of the vector control cells was arrested. The reactive oxygen species and total glutathione levels of the S. pombe cells harboring pYSTP were lower and higher than those of the vector control cells under the same stressful conditions, respectively. They exhibited a lower nitric oxide level than the vector control cells when subjected to sodium nitroprusside. Taken together, the sdu1⁺ gene encodes an actual protein having deubiquitinating activity and is involved in the response against oxidative and nitrosative stresses in S. pombe.

  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. Saturation mapping of QTL regions and identification of putative candidate genes for drought tolerance in rice.

    PubMed

    Nguyen, T T T; Klueva, N; Chamareck, V; Aarti, A; Magpantay, G; Millena, A C M; Pathan, M S; Nguyen, H T

    2004-08-01

    We have developed 85 new markers (50 RFLPs, 5 SSRs, 12 DD cDNAs, 9 ESTs, 8 HSP-encoding cDNAs and one BSA-derived AFLP marker) for saturation mapping of QTL regions for drought tolerance in rice, in our efforts to identify putative candidate genes. Thirteen of the markers were localized in the close vicinity of the targeted QTL regions. Fifteen of the additional markers mapped, respectively, inside one QTL region controlling osmotic adjustment on chromosome 3 ( oa3.1) and 14 regions that affect root traits on chromosomes 1, 2, 4, 5, 6, 7, 8, 9, 10 and 12. Differential display was used to identify more putative candidate genes and to saturate the QTL regions of the genetic map. Eleven of the isolated cDNA clones were found to be derived from drought-inducible genes. Two of them were unique and did not match any genes in the GenBank, while nine were highly similar to cDNAs encoding known proteins, including a DnaJ-related protein, a zinc-finger protein, a protease inhibitor, a glutathione-S-transferase, a DNA recombinase, and a protease. Twelve new cDNA fragments were mapped onto the genetic linkage map; seven of these mapped inside, or in close proximity to, the targeted QTL regions determining root thickness and osmotic adjustment capacity. The gene I12A1, which codes for a UDP-glucose 4-epimerase homolog, was identified as a putative target gene within the prt7.1/brt7.1 QTL region, as it is involved in the cell wall biogenesis pathway and hence may be implicated in modulating the ability of rice roots to penetrate further into the substratum when exposed to drought conditions. RNAs encoding elongation factor 1beta, a DnaJ-related protein, and a homolog of wheat zinc-finger protein were more prominently induced in the leaves of IR62266 (the lowland rice parent of the mapping materials used) than in those of CT9993 (the upland rice parent) under drought conditions. Homologs of 18S ribosomal RNA, and mRNAs for a multiple-stress induced zinc-finger protein, a protease

  5. Saturation mapping of QTL regions and identification of putative candidate genes for drought tolerance in rice.

    PubMed

    Nguyen, T T T; Klueva, N; Chamareck, V; Aarti, A; Magpantay, G; Millena, A C M; Pathan, M S; Nguyen, H T

    2004-08-01

    We have developed 85 new markers (50 RFLPs, 5 SSRs, 12 DD cDNAs, 9 ESTs, 8 HSP-encoding cDNAs and one BSA-derived AFLP marker) for saturation mapping of QTL regions for drought tolerance in rice, in our efforts to identify putative candidate genes. Thirteen of the markers were localized in the close vicinity of the targeted QTL regions. Fifteen of the additional markers mapped, respectively, inside one QTL region controlling osmotic adjustment on chromosome 3 ( oa3.1) and 14 regions that affect root traits on chromosomes 1, 2, 4, 5, 6, 7, 8, 9, 10 and 12. Differential display was used to identify more putative candidate genes and to saturate the QTL regions of the genetic map. Eleven of the isolated cDNA clones were found to be derived from drought-inducible genes. Two of them were unique and did not match any genes in the GenBank, while nine were highly similar to cDNAs encoding known proteins, including a DnaJ-related protein, a zinc-finger protein, a protease inhibitor, a glutathione-S-transferase, a DNA recombinase, and a protease. Twelve new cDNA fragments were mapped onto the genetic linkage map; seven of these mapped inside, or in close proximity to, the targeted QTL regions determining root thickness and osmotic adjustment capacity. The gene I12A1, which codes for a UDP-glucose 4-epimerase homolog, was identified as a putative target gene within the prt7.1/brt7.1 QTL region, as it is involved in the cell wall biogenesis pathway and hence may be implicated in modulating the ability of rice roots to penetrate further into the substratum when exposed to drought conditions. RNAs encoding elongation factor 1beta, a DnaJ-related protein, and a homolog of wheat zinc-finger protein were more prominently induced in the leaves of IR62266 (the lowland rice parent of the mapping materials used) than in those of CT9993 (the upland rice parent) under drought conditions. Homologs of 18S ribosomal RNA, and mRNAs for a multiple-stress induced zinc-finger protein, a protease

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

    PubMed Central

    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

    2016-01-01

    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

  7. Four transcripts encode glucose 6-phosphate dehydrogenase (G6PDH) 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 characterization of four transcripts (G6PDH-A, -B, -C, and -D) that putatively encode the enzyme in the southern cattle tick, Rhipicephalus (Boophilus) microplus. The ...

  8. 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...

  9. In silicio search for genes encoding peroxisomal proteins in Saccharomyces cerevisiae.

    PubMed

    Kal, A J; Hettema, E H; van den Berg, M; Koerkamp, M G; van Ijlst, L; Distel, B; Tabak, H F

    2000-01-01

    The biogenesis of peroxisomes involves the synthesis of new proteins that after, completion of translation, are targeted to the organelle by virtue of peroxisomal targeting signals (PTS). Two types of PTSs have been well characterized for import of matrix proteins (PTS1 and PTS2). Induction of the genes encoding these matrix proteins takes place in oleate-containing medium and is mediated via an oleate response element (ORE) present in the region preceding these genes. The authors have searched the yeast genome for OREs preceding open reading frames (ORFs), and for ORFs that contain either a PTS1 or PTS2. Of the ORFs containing an ORE, as well as either a PTS1 or a PTS2, many were known to encode bona fide peroxisomal matrix proteins. In addition, candidate genes were identified as encoding putative new peroxisomal proteins. For one case, subcellular location studies validated the in silicio prediction. This gene encodes a new peroxisomal thioesterase.

  10. The melanoma proteoglycan: restricted expression on microspikes, a specific microdomain of the cell surface

    PubMed Central

    1986-01-01

    A cell surface chondroitin sulfate proteoglycan associated with human melanomas and defined by mAb's F24.47 and 48.7 has been characterized biochemically and localized by indirect immunogold electron microscopy. These antibodies recognize distinct epitopes on the intact proteoglycan. In addition, mAb 48.7 also recognizes an epitope on a 250,000-D glycoprotein and is therefore similar to antibody 9.2.27 (described by Bumol, T.F., and R.A. Reisfeld, 1982, Proc. Natl. Acad. Sci. USA., 79:1245-1249). Furthermore, it was shown that the glycosaminoglycan chains released by alkaline borohydride treatment of the proteoglycan recognized by mAb 48.7 had a size of approximately 60,000 D. Since the intact proteoglycan was estimated to be 420,000 D, there are probably three chondroitin sulfate chains attached to the 250,000-D core glycoprotein. Furthermore, an oligosaccharide fraction containing 42% of the 3H activity (glucosamine as precursor) was isolated. Immunolocalization studies using whole-mount electron microscopy revealed that the chondroitin sulfate proteoglycan was present almost exclusively on microspikes, a microdomain of the melanoma cell surface. These processes were present as 1-2-micron structures on the upper cell surface and as longer (up to 20 micron) structures at the cell periphery. Peripheral microspikes were involved in the initial interactions between adjacent cells and formed complex footpads that made contact with the substratum. Immunogold-labeled cells were also thin sectioned and the specific localization of the chondroitin sulfate proteoglycan antigen was quantitated. The data confirmed the results of whole-mount microscopy and demonstrated a statistically significant association of the antigen with the microspike processes as compared with other areas of the cell surface. By using two different mAb's (48.7 and F24.47) that recognize epitopes on either the core glycoprotein or the intact proteoglycan, respectively, we have demonstrated that both

  11. 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

  12. The post-translational modification of the Clostridium difficile flagellin affects motility, cell surface properties and virulence.

    PubMed

    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

    2014-10-01

    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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. Optimum design of amphiphilic polymers bearing hydrophobic groups for both cell surface ligand presentation and intercellular cross-linking.

    PubMed

    Takeo, Masafumi; Li, Cuicui; Matsuda, Masayoshi; Nagai, Hiroko; Hatanaka, Wataru; Yamamoto, Tatsuhiro; Kishimura, Akihiro; Mori, Takeshi; Katayama, Yoshiki

    2015-01-01

    Amphiphilic polymers bearing hydrophobic alkyl groups are expected to be applicable for both ligand presentation on the cell surface and intercellular crosslinking. To explore the optimum design for each application, we synthesized eight different acyl-modified dextrans with varying molecular weight, alkyl length, and alkyl modification degree. We found that the behenate-modified polymers retained on the cell surface longer than the palmitate-modified ones. Since the polymers were also modified with biotin, streptavidin can be presented on the cell surface through biotin-streptavidin recognition. The duration of streptavidin on the cell surface is longer in the behenate-modified polymer than the palmitate-modified one. As for the intercellular crosslinking, the palmitate-modified polymers were more efficient than the behenate-modified polymers. The findings in this research will be helpful to design the acyl-modified polymers for the cell surface engineering.

  19. Multidimensionally encoded magnetic resonance imaging.

    PubMed

    Lin, Fa-Hsuan

    2013-07-01

    Magnetic resonance imaging (MRI) typically achieves spatial encoding by measuring the projection of a q-dimensional object over q-dimensional spatial bases created by linear spatial encoding magnetic fields (SEMs). Recently, imaging strategies using nonlinear SEMs have demonstrated potential advantages for reconstructing images with higher spatiotemporal resolution and reducing peripheral nerve stimulation. In practice, nonlinear SEMs and linear SEMs can be used jointly to further improve the image reconstruction performance. Here, we propose the multidimensionally encoded (MDE) MRI to map a q-dimensional object onto a p-dimensional encoding space where p > q. MDE MRI is a theoretical framework linking imaging strategies using linear and nonlinear SEMs. Using a system of eight surface SEM coils with an eight-channel radiofrequency coil array, we demonstrate the five-dimensional MDE MRI for a two-dimensional object as a further generalization of PatLoc imaging and O-space imaging. We also present a method of optimizing spatial bases in MDE MRI. Results show that MDE MRI with a higher dimensional encoding space can reconstruct images more efficiently and with a smaller reconstruction error when the k-space sampling distribution and the number of samples are controlled.

  20. Cell surface localised Hsp70 is a cancer specific regulator of clathrin-independent endocytosis.

    PubMed

    Nimmervoll, Benedikt; Chtcheglova, Lilia A; Juhasz, Kata; Cremades, Nunilo; Aprile, Francesco A; Sonnleitner, Alois; Hinterdorfer, Peter; Vigh, Laszlo; Preiner, Johannes; Balogi, Zsolt

    2015-09-14

    The stress inducible heat shock protein 70 (Hsp70) is present specifically on the tumour cell surface yet without a pro-tumour function revealed. We show here that cell surface localised Hsp70 (sHsp70) supports clathrin-independent endocytosis (CIE) in melanoma models. Remarkably, ability of Hsp70 to cluster on lipid rafts in vitro correlated with larger nano-domain sizes of sHsp70 in high sHsp70 expressing cell membranes. Interfering with Hsp70 oligomerisation impaired sHsp70-mediated facilitation of endocytosis. Altogether our findings suggest that a sub-fraction of sHsp70 co-localising with lipid rafts enhances CIE through oligomerisation and clustering. Targeting or utilising this tumour specific mechanism may represent an additional benefit for anti-cancer therapy.

  1. 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.

  2. 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

  3. 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

  4. 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

  5. Cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans

    PubMed Central

    1992-01-01

    The role of cell surface heparan sulfate in herpes simplex virus (HSV) infection was investigated using CHO cell mutants defective in various aspects of glycosaminoglycan synthesis. Binding of radiolabeled virus to the cells and infection were assessed in mutant and wild-type cells. Virus bound efficiently to wild-type cells and initiated an abortive infection in which immediate-early or alpha viral genes were expressed, despite limited production of late viral proteins and progeny virus. Binding of virus to heparan sulfate-deficient mutant cells was severely impaired and mutant cells were resistant to HSV infection. Intermediate levels of binding and infection were observed for a CHO cell mutant that produced undersulfated heparan sulfate. These results show that heparan sulfate moieties of cell surface proteoglycans serve as receptors for HSV. PMID:1310996

  6. Bacterial cell surface display for epitope mapping of hepatitis C virus core antigen.

    PubMed

    Kang, Su-Min; Rhee, Jin-Kyu; Kim, Eui-Joong; Han, Kwang-Hyub; Oh, Jong-Won

    2003-09-26

    Cell surface expression of protein has been widely used to display enzymes and antigens. Here we show that Pseudomonas syringae ice nucleation protein with a deletion of internal repeating domain (INC) can be used in Escherichia coli to display peptide in a conformationally active form on the outside of the folded protein by fusing to the C-terminus of INC. Diagnostic potential of this technology was demonstrated by effective mapping of antigenic epitopes derived from hepatitis C virus (HCV) core protein. Amino acids 1-38 and 26-53 of HCV core protein were found to react more sensitively in a native conformation with the HCV patient sera than commercial diagnostic antigen, c22p (amino acids 10-53) by display-ELISA. These results demonstrate that the bacterial cell surface display using INC is useful for peptide presentation and thus epitope mapping of antigen. PMID:14553932

  7. 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.

  8. Internalization and Trafficking of Cell Surface Proteoglycans and Proteoglycan-Binding Ligands

    PubMed Central

    Payne, Christine K.; Jones, Sara A.; Chen, Chen; Zhuang, Xiaowei

    2009-01-01

    Using multi-color live cell imaging in combination with biochemical assays we have investigated an endocytic pathway mediated by cell surface proteoglycans, primary receptors for many cationic ligands. We have characterized this pathway for a variety of proteoglycan-binding ligands including cationic polymers, lipids, and polypeptides. Following clathrin- and caveolin-independent, but flotillin- and dynamin-dependent internalization, proteoglycan-bound ligands associate with flotillin-1-positive vesicles and are efficiently trafficked to late endosomes. The route to late endosomes differs considerably from that following clathrin-mediated endocytosis. The proteoglycan-dependent pathway to late endosomes does not require microtubule-dependent transport or PI(3)K-dependent sorting from early endosomes. The pathway taken by these ligands is identical to that taken by an antibody against heparan sulfate proteoglycans, suggesting this mechanism may be used generally by cell surface proteoglycans and proteoglycan-binding ligands without secondary receptors. PMID:17394486

  9. 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.

  10. 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.

  11. tincar encodes a novel transmembrane protein expressed in the Tinman-expressing cardioblasts of Drosophila.

    PubMed

    Hirota, Yuki; Sawamoto, Kazunobu; Okano, Hideyuki

    2002-12-01

    We cloned and characterized the Drosophila gene, tincar (tinc), which encodes a novel protein with eight putative transmembrane domains. The tinc mRNA was expressed specifically in four of the six pairs of cardioblasts in each segment, in a pattern identical to that of tinman (tin), a homeobox gene required for the specification of the dorsal vessel. In the non-Tin-expressing pairs of cardioblasts, tinc transcription seemed to be repressed by Seven-up. PMID:14516698

  12. tincar encodes a novel transmembrane protein expressed in the Tinman-expressing cardioblasts of Drosophila.

    PubMed

    Hirota, Yuki; Sawamoto, Kazunobu; Okano, Hideyuki

    2002-12-01

    We cloned and characterized the Drosophila gene, tincar (tinc), which encodes a novel protein with eight putative transmembrane domains. The tinc mRNA was expressed specifically in four of the six pairs of cardioblasts in each segment, in a pattern identical to that of tinman (tin), a homeobox gene required for the specification of the dorsal vessel. In the non-Tin-expressing pairs of cardioblasts, tinc transcription seemed to be repressed by Seven-up. PMID:12617821

  13. 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

  14. 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

  15. Sweets for a bitter end: lung cancer cell surface protein glycosylation mediates metastatic colonization

    PubMed Central

    Arnal-Estapé, Anna; Nguyen, Don X.

    2015-01-01

    Summary Glycosylation is one of the most predominant forms of cell surface protein modifications and yet its de-regulation in cancer and contribution to tumor microenvironment interactions remains poorly understood. In this issue of Cancer Discovery, Reticker-Flynn and Bhatia characterize an enzymatic switch in lung cancer cells that triggers aberrant surface protein glycosylation patterns, adhesion to lectins on the surface of inflammatory cells, and subsequent metastatic colonization of the liver. PMID:25656895

  16. Revealing the fate of cell surface human P-glycoprotein (ABCB1): The lysosomal degradation pathway.

    PubMed

    Katayama, Kazuhiro; Kapoor, Khyati; Ohnuma, Shinobu; Patel, Atish; Swaim, William; Ambudkar, Indu S; Ambudkar, Suresh V

    2015-10-01

    P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7±1.1 h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1±0.5 h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50 h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.

  17. 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

  18. Adhesion of phospholipid vesicles to Chinese hamster fibroblasts: Role of cell surface proteins

    PubMed Central

    Pagano, RE; Takeichi, M

    1977-01-01

    The adhesion of artificially generated lipid membrane vesicles to Chinese hamster V79 fibroblasts in suspension was used as a model system for studying membrane interactions. Below their gel-liquid crystalline phase transition temperature, vesicles comprised of dipalmitoyl lecithin (DPL) or dimyristoyl lecithin (DML) absorbed to the surfaces of EDTA- dissociated cells. These adherent vesicles could not be removed by repeated washings of the treated cells but could be released into the medium by treatment with trypsin. EM autoradiographic studies of cells treated with[(3)H]DML or [(3)H]DPL vesicles showed that most of the radioactive lipids were confined to the cell periphery. Scanning electron microscopy and fluorescence microscopy further confirmed the presence of adherent vesicles at the cell surface. Adhesion of DML or DPL vesicles to EDTA-dissociated cells modified the lactoperoxidase-catalyzed iodination pattern of the cell surface proteins; the inhibition of labeling of two proteins with an approximately 60,000- dalton mol wt was particularly evident. Incubation of cells wit h (3)H-lipid vesicles followed by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis showed that some of the (3)H-lipid migrated preferentially with these approximately 60,000-mol wt proteins. Studies of the temperature dependence of vesicle uptake and subsequent release by trypsin showed that DML or DPL vesicle adhesion to EDTA- dissociated cells increased with decreasing temperatures. In contrast, cells trypsinized before incubation with vesicles showed practically no temperature dependence of vesicle uptake. These results suggest two pathways for adhesion of lipid vesicles to the cell surface-a temperature-sensitive one involving cell surface proteins, and a temperature-independent one. These findings are discussed in terms of current models for cell-cell interactions. PMID:407233

  19. Isolation of pigmented and nonpigmented mutants of Serratia marcescens with reduced cell surface hydrophobicity

    SciTech Connect

    Rosenberg, M.

    1984-10-01

    Enrichment for nonhydrophobic mutants of Serratia marcescens yielded two types: (i) a nonpigmented mutant which exhibited partial hydrophobic characteristics compared with the wild type, as determined by adherence to hexadecane and polystyrene; and (ii) a pigmented, nonhydrophobic mutant whose colonies were translucent with respect to those of the wild type. The data suggest that the pronounced cell surface hydrophobicity of the wild type is mediated by a combination of several surface f

  20. Trafficking of cell surface beta-amyloid precursor protein: retrograde and transcytotic transport in cultured neurons

    PubMed Central

    1995-01-01

    Amyloid beta-protein (A beta), the principal constituent of senile plaques seen in Alzheimer's disease (AD), is derived by proteolysis from the beta-amyloid precursor protein (beta PP). The mechanism of A beta production in neurons, which are hypothesized to be a rich source of A beta in brain, remains to be defined. In this study, we describe a detailed localization of cell surface beta PP and its subsequent trafficking in primary cultured neurons. Full-length cell surface beta PP was present primarily on perikarya and axons, the latter with a characteristic discontinuous pattern. At growth cones, cell surface beta PP was inconsistently detected. By visualizing the distribution of beta PP monoclonal antibodies added to intact cultures, beta PP was shown to be internalized from distal axons or terminals and retrogradely transported back to perikarya in organelles which colocalized with fluid-phase endocytic markers. Retrograde transport of beta PP was shown in both hippocampal and peripheral sympathetic neurons, the latter using a compartment culture system that isolated cell bodies from distal axons and terminals. In addition, we demonstrated that beta PP from distal axons was transcytotically transported to the surface of perikarya from distal axons in sympathetic neurons. Indirect evidence of this transcytotic pathway was obtained in hippocampal neurons using antisense oligonucleotide to the kinesin heavy chain to inhibit anterograde beta PP transport. Taken together, these results demonstrate novel aspects of beta PP trafficking in neurons, including retrograde axonal transport and transcytosis. Moreover, the axonal predominance of cell surface beta PP is unexpected in view of the recent report of polarized sorting of beta PP to the basolateral domain of MDCK cells. PMID:7721945

  1. 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

  2. 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

  3. A putative porin gene of Burkholderia sp. NK8 involved in chemotaxis toward β-ketoadipate.

    PubMed

    Yamamoto-Tamura, Kimiko; Kawagishi, Ikuro; Ogawa, Naoto; Fujii, Takeshi

    2015-01-01

    Burkholderia sp. NK8 can utilize 3-chlorobenzoate (3CB) as a sole source of carbon because it has a megaplasmid (pNK8) that carries the gene cluster (tfdT-CDEF) encoding chlorocatechol-degrading enzymes. The expression of tfdT-CDEF is induced by 3CB. In this study, we found that NK8 cells were attracted to 3CB and its degradation products, 3- and 4-chlorocatechol, and β-ketoadipate. Capillary assays revealed that a pNK8-eliminated strain (NK82) was defective in chemotaxis toward β-ketoadipate. The introduction of a plasmid carrying a putative outer membrane porin gene, which we name ompNK8, into strain NK82 restored chemotaxis toward β-ketoadipate. RT-PCR analyses demonstrated that the transcription of the ompNK8 gene was enhanced in the presence of 3CB.

  4. Identification of calcium-transporting ATPases of Entamoeba histolytica and cellular localization of the putative SERCA.

    PubMed

    Martinez-Higuera, Aarón; Salas-Casas, Andrés; Calixto-Gálvez, Mercedes; Chávez-Munguía, Bibiana; Pérez-Ishiwara, D Guillermo; Ximénez, Cecilia; Rodríguez, Mario A

    2013-09-01

    Calcium has an important role on signaling of different cellular processes in the protozoa parasite Entamoeba histolytica, including development and pathogenesis. However, the systems that control calcium responses in this parasite are incompletely understood. Calcium-ATPases (Ca(2+)-ATPases) are proteins that play an important role in calcium homeostasis by catalyzing the active efflux of this ion from cytoplasm and are essential to the correct functioning of the cell machinery. Here, we reported the identification of five E. histolytica genes encoding putative Ca(2+)-ATPases, three related to PMCA, and two related to organellar ATPases. RT-PCR assays showed that all those genes are expressed in trophozoites and specific antibodies against the SERCA-like member located this protein in a continuous cytoplasmic network, supporting the hypothesis that it corresponds to the Ca(2+)-ATPase responsible to sequester calcium in the endoplasmic reticulum of this parasite.

  5. Identification of a putative tetrasporophyte-specific gene in Gracilaria lemaneiformis (Gracilariales, Rhodophyte)

    NASA Astrophysics Data System (ADS)

    Ren, Xueying; Zhang, Xuecheng

    2008-08-01

    A putative tetrasporophyte-specific gene, designated as SSH466 (GenBank accession No. DQ019223), was one of the genes identified in this work using suppression subtractive hybridization (SSH) method in Gracilaria lemaneiformis. The full length of the gene was obtained using SMART RACE strategy. Sequence analysis revealed that the gene had 1 019 nucleotides, including an open reading frame of 498 nucleotides encoding 166 amino acid residues, 158 nucleotides of 5' untranslated region and 363 nucleotides of 3' non-coding region. Protein motif and secondary structure prediction showed that there existed a transmembrane domain with a unique β-sheet. Thus, SSH466 protein might be a cross-membrane protein. Sequence homology search in the public GenBank databases did not reveal any significant match with SSH466. Virtual Northern blot analysis confirmed that it was a tetrasporophyte-specific gene.

  6. Lipopolysaccharide transport to the cell surface: biosynthesis and extraction from the inner membrane

    PubMed Central

    Simpson, Brent W.; May, Janine M.; Sherman, David J.; Kahne, Daniel; Ruiz, Natividad

    2015-01-01

    The cell surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS). The network of charges and sugars provided by the dense packing of LPS molecules in the outer leaflet of the outer membrane interferes with the entry of hydrophobic compounds into the cell, including many antibiotics. In addition, LPS can be recognized by the immune system and plays a crucial role in many interactions between bacteria and their animal hosts. LPS is synthesized in the inner membrane of Gram-negative bacteria, so it must be transported across their cell envelope to assemble at the cell surface. Over the past two decades, much of the research on LPS biogenesis has focused on the discovery and understanding of Lpt, a multi-protein complex that spans the cell envelope and functions to transport LPS from the inner membrane to the outer membrane. This paper focuses on the early steps of the transport of LPS by the Lpt machinery: the extraction of LPS from the inner membrane. The accompanying paper (May JM, Sherman DJ, Simpson BW, Ruiz N, Kahne D. 2015 Phil. Trans. R. Soc. B 370, 20150027. (doi:10.1098/rstb.2015.0027)) describes the subsequent steps as LPS travels through the periplasm and the outer membrane to its final destination at the cell surface. PMID:26370941

  7. Dependence of technetium-99m red blood cell labeling efficiency on red cell surface charge.

    PubMed

    Seldin, D W; Simchon, S; Jan, K M; Chien, S; Alderson, P O

    1988-10-01

    The mechanisms by which [99mTc]pertechnetate becomes attached to stannous-primed red blood cells are not known in detail. To study the problem further, the effect of red cell surface charge on labeling efficiency was evaluated. Red cell surface charge was reduced by using the enzyme neuraminidase to remove the terminal charge-bearing sialic acid moiety of the membrane glycoprotein. Forty-five blood samples from six volunteers were treated with neuraminidase for varying lengths of time, resulting in the removal of from 11% to 99% of the normal negative surface charge, as determined from electrophoretic mobility measurements. There was excellent linear correlation between labeling efficiency and the remaining red cell surface charge for values down to 20% of normal (r = 0.89). When surface charge was less than 20% of normal, labeling efficiency was constant at 30%. Eleven blood samples from three donors were divided into two groups that were treated with neuraminidase either before or after they were labeled. The labeling efficiency was independent of the order in which the steps were performed. No evidence for shifting of the radiolabel from the cell membrane to hemoglobin was found. The results suggest that clinical conditions associated with a reduction of sialic acid on the erythrocyte membrane may be one cause of decreased red blood cell labeling efficiency, and that increased membrane permeability for reduced technetium species may be responsible for the decrease.

  8. Cell Surface Epidermal Growth Factor Receptors Increase Src and c-Cbl Activity and Receptor Ubiquitylation*

    PubMed Central

    Parks, Eileen E.; Ceresa, Brian P.

    2014-01-01

    There is an established role for the endocytic pathway in regulation of epidermal growth factor receptor (EGFR) signaling to downstream effectors. However, because ligand-mediated EGFR endocytosis utilizes multiple “moving parts,” dissecting the spatial versus temporal contributions has been challenging. Blocking all endocytic trafficking can have unintended effects on other receptors as well as give rise to compensatory mechanisms, both of which impact interpretation of EGFR signaling. To overcome these limitations, we used epidermal growth factor (EGF) conjugated to polystyrene beads (EGF beads). EGF beads simultaneously activate the EGFR while blocking its endocytosis and allow analysis of EGFR signaling from the plasma membrane. Human telomerase immortalized corneal epithelial (hTCEpi) cells were used to model normal epithelial cell biology. In hTCEpi cells, both cell surface and intracellular EGFRs exhibited dose-dependent increases in effector activity after 15 min of ligand stimulation, but only the serine phosphorylation of signal transducer and activator of transcription 3 (STAT3) was statistically significant when accounting for receptor phosphorylation. However, over time with physiological levels of receptor phosphorylation, cell surface receptors produced either enhanced or sustained mitogen-activated protein kinase kinase (MEK), Casitas B-lineage lymphoma (c-Cbl), and the pro-oncogene Src activity. These increases in effector communication by cell surface receptors resulted in an increase in EGFR ubiquitylation with sustained ligand incubation. Together, these data indicate that spatial regulation of EGFR signaling may be an important regulatory mechanism in receptor down-regulation. PMID:25074934

  9. Brown spider venom toxins interact with cell surface and are endocytosed by rabbit endothelial cells.

    PubMed

    Nowatzki, Jenifer; de Sene, Reginaldo Vieira; Paludo, Katia Sabrina; Veiga, Silvio Sanches; Oliver, Constance; Jamur, Maria Célia; Nader, Helena Bonciani; Trindade, Edvaldo S; Franco, Célia Regina C

    2010-09-15

    Bites from the Loxosceles genus (brown spiders) cause severe clinical symptoms, including dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of dermonecrotic lesions in animals exposed to Loxosceles intermedia venom show numerous vascular alterations. Study of the hemorrhagic consequences of the venom in endothelial cells has demonstrated that the degeneration of blood vessels results not only from degradation of the extracellular matrix molecule or massive leukocyte infiltration, but also from a direct and primary activity of the venom on endothelial cells. Exposure of an endothelial cell line in vitro to L. intermedia venom induce morphological alterations, such as cell retraction and disadhesion to the extracellular matrix. The aim of the present study was to investigate the interaction between the venom toxins and the endothelial cell surface and their possible internalization, in order to illuminate the information about the deleterious effect triggered by venom. After treating endothelial cells with venom toxins, we observed that the venom interacts with cell surface. Venom treatment also can cause a reduction of cell surface glycoconjugates. When cells were permeabilized, it was possible to verify that some venom toxins were internalized by the endothelial cells. The venom internalization involves endocytic vesicles and the venom was detected in the lysosomes. However, no damage to lysosomal integrity was observed, suggesting that the cytotoxic effect evoked by L. intermedia venom on endothelial cells is not mediated by venom internalization.

  10. 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.

  11. Sialyl-lactotetra, a novel cell surface marker of undifferentiated human pluripotent stem cells.

    PubMed

    Barone, Angela; Säljö, Karin; Benktander, John; Blomqvist, Maria; Månsson, Jan-Eric; Johansson, Bengt R; Mölne, Johan; Aspegren, Anders; Björquist, Petter; Breimer, Michael E; Teneberg, Susann

    2014-07-01

    Cell surface glycoconjugates are used as markers for undifferentiated pluripotent stem cells. Here, antibody binding and mass spectrometry characterization of acid glycosphingolipids isolated from a large number (1 × 10(9) cells) of human embryonic stem cell (hESC) lines allowed identification of several novel acid glycosphingolipids, like the gangliosides sialyl-lactotetraosylceramide and sialyl-globotetraosylceramide, and the sulfated glycosphingolipids sulfatide, sulf-lactosylceramide, and sulf-globopentaosylceramide. A high cell surface expression of sialyl-lactotetra on hESC and human induced pluripotent stem cells (hiPSC) was demonstrated by flow cytometry, immunohistochemistry, and electron microscopy, whereas sulfated glycosphingolipids were only found in intracellular compartments. Immunohistochemistry showed distinct cell surface anti-sialyl-lactotetra staining on all seven hESC lines and three hiPSC lines analyzed, whereas no staining of hESC-derived hepatocyte-like or cardiomyocyte-like cells was obtained. Upon differentiation of hiPSC into hepatocyte-like cells, the sialyl-lactotetra epitope was rapidly down-regulated and not detectable after 14 days. These findings identify sialyl-lactotetra as a promising marker of undifferentiated human pluripotent stem cells.

  12. Induction kinetics and cell surface distribution of Escherichia coli lipoprotein under lac promoter control.

    PubMed Central

    Hiemstra, H; de Hoop, M J; Inouye, M; Witholt, B

    1986-01-01

    The induction kinetics and surface accessibility of the outer membrane lipoprotein were studied in an Escherichia coli strain with the lpp gene under control of the lac promoter. Free lipoprotein appeared rapidly after induction with isopropyl-beta-D-thiogalactopyranoside and reached a steady-state level after 30 min. The newly induced lipoprotein was slowly bound to the peptidoglycan layer. Immunological methods were developed to detect lipoprotein accessible at the cell surface after various pretreatments as well as peptidoglycan-bound lipoprotein at the surface of isolated peptidoglycan sacculi with specific antibodies in combination with 125I-protein A. With these methods an increase in lipoprotein molecules at the cell surface and bound to the peptidoglycan sacculus could be detected following induction. The topology of newly synthesized lipoprotein was examined in thin sections as well as at the cell surface and the surface of the peptidoglycan sacculus with immunoelectron microscopy. Ultrathin cell sections, whole cells, and isolated peptidoglycan sacculi showed lipoprotein distributed homogeneously over the entire surface. Images PMID:3531164

  13. GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export

    PubMed Central

    Zhang, Maoxiang; Davis, Jason E.; Li, Chunman; Gao, Jie; Huang, Wei; Lambert, Nevin A.; Terry, Alvin V.

    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

  14. 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.

  15. Large-scale co-aggregation of fluorescent lipid probes with cell surface proteins

    PubMed Central

    1994-01-01

    Large scale aggregation of fluorescein-labeled immunoglobulin E (IgE) receptor complexes on the surface of RBL cells results in the co- aggregation of a large fraction of the lipophilic fluorescent probe 3,3'-dihexadecylindocarbocyanine (diI) that labels the plasma membranes much more uniformly in the absence of receptor aggregation. Most of the diI molecules that are localized in patches of aggregated receptors have lost their lateral mobility as determined by fluorescence photobleaching recovery. The diI outside of patches is mobile, and its mobility is similar to that in control cells without receptor aggregates. It is unlikely that the co-aggregation of diI with IgE receptors is due to specific interactions between these components, as two other lipophilic probes of different structures are also observed to redistribute with aggregated IgE receptors, and aggregation of two other cell surface antigens also results in the coredistribution of diI at the RBL cell surface. Quantitative analysis of CCD images of labeled cells reveals some differences in the spatial distributions of co- aggregated diI and IgE receptors. The results indicate that cross- linking of specific cell surface antigens causes a substantial change in the organization of the plasma membrane by redistributing pre- existing membrane domains or causing their formation. PMID:8188747

  16. 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.

  17. Effect of direct electric current on the cell surface properties of phenol-degrading bacteria.

    PubMed

    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.

  18. Lipopolysaccharide transport to the cell surface: biosynthesis and extraction from the inner membrane.

    PubMed

    Simpson, Brent W; May, Janine M; Sherman, David J; Kahne, Daniel; Ruiz, Natividad

    2015-10-01

    The cell surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS). The network of charges and sugars provided by the dense packing of LPS molecules in the outer leaflet of the outer membrane interferes with the entry of hydrophobic compounds into the cell, including many antibiotics. In addition, LPS can be recognized by the immune system and plays a crucial role in many interactions between bacteria and their animal hosts. LPS is synthesized in the inner membrane of Gram-negative bacteria, so it must be transported across their cell envelope to assemble at the cell surface. Over the past two decades, much of the research on LPS biogenesis has focused on the discovery and understanding of Lpt, a multi-protein complex that spans the cell envelope and functions to transport LPS from the inner membrane to the outer membrane. This paper focuses on the early steps of the transport of LPS by the Lpt machinery: the extraction of LPS from the inner membrane. The accompanying paper (May JM, Sherman DJ, Simpson BW, Ruiz N, Kahne D. 2015 Phil. Trans. R. Soc. B 370, 20150027. (doi:10.1098/rstb.2015.0027)) describes the subsequent steps as LPS travels through the periplasm and the outer membrane to its final destination at the cell surface.

  19. Bacterial Cell Surface Display of a Multifunctional Cellulolytic Enzyme Screened from a Bovine Rumen Metagenomic Resource.

    PubMed

    Ko, Kyong-Cheol; Lee, Binna; Cheong, Dae-Eun; Han, Yunjon; Choi, Jong Hyun; Song, Jae Jun

    2015-11-01

    A cell surface display system for heterologous expression of the multifunctional cellulase, CelEx-BR12, in Escherichia coli was developed using truncated E. coli outer membrane protein C (OmpC) as an anchor motif. Cell surface expression of CelEx-BR12 cellulase in E. coli harboring OmpC-fused CelEx-BR12, designated MC4100 (pTOCBR12), was confirmed by fluorescence-activated cell sorting and analysis of outer membrane fractions by western blotting, which verified the expected molecular mass of OmpC-fused CelEx-BR12 (~72 kDa). Functional evidence for exocellulase activity was provided by enzymatic assays of whole cells and outer membrane protein fractions from E. coli MC4100 (pTOCBR12). The stability of E. coli MC4100 (pTOCBR12) cellulase activity was tested by carrying out repeated reaction cycles, which demonstrated the reusability of recombinant cells. Finally, we showed that recombinant E. coli cells displaying the CelEx-BR12 enzyme on the cell surface were capable of growth using carboxymethyl cellulose as the sole carbon source.

  20. Activation of Cell Surface Bound 20S Proteasome Inhibits Vascular Cell Growth and Arteriogenesis

    PubMed Central

    Ito, Wulf D.; Lund, Natalie; Zhang, Ziyang; Buck, Friedrich; Lellek, Heinrich; Horst, Andrea; Machens, Hans-Günther; Schunkert, Heribert; Schaper, Wolfgang; Meinertz, Thomas

    2015-01-01

    Arteriogenesis is an inflammatory process associated with rapid cellular changes involving vascular resident endothelial progenitor cells (VR-EPCs). Extracellular cell surface bound 20S proteasome has been implicated to play an important role in inflammatory processes. In our search for antigens initially regulated during collateral growth mAb CTA 157-2 was generated against membrane fractions of growing collateral vessels. CTA 157-2 stained endothelium of growing collateral vessels and the cell surface of VR-EPCs. CTA 157-2 bound a protein complex (760 kDa) that was identified as 26 kDa α7 and 21 kDa β3 subunit of 20S proteasome in mass spectrometry. Furthermore we demonstrated specific staining of 20S proteasome after immunoprecipitation of VR-EPC membrane extract with CTA 157-2 sepharose beads. Functionally, CTA 157-2 enhanced concentration dependently AMC (7-amino-4-methylcoumarin) cleavage from LLVY (N-Succinyl-Leu-Leu-Val-Tyr) by recombinant 20S proteasome as well as proteasomal activity in VR-EPC extracts. Proliferation of VR-EPCs (BrdU incorporation) was reduced by CTA 157-2. Infusion of the antibody into the collateral circulation reduced number of collateral arteries, collateral proliferation, and collateral conductance in vivo. In conclusion our results indicate that extracellular cell surface bound 20S proteasome influences VR-EPC function in vitro and collateral growth in vivo. PMID:26146628

  1. Quantitative immunoferritin localization of [Na+,K+]ATPase on canine hepatocyte cell surface

    PubMed Central

    1984-01-01

    Distribution of [Na+,K+]ATPase on the cell surface of canine hepatocytes was investigated quantitatively by incubating prefixed and dissociated liver cells with ferritin antibody conjugates against canine kidney holo[Na+,K+]ATPase. We found that [Na+,K+]-ATPase exists bilaterally both on the bile canalicular and sinusoid-lateral surfaces. The particle density on the bile canalicular surface was much higher (approximately 2.5 times) than that on the sinusoid-lateral surface. In the latter region, the enzyme was detected almost equally both on the sinusoidal and lateral surfaces. On all the surfaces, the distribution of the enzyme was homogeneous and no clustering of the enzyme was detected. Total number of the enzyme on the sinusoid-lateral surface was, however, approximately three times higher than that on the bile canalicular region, because the sinusoid-lateral surface represents approximately 87% of the total cell surface of a hepatocyte. We suggest that the [Na+, K+]ATPase on the bile canalicular surface is responsible for the bile acid-independent bile flow and the other transport processes on the bile canalicular cell surface, while that on the sinusoid-lateral surface is responsible not only for the active transport of Na+ but also for the secondary active transport of various substances in this region. PMID:6090472

  2. Alternative splicing of agrin regulates its binding to heparin alpha-dystroglycan, and the cell surface.

    PubMed Central

    O'Toole, J J; Deyst, K A; Bowe, M A; Nastuk, M A; McKechnie, B A; Fallon, J R

    1996-01-01

    Agrin is a basal lamina molecule that directs key events in postsynaptic differentiation, most notably the aggregation of acetylcholine receptors (AChRs) on the muscle cell surface. Agrin's AChR clustering activity is regulated by alternative mRNA splicing. Agrin splice forms having inserts at two sites (y and z) in the C-terminal region are highly active, but isoforms lacking these inserts are weakly active. The biochemical consequences of this alternative splicing are unknown. Here, the binding of four recombinant agrin isoforms to heparin, to alpha-dystroglycan (a component of an agrin receptor), and to myoblasts was tested. The presence of a four-amino acid insert at the y site is necessary and sufficient to confer heparin binding ability to agrin. Moreover, the binding of agrin to alpha-dystroglycan is inhibited by heparin when this insert is present. Agrin binding to the cell surface showed analogous properties: heparin inhibits the binding of only those agrin isoforms containing this four-amino acid insert. The results show that alternative splicing of agrin regulates its binding to heparin and suggest that agrin's interaction with alpha-dystroglycan may be modulated by cell surface glycosaminoglycans in an isoform-dependent manner. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8693000

  3. Sialyl-lactotetra, a Novel Cell Surface Marker of Undifferentiated Human Pluripotent Stem Cells*

    PubMed Central

    Barone, Angela; Säljö, Karin; Benktander, John; Blomqvist, Maria; Månsson, Jan-Eric; Johansson, Bengt R.; Mölne, Johan; Aspegren, Anders; Björquist, Petter; Breimer, Michael E.; Teneberg, Susann

    2014-01-01

    Cell surface glycoconjugates are used as markers for undifferentiated pluripotent stem cells. Here, antibody binding and mass spectrometry characterization of acid glycosphingolipids isolated from a large number (1 × 109 cells) of human embryonic stem cell (hESC) lines allowed identification of several novel acid glycosphingolipids, like the gangliosides sialyl-lactotetraosylceramide and sialyl-globotetraosylceramide, and the sulfated glycosphingolipids sulfatide, sulf-lactosylceramide, and sulf-globopentaosylceramide. A high cell surface expression of sialyl-lactotetra on hESC and human induced pluripotent stem cells (hiPSC) was demonstrated by flow cytometry, immunohistochemistry, and electron microscopy, whereas sulfated glycosphingolipids were only found in intracellular compartments. Immunohistochemistry showed distinct cell surface anti-sialyl-lactotetra staining on all seven hESC lines and three hiPSC lines analyzed, whereas no staining of hESC-derived hepatocyte-like or cardiomyocyte-like cells was obtained. Upon differentiation of hiPSC into hepatocyte-like cells, the sialyl-lactotetra epitope was rapidly down-regulated and not detectable after 14 days. These findings identify sialyl-lactotetra as a promising marker of undifferentiated human pluripotent stem cells. PMID:24841197

  4. 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

  5. Role of TI-VAMP and CD82 in EGFR cell-surface dynamics and signaling.

    PubMed

    Danglot, Lydia; Chaineau, Mathilde; Dahan, Maxime; Gendron, Marie-Claude; Boggetto, Nicole; Perez, Franck; Galli, Thierry

    2010-03-01

    The v-SNARE TI-VAMP (VAMP7) mediates exocytosis during neuritogenesis, phagocytosis and lysosomal secretion. It localizes to endosomes and lysosomes but also to the trans-Golgi network. Here we show that depletion of TI-VAMP enhances the endocytosis of activated EGF receptor (EGFR) without affecting constitutive endocytosis of EGFR, or transferrin uptake. This increased EGFR internalization is mainly clathrin dependent. Searching for defects in EGFR regulators, we found that TI-VAMP depletion reduces the cell surface amount of CD82, a tetraspanin known to control EGFR localization in microdomains. We further show that TI-VAMP is required for secretion from the Golgi apparatus to the cell surface, and that TI-VAMP-positive vesicles transport CD82. Quantum dots video-microscopy indicates that depletion of TI-VAMP, or its cargo CD82, restrains EGFR diffusion and the area explored by EGFR at the cell surface. Both depletions also impair MAPK signaling and enhance endocytosis of activated EGFR by increased recruitment of AP-2. These results highlight the role of TI-VAMP in the secretory pathway of a tetraspanin, and support a model in which CD82 allows EGFR entry in microdomains that control its clathrin-dependent endocytosis and signaling.

  6. 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

  7. Molecular polymorphism of a cell surface proteoglycan: distinct structures on simple and stratified epithelia.

    PubMed

    Sanderson, R D; Bernfield, M

    1988-12-01

    Epithelial cells are organized into either a single layer (simple epithelia) or multiple layers (stratified epithelia). Maintenance of these cellular organizations requires distinct adhesive mechanisms involving many cell surface molecules. One such molecule is a cell surface proteoglycan, named syndecan, that contains both heparan sulfate and chondroitin sulfate chains. This proteoglycan binds cells to fibrillar collagens and fibronectin and thus acts as a receptor for interstitial matrix. The proteoglycan is restricted to the basolateral surface of simple epithelial cells, but is located over the entire surface of stratified epithelial cells, even those surfaces not contacting matrix. We now show that the distinct localization in simple and stratified epithelia correlates with a distinct proteoglycan structure. The proteoglycan from simple epithelia (modal molecular size, 160 kDa) is larger than that from stratified epithelia (modal molecular size, 92 kDa), but their core proteins are identical in size and immunoreactivity. The proteoglycan from simple epithelia has more and larger heparan sulfate and chondroitin sulfate chains than the proteoglycan from stratified epithelia. Thus, the cell surface proteoglycan shows a tissue-specific structural polymorphism due to distinct posttranslational modifications. This polymorphism likely reflects distinct proteoglycan functions in simple and stratified epithelia, potentially meeting the different adhesive requirements of the cells in these different organizations.

  8. Changes in immunologic cell surface markers during cocaine withdrawal in pregnant women.

    PubMed

    Johnson, T R; Knisely, J S; Christmas, J T; Schnoll, S H; Ruddy, S

    1996-12-01

    To evaluate the effects of acute cocaine withdrawal on the immune system of pregnant women, we analyzed changes in a panel of cell surface markers and plasma proteins that have immunological importance. The cell surface markers included complement receptors [CR1 (CD35), CR2 (CD21), CR3 (CD11b, CD18)], immunoglobulin Fc receptors [FcgammaRII (CD32), FcgammaRIII (CD16)], proteins important for lymphocyte function [CD3, CD4, CD8, CD19, CD25, CD45RA], and the framework antigen HLA-ABC. We also measured levels of the plasma proteins C3, C4, IgG, IgM, and IgA, along with the cytokine interleukin-2, soluble lymphocyte markers sCD25, sCD4, sCD8, IL-2, and soluble immune complexes. While no significant changes were seen in the levels of plasma proteins, changes paralleling the course of acute withdrawal were seen in complement receptors and immunoglobulin Fc receptors on leukocyte subpopulations. By contrast, proteins important for lymphocyte function were relatively unperturbed. There was an increase in receptor expression at the onset of withdrawal that peaked 3-5 days after last cocaine use, followed by a decrease in expression to initial (pre-withdrawal) levels. These changes in cell surface receptors may reflect altered immune function in the women who were withdrawing from cocaine.

  9. Synaptic encoding of temporal contiguity

    PubMed Central

    Ostojic, Srdjan; Fusi, Stefano

    2013-01-01

    Often we need to perform tasks in an environment that changes stochastically. In these situations it is important to learn the statistics of sequences of events in order to predict the future and the outcome of our actions. The statistical description of many of these sequences can be reduced to the set of probabilities that a particular event follows another event (temporal contiguity). Under these conditions, it is important to encode and store in our memory these transition probabilities. Here we show that for a large class of synaptic plasticity models, the distribution of synaptic strengths encodes transitions probabilities. Specifically, when the synaptic dynamics depend on pairs of contiguous events and the synapses can remember multiple instances of the transitions, then the average synaptic weights are a monotonic function of the transition probabilities. The synaptic weights converge to the distribution encoding the probabilities also when the correlations between consecutive synaptic modifications are considered. We studied how this distribution depends on the number of synaptic states for a specific model of a multi-state synapse with hard bounds. In the case of bistable synapses, the average synaptic weights are a smooth function of the transition probabilities and the accuracy of the encoding depends on the learning rate. As the number of synaptic states increases, the average synaptic weights become a step function of the transition probabilities. We finally show that the information stored in the synaptic weights can be read out by a simple rate-based neural network. Our study shows that synapses encode transition probabilities under general assumptions and this indicates that temporal contiguity is likely to be encoded and harnessed in almost every neural circuit in the brain. PMID:23641210

  10. 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.

  11. 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

  12. 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

  13. Epstein-Barr virus BILF1 evolved to downregulate cell surface display of a wide range of HLA class I molecules through their cytoplasmic tail

    PubMed Central

    Griffin, Bryan D.; Gram, Anna M.; Mulder, Arend; Van Leeuwen, Daphne; Claas, Frans H. J.; Wang, Fred; Ressing, Maaike E.; Wiertz, Emmanuel

    2013-01-01

    Co-evolution of herpesviruses and their hosts has driven the development of both host antiviral mechanisms to detect and eliminate infected cells, and viral ploys to escape immune surveillance. Among the immune evasion strategies employed by the lymphocryptovirus (γ1-herpesvirus) EBV is the downregulation of surface HLA class I expression by the virally-encoded G-protein coupled receptor BILF1, thereby impeding presentation of viral antigens and cytotoxic T cell recognition of the infected cell. Here, we show EBV BILF1 to be expressed early in the viral lytic cycle. BILF1 targets a broad range of HLA class I molecules, including multiple HLA-A and -B types, and HLA-E. In contrast, HLA-C was only marginally affected. We advance the mechanistic understanding of the process by showing the cytoplasmic C-terminal tail of EBV BILF1 to be required for reducing surface HLA class I expression. Susceptibility to BILF1-mediated downregulation is in turn conferred by specific residues in the intracellular tail of the HLA class I heavy chain. Finally, we explore the evolution of BILF1 within the lymphocryptovirus genus. While the homolog of BILF1 encoded by the lymphocryptovirus infecting Old World rhesus primates shares the ability of EBV to downregulate cell surface HLA class I expression, this function is not possessed by New World marmoset lymphocryptovirus BILF1. This study therefore furthers our knowledge on the evolution of immunoevasive functions by the lymphocryptovirus genus of herpesviruses. PMID:23315076

  14. 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.

  15. A novel putative insect chitinase with multiple catalytic domains: hormonal regulation during metamorphosis.

    PubMed Central

    Royer, Véronique; Fraichard, Stéphane; Bouhin, Hervé

    2002-01-01

    We have used differential display to identify genes that are regulated by juvenile hormone in the epidermis of the beetle Tenebrio molitor. One of the genes encodes T. molitor chitinase 5 (TmChit5), a chitinase possessing an unusual structure. Sequence analysis of TmChit5 identified five 'chitinase units' of approx. 480 amino acids with similarity to chitinase family 18. These units are separated by less conserved regions containing putative PEST (rich in proline, glutamic acid, serine and threonine) sequences, putative chitin-binding domains and mucin domains. Northern-blot analysis identified a single transcript of approx. 9 kb, whose abundance correlated with that of 20-hydroxyecdysone during metamorphosis. Injection of pupae with 20-hydroxyecdysone alone, or in combination with cycloheximide, indicated that TmChit5 expression is directly induced by the hormone. Further experiments indicated that methoprene (a juvenile hormone analogue) indirectly induced TmChit5 mRNA expression. On the basis of the present results and previous studies, we propose a molecular mechanism for cuticle digestion during the moulting process. PMID:12059786

  16. Duplications and losses in gene families of rust pathogens highlight putative effectors.

    PubMed

    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.

  17. 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

  18. 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

  19. A novel putative insect chitinase with multiple catalytic domains: hormonal regulation during metamorphosis.

    PubMed

    Royer, Véronique; Fraichard, Stéphane; Bouhin, Hervé

    2002-09-15

    We have used differential display to identify genes that are regulated by juvenile hormone in the epidermis of the beetle Tenebrio molitor. One of the genes encodes T. molitor chitinase 5 (TmChit5), a chitinase possessing an unusual structure. Sequence analysis of TmChit5 identified five 'chitinase units' of approx. 480 amino acids with similarity to chitinase family 18. These units are separated by less conserved regions containing putative PEST (rich in proline, glutamic acid, serine and threonine) sequences, putative chitin-binding domains and mucin domains. Northern-blot analysis identified a single transcript of approx. 9 kb, whose abundance correlated with that of 20-hydroxyecdysone during metamorphosis. Injection of pupae with 20-hydroxyecdysone alone, or in combination with cycloheximide, indicated that TmChit5 expression is directly induced by the hormone. Further experiments indicated that methoprene (a juvenile hormone analogue) indirectly induced TmChit5 mRNA expression. On the basis of the present results and previous studies, we propose a molecular mechanism for cuticle digestion during the moulting process.

  20. Putative Monofunctional Type I Polyketide Synthase Units: A Dinoflagellate-Specific Feature?

    PubMed Central

    Eichholz, Karsten; Beszteri, Bánk; John, Uwe

    2012-01-01

    Marine dinoflagellates (alveolata) are microalgae of which some cause harmful algal blooms and produce a broad variety of most likely polyketide synthesis derived phycotoxins. Recently, novel polyketide synthesase (PKS) transcripts have been described from the Florida red tide dinoflagellate Karenia brevis (gymnodiniales) which are evolutionarily related to Type I PKS but were apparently expressed as monofunctional proteins, a feature typical of Type II PKS. Here, we investigated expression units of PKS I-like sequences in Alexandrium ostenfeldii (gonyaulacales) and Heterocapsa triquetra (peridiniales) at the transcript and protein level. The five full length transcripts we obtained were all characterized by polyadenylation, a 3′ UTR and the dinoflagellate specific spliced leader sequence at the 5′end. Each of the five transcripts encoded a single ketoacylsynthase (KS) domain showing high similarity to K. brevis KS sequences. The monofunctional structure was also confirmed using dinoflagellate specific KS antibodies in Western Blots. In a maximum likelihood phylogenetic analysis of KS domains from diverse PKSs, dinoflagellate KSs formed a clade placed well within the protist Type I PKS clade between apicomplexa, haptophytes and chlorophytes. These findings indicate that the atypical PKS I structure, i.e., expression as putative monofunctional units, might be a dinoflagellate specific feature. In addition, the sequenced transcripts harbored a previously unknown, apparently dinoflagellate specific conserved N-terminal domain. We discuss the implications of this novel region with regard to the putative monofunctional organization of Type I PKS in dinoflagellates. PMID:23139807

  1. 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

  2. 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.

  3. 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.

  4. Role of a putative tungsten-dependent formylmethanofuran dehydrogenase in Methanosarcina acetivorans.

    PubMed

    Matschiavelli, Nicole; Rother, Michael

    2015-04-01

    Methanogenesis, the biological production of methane, is the sole means for energy conservation for methanogenic archaea. Among the few methanogens shown to grow on carbon monoxide (CO) is Methanosarcina acetivorans, which produces, beside methane, acetate and formate in the process. Since CO-dependent methanogenesis proceeds via formation of formylmethanofuran from CO2 and methanofuran, catalyzed by formylmethanofuran dehydrogenase, we were interested whether this activity could participate in the formate formation from CO. The genome of M. acetivorans encodes four putative formylmethanofuran dehydrogenases, two annotated as molybdenum-dependent and the remaining two as tungsten-dependent enzymes. A mutant lacking one of the putative tungsten enzymes grew very slowly on CO and only after a prolonged adaptation period, which suggests an important role for this isoform during growth on CO. Methanol- and CO-dependent growth of the mutant required the presence of molybdenum indicating an indispensable function of this metal in the remaining isoforms. CO-dependent formate formation could not be observed in the mutant indicating involvement of the respective isoform in the process. However, addition of formaldehyde, which spontaneously reacts with tetrahydrosarcinapterin (H4SPT) to methenyl-H4SPT, led to near-wild-type formate production rates, which argues for an alternative route of formate formation in this organism.

  5. 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

  6. Crystallization and preliminary X-ray diffraction analysis of Lin1840, a putative β-glucosidase from Listeria innocua.

    PubMed

    Nakajima, Masahiro; Yoshida, Ryuta; Miyanaga, Akimasa; Taguchi, Hayao

    2014-10-01

    Lin1840 is a putative β-glucosidase that is predicted to be involved in 1,2-β-glucan metabolism since the lin1839 gene encoding a 1,2-β-oligoglucan phosphorylase and the lin1840 gene are located in the same gene cluster. Here, Lin1840 was crystallized. The crystals of Lin1840 diffracted to beyond 1.8 Å resolution. The crystal belonged to space group I121, with unit-cell parameters a = 89.75, b = 95.10, c = 215.00 Å, α = 90.00, β = 96.34, γ = 90.00°.

  7. [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.

  8. 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.

  9. Cloning and sequencing of the leu C and npr M genes and a putative spo IV gene from Bacillus megaterium DSM319.

    PubMed

    Meinhardt, F; Busskamp, M; Wittchen, K D

    1994-05-01

    The leuC gene, encoding 3-isopropylmalate dehydrogenase, the nprM gene (neutral protease) and a sporulation gene coding for a putative spoIV protein (spoIV) from Bacillus megaterium DSM 319 were cloned and the nucleotide sequences were determined. The leuC gene is 1101 bp in length, preceded by a ribosome binding site; no promoter consensus sequence could be found. The nucleotide sequence from nprM when compared to the recently published gene from B. megaterium ATCC 14581 exhibited only a 17-base pair deviation. From a sporulation mutant isolated after transposon-mutagenesis with transposon Tn917 the insertion site of the transposon was cloned and adjacent chromosomal fragments were characterized. An open reading frame that encodes for a putative spo protein of 247 amino-acid residues was identified. PMID:7764969

  10. 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…

  11. 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…

  12. 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

  13. Nucleus-encoded periplastid-targeted EFL in chlorarachniophytes.

    PubMed

    Gile, Gillian H; Keeling, Patrick J

    2008-09-01

    Chlorarachniophytes are cercozoan amoeboflagellates that acquired photosynthesis by enslaving a green alga, which has retained a highly reduced nucleus called a nucleomorph. The nucleomorph lacks many genes necessary for its own maintenance and expression, suggesting that some genes have been moved to the host nucleus and their products are now targeted back to the periplastid compartment (PPC), the reduced eukaryotic cytoplasm of the endosymbiont. Protein trafficking in chlorarachniophytes is therefore complex, including nucleus-encoded plastid-targeted proteins, nucleomorph-encoded plastid-targeted proteins, and nucleus-encoded periplastid-targeted proteins. A major gap in our understanding of this system is the PPC-targeted proteins because none have been described in any chlorarachniophytes. Here we describe the first such protein, the GTPase EFL. EFL was characterized from 7 chlorarachniophytes, and 2 distinct types were found. One is related to foraminiferan EFL and lacks an amino-terminal extension. The second, distantly related, type encodes an amino-terminal extension consisting of a signal peptide followed by sequence sharing many characteristics with transit peptides from nucleus-encoded plastid-targeted proteins and which we conclude is most likely PPC targeted. Western blotting with antibodies specific to putative host and PPC-targeted EFL from the chlorarachniophytes Bigelowiella natans and Gymnochlora stellata is consistent with posttranslational cleavage of the leaders from PPC-targeted proteins. Immunolocalization of both proteins in B. natans confirmed the cytosolic location of the leaderless EFL and a distinct localization pattern for the PPC-targeted protein but could not rule out a plastid location (albeit very unlikely). We sought other proteins with a similar leader and identified a eukaryotic translation initiation factor 1 encoding a bipartite extension with the same properties. Transit peptide sequences were characterized from all 3

  14. 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

  15. Putative Domain-Domain Interactions in the Vesicular Stomatitis Virus L Polymerase Protein Appendage Region

    PubMed Central

    Ruedas, John B.

    2014-01-01

    ABSTRACT The multidomain polymerase protein (L) of nonsegmented negative-strand (NNS) RNA viruses catalyzes transcription and replication of the virus genome. The N-terminal half of the protein forms a ring-like polymerase structure, while the C-terminal half encoding viral mRNA transcript modifications consists of a flexible appendage with three distinct globular domains. To gain insight into putative transient interactions between L domains during viral RNA synthesis, we exchanged each of the four distinct regions encompassing the appendage region of vesicular stomatitis virus (VSV) Indiana serotype L protein with their counterparts from VSV New Jersey and analyzed effects on virus polymerase activity in a minigenome system. The methyltransferase domain exchange yielded a fully active polymerase protein, which functioned as well as wild-type L in the context of a recombinant virus. Exchange of the downstream C-terminal nonconserved region abolished activity, but coexchanging it with the methyltransferase domain generated a polymerase favoring replicase over transcriptase activity, providing strong evidence of interaction between these two regions. Exchange of the capping enzyme domain or the adjacent nonconserved region thought to function as an “unstructured” linker also abrogated polymerase activity even when either domain was coexchanged with other appendage domains. Further probing of the putative linker segment using in-frame enhanced green fluorescent protein (EGFP) insertions similarly abrogated activity. We discuss the implications of these findings with regard to L protein appendage domain structure and putative domain-domain interactions required for polymerase function. IMPORTANCE NNS viruses include many well-known human pathogens (e.g., rabies, measles, and Ebola viruses), as well as emerging viral threats (e.g., Nipah and Hendra viruses). These viruses all encode a large L polymerase protein similarly organized into multiple domains that work in

  16. 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.

  17. 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.

  18. 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

  19. Escherichia coli kgtP encodes an. alpha. -ketoglutarate transporter

    SciTech Connect

    Seol, Wongi; Shatkin, A.J. )

    1991-05-01

    The witA gene located between pss and rrnG on the Escherichia coli chromosome encodes a 432-amino acid protein. It is homologous to a human hepatoma glucose transporter and to E. coli membrane proteins that transport citrate (CitA), arabinose (AraE), and xylose (XylE), and, like these carrier proteins, WitA also contains 12 highly hydrophobic putative membrane-spanning regions. Gene disruption mutants constructed in two E. coli strains grew slowly or not at all, depending on genetic background, in M9 minimal medium containing {alpha}-ketoglutarate and uptake of {alpha}-({sup 14}C)ketoglutarate were restored by transformation with plasmids containing witA. These complementation studies indicate that WitA is an {alpha}-ketoglutarate transporter and should be renamed kgtP({alpha}-ketoglutarate permease).

  20. A preliminary crystallographic analysis of the putative mevalonate diphosphate decarboxylase from Trypanosoma brucei

    SciTech Connect

    Byres, Emma; Martin, David M. A.; Hunter, William N.

    2005-06-01

    The gene encoding the putative mevalonate diphosphate decarboxylase, an enzyme from the mevalonate pathway of isoprenoid precursor biosynthesis, has been cloned from T. brucei. Recombinant protein has been expressed, purified and highly ordered crystals obtained and characterized to aid the structure–function analysis of this enzyme. Mevalonate diphosphate decarboxylase catalyses the last and least well characterized step in the mevalonate pathway for the biosynthesis of isopentenyl pyrophosphate, an isoprenoid precursor. A gene predicted to encode the enzyme from Trypanosoma brucei has been cloned, a highly efficient expression system established and a purification protocol determined. The enzyme gives monoclinic crystals in space group P2{sub 1}, with unit-cell parameters a = 51.5, b = 168.7, c = 54.9 Å, β = 118.8°. A Matthews coefficient V{sub M} of 2.5 Å{sup 3} Da{sup −1} corresponds to two monomers, each approximately 42 kDa (385 residues), in the asymmetric unit with 50% solvent content. These crystals are well ordered and data to high resolution have been recorded using synchrotron radiation.

  1. The putative oligosaccharide translocase SypK connects biofilm formation with quorum signaling in Vibrio fischeri

    PubMed Central

    Miyashiro, Tim; Oehlert, Dane; Ray, Valerie A; Visick, Karen L; Ruby, Edward G

    2014-01-01

    Quorum signaling (QS) describes how bacteria can use small signaling molecules (autoinducers) to coordinate group-level behaviors. In Vibrio fischeri, QS is achieved through a complex regulatory network that ultimately controls bioluminescence, motility, and host colonization. We conducted a genetic screen focused on qrr1, which encodes a small regulatory RNA that is necessary for the core quorum-signaling cascade to transduce autoinducer information into cellular responses. We isolated unique mutants with a transposon inserted into one of two genes within the syp locus, which is involved in biofilm formation. We found that overexpression of sypK, which encodes a putative oligosaccharide translocase, is sufficient to activate qrr1, and, in addition, this effect appears to depend on the kinase activity of the sensor LuxQ. Consistent with the established model for QS in V. fischeri, enhanced expression of qrr1 by the overexpression of sypK resulted in reduced bioluminescence and increased motility. Finally, we found that induction of the syp locus by overexpression of sypG was sufficient to activate qrr1 levels. Together, our results show how conditions that promote biofilm formation impact the quorum-signaling network in V. fischeri, and further highlight the integrated nature of the regulatory circuits involved in complex bacterial behaviors. PMID:25257018

  2. Desulfation of cell surface HSPG is an effective strategy for the treatment of gallbladder carcinoma.

    PubMed

    Yi, Bin; Qiu, Yinghe; Ji, Weidan; Wei, Miaoyan; Liu, Chunying; Peng, Zhangxiao; Zhang, Yongjie; Quan, Zhiwei; Tang, Zhaohui; Su, Changqing

    2016-10-28

    Cell surface heparan sulfate proteoglycan (HSPG) is a group of critical glycoproteins that mediates signal transduction. Sulfated HSPG can mediate the activation of a variety of cell growth factor signal pathway to promote the progression of gallbladder carcinoma (GBC). This study analyzed 527 clinical GBC specimens and confirmed that the HSPG sulfation level was significantly higher in GBC tissues than in gallbladder mucosa (GBM) tissues. The high HSPG sulfation level was closely associated with poor differentiation, local metastasis, and advanced clinical stage of GBC; it was also associated with the shortening of disease-free survival (DFS) and overall survival (OS) and influenced the outcome of chemotherapy or radio-chemotherapy in patients with GBC recurrence. Inhibition of HSPG sulfation on the GBC cell surface using human sulfatase 1 (hSulf-1) significantly reduced the phosphorylation levels of growth factor receptors and signaling protein kinases in GBC cells, decreased cell responses to growth factors, and inhibited cell proliferation and migration abilities. In a nude mouse model with GBC xenografts, we observed that the xenograft tumor growth was suppressed and the phosphorylation levels of signaling proteins were downregulated, together with decreased expression of Ki67 and reduced sensitivity to bFGF (basic fibroblast growth factor) induction after inhibition of HSPG sulfation. Our study demonstrated that a high HSPG sulfation endows GBC with high malignant biological behaviors and a poor prognosis. Desulfation of cell surface HSPG can inhibit the kinase activities of a variety of signaling proteins, hinder the cell response to growth factors, and effectively inhibit the malignant biological behaviors of GBC cells.

  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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. Equilibrium of sortase A dimerization on Staphylococcus aureus cell surface mediates its cell wall sorting activity

    PubMed Central

    Zhu, Jie; Xiang, Liang; Jiang, Faqin

    2015-01-01

    Staphylococcus aureus sortase A (SrtA) transpeptidase is a therapeutically important membrane-bound enzyme in Gram-positive bacteria, which organizes the covalently attached cell surface proteins on the peptidoglycan cell wall of the organism. Here, we report the direct observation of the highly selective homo-dimerization of SrtA on the cell membrane. To address the biological significance of the dimerization towards enzyme function, site-directed mutagenesis was performed to generate a SrtA mutant, which exists as monomer on the cell membrane. We observed that the cell surface display of adhesive proteins in S. aureus cells expressing monomeric SrtA mutant is more prominent than the cells expressing the wild-type enzyme. A cell-based invasion assay was also performed to evaluate the activities of wild-type SrtA and its monomeric mutant as well. Our data demonstrated that S. aureus cells expressing SrtA in monomeric form invade host mammalian cells more efficiently than those expressing wild-type SrtA in dimer-monomer equilibrium. The results suggested that the monomeric form of SrtA is more active than the dimeric form of the enzyme in terms of cell surface display of virulence factors for infection. This is the first study to present the oligomerization of SrtA and its related biological function on the cell membrane. Study of SrtA dimerization has implications for understanding its catalytic mechanism at the cellular level as well as the development of novel anti-infective agents. PMID:26129884

  10. 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.

  11. 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.

  12. 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.

  13. Cell-surface Attachment of Bacterial Multienzyme Complexes Involves Highly Dynamic Protein-Protein Anchors*

    PubMed Central

    Cameron, Kate; Najmudin, Shabir; Alves, Victor D.; Bayer, Edward A.; Smith, Steven P.; Bule, Pedro; Waller, Helen; Ferreira, Luís M. A.; Gilbert, Harry J.; Fontes, Carlos M. G. A.

    2015-01-01

    Protein-protein interactions play a pivotal role in the assembly of the cellulosome, one of nature's most intricate nanomachines dedicated to the depolymerization of complex carbohydrates. The integration of cellulosomal components usually occurs through the binding of type I dockerin modules located at the C terminus of the enzymes to cohesin modules located in the primary scaffoldin subunit. Cellulosomes are typically recruited to the cell surface via type II cohesin-dockerin interactions established between primary and cell-surface anchoring scaffoldin subunits. In contrast with type II interactions, type I dockerins usually display a dual binding mode that may allow increased conformational flexibility during cellulosome assembly. Acetivibrio cellulolyticus produces a highly complex cellulosome comprising an unusual adaptor scaffoldin, ScaB, which mediates the interaction between the primary scaffoldin, ScaA, through type II cohesin-dockerin interactions and the anchoring scaffoldin, ScaC, via type I cohesin-dockerin interactions. Here, we report the crystal structure of the type I ScaB dockerin in complex with a type I ScaC cohesin in two distinct orientations. The data show that the ScaB dockerin displays structural symmetry, reflected by the presence of two essentially identical binding surfaces. The complex interface is more extensive than those observed in other type I complexes, which results in an ultra-high affinity interaction (Ka ∼1012 m). A subset of ScaB dockerin residues was also identified as modulating the specificity of type I cohesin-dockerin interactions in A. cellulolyticus. This report reveals that recruitment of cellulosomes onto the cell surface may involve dockerins presenting a dual binding mode to incorporate additional flexibility into the quaternary structure of highly populated multienzyme complexes. PMID:25855788

  14. 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.

  15. 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

  16. Encoding information into precipitation structures

    NASA Astrophysics Data System (ADS)

    Martens, Kirsten; Bena, Ioana; Droz, Michel; Rácz, Zoltan

    2008-12-01

    Material design at submicron scales would be profoundly affected if the formation of precipitation patterns could be easily controlled. It would allow the direct building of bulk structures, in contrast to traditional techniques which consist of removing material in order to create patterns. Here, we discuss an extension of our recent proposal of using electrical currents to control precipitation bands which emerge in the wake of reaction fronts in A+ + B- → C reaction-diffusion processes. Our main result, based on simulating the reaction-diffusion-precipitation equations, is that the dynamics of the charged agents can be guided by an appropriately designed time-dependent electric current so that, in addition to the control of the band spacing, the width of the precipitation bands can also be tuned. This makes straightforward the encoding of information into precipitation patterns and, as an amusing example, we demonstrate the feasibility by showing how to encode a musical rhythm.

  17. Fluorescence resonance energy transfers measurements on cell surfaces via fluorescence polarization.

    PubMed Central

    Cohen-Kashi, Meir; Moshkov, Sergey; Zurgil, Naomi; Deutsch, Mordechai

    2002-01-01

    A method has been developed for the determination of the efficiency of fluorescence resonance energy transfer efficiency between moieties located on cell surfaces by performing individual cell fluorescence polarization (FP) measurements. The absolute value of energy transfer efficiency (E) is calculated on an individual cell basis. The examination of this methodology was carried out using model experiments on human T lymphocyte cells. The cells were labeled with fluorescein-conjugated Concanavalin A (ConA) as donor, or rhodamine-conjugated ConA as acceptor. The experiments and results clearly indicate that determination of E via FP measurements is possible, efficient, and more convenient than other methods. PMID:12202365

  18. Glycoprotein mucin molecular brush on cancer cell surface acting as mechanical barrier against drug delivery

    NASA Astrophysics Data System (ADS)

    Wang, Xin; Shah, Aalok A.; Campbell, Robert B.; Wan, Kai-tak

    2010-12-01

    Uptake of cytotoxic drugs by typical tumor cells is limited by the dense dendritic network of oligosaccharide mucin chains that forms a mechanical barrier. Atomic force microscopy is used to directly measure the force needed to pierce the mucin layer to reach the cell surface. Measurements are analyzed by de Gennes' steric reptation theory. Multidrug resistant ovarian tumor cells shows significantly larger penetration load compared to the wide type. A pool of pancreatic, lung, colorectal, and breast cells are also characterized. The chemotherapeutic agent, benzyl-α-GalNac, for inhibiting glycosylation is shown to be effective in reducing the mechanical barrier.

  19. Absence of cell-surface EpCAM in congenital tufting enteropathy

    PubMed Central

    Schnell, Ulrike; Kuipers, Jeroen; Mueller, James L.; Veenstra-Algra, Anneke; Sivagnanam, Mamata; Giepmans, Ben N.G.

    2013-01-01

    Mutations in the epithelial cell adhesion molecule (EpCAM; CD326) gene are causal for congenital tufting enteropathy (CTE), a disease characterized by intestinal abnormalities resulting in lethal diarrhea in newborns. Why the different mutations all lead to the same disease is not clear. Here, we report that most mutations, including a novel intronic variant, will result in lack of EpCAM's transmembrane domain, whereas two mutations allow transmembrane localization. We find that these mutants are not routed to the plasma membrane, and that truncated mutants are secreted or degraded. Thus, all epcam mutations lead to loss of cell-surface EpCAM, resulting in CTE. PMID:23462293

  20. Studies on thyroid cell surface antigens using cultured human thyroid cells.

    PubMed Central

    Fenzi, G F; Bartalena, L; Chiovato, L; Marcocci, C; Rotella, C M; Zonefrati, R; Toccafondi, R; Pinchera, A

    1982-01-01

    Human thyroid cells in primary culture were used for studies of thyroid cell surface antibodies in patients with thyroid autoimmune disorders. Radioiodinated IgG preparations containing thyroid microsomal antibody (TMAb), thyroid stimulating antibody (TSAb) and/or thyroglobulin antibody (TgAb) were tested for binding to thyroid cells. Binding was observed with radioiodinated IgG from patients with Graves' disease, Hashimoto's thyroiditis and idiopathic myxoedema containing TMAb, irrespective of the presence of TSAb and TgAb, while negative results were obtained with normal IgG. A dose-dependent inhibition of binding to thyroid cells was produced by the addition of the corresponding unlabelled IgG preparations. Evidence for tissue specificity was provided by the absence of binding to human skin fibroblasts used as controls. Preabsorption with human thyroid microsomes completely abolished the binding to thyroid cells of a radioiodinated TMAb positive IgG preparation, while only incomplete removal of the reactivity to thyroid microsomes was produced by preabsorption with thyroid cells. These data suggest that some but not all microsomal antigenic determinants are expressed on the thyroid cell surface. Binding to thyroid cells was also observed with purified TgAb, indicating that thyroglobulin antigenic determinants are present on the surface of thyroid cells. No evidence of binding was obtained with a TSAb positive Graves' IgG preparation with undetectable TMAb and TgAb. Unlabelled IgG preparations containing TMAb from patients with either Hashimoto's thyroiditis or idiopathic myxoedema were shown to inhibit the binding to thyroid cells of radioiodinated TMAb positive Graves' IgG and vice versa. These data indicate that antibodies present in these thyroid autoimmune disorders share common thyroid cell surface antigens. However, the binding of radioiodinated IgG from a patient with idiopathic myxoedema was only partially inhibited by Graves' or Hashimoto's Ig

  1. 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

  2. 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...

  3. 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

  4. 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.

  5. 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

  6. 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

  7. Molecular cloning and characterization of a cDNA encoding endonuclease from potato (Solanum tuberosum).

    PubMed

    Larsen, Knud

    2005-11-01

    A cDNA, StEN1, encoding a potato (Solanum tuberosum) endonuclease was cloned and sequenced. The nucleotide sequence of this clone contains an open reading frame of 906 nucleotides encoding a protein of 302 amino acids, and with a calculated molecular mass of 34.4kDa and a Pi of 5.6. The deduced StEN1 protein contains a putative signal sequence of 25 amino acid residues. The StEN1 encoded protein shows substantial homology to both plant and fungal endonucleases isolated and cloned from other sources. The highest identity (73%) was observed with AgCEL I from celery, Apium graveolens, ZEN1 from Zinnia elegans (69%) and DSA6 from daylily, Hemerocallis (68%). RT-PCR expression analysis demonstrated that the potato StEN1 gene is constitutively expressed in potato, although minor differences in expression level in different tissues were observed. PMID:16323278

  8. Epigenetics, chromatin and genome organization: recent advances from the ENCODE project.

    PubMed

    Siggens, L; Ekwall, K

    2014-09-01

    The organization of the genome into functional units, such as enhancers and active or repressed promoters, is associated with distinct patterns of DNA and histone modifications. The Encyclopedia of DNA Elements (ENCODE) project has advanced our understanding of the principles of genome, epigenome and chromatin organization, identifying hundreds of thousands of potential regulatory regions and transcription factor binding sites. Part of the ENCODE consortium, GENCODE, has annotated the human genome with novel transcripts including new noncoding RNAs and pseudogenes, highlighting transcriptional complexity. Many disease variants identified in genome-wide association studies are located within putative enhancer regions defined by the ENCODE project. Understanding the principles of chromatin and epigenome organization will help to identify new disease mechanisms, biomarkers and drug targets, particularly as ongoing epigenome mapping projects generate data for primary human cell types that play important roles in disease.

  9. Biodegradation and surfactant-mediated biodegradation of diesel fuel by 218 microbial consortia are not correlated to cell surface hydrophobicity.

    PubMed

    Owsianiak, Mikołaj; Szulc, Alicja; Chrzanowski, Łukasz; Cyplik, Paweł; Bogacki, Mariusz; Olejnik-Schmidt, Agnieszka K; Heipieper, Hermann J

    2009-09-01

    In this study, we elucidated the role of cell surface hydrophobicity (microbial adhesion to hydrocarbons method, MATH) and the effect of anionic rhamnolipids and nonionic Triton X-100 surfactants on biodegradation of diesel fuel employing 218 microbial consortia isolated from petroleum-contaminated soils. Applied enrichment procedure with floating diesel fuel as a sole carbon source in liquid cultures resulted in consortia of varying biodegradation potential and diametrically different cell surface properties, suggesting that cell surface hydrophobicity is a conserved parameter. Surprisingly, no correlations between cell surface hydrophobicity and biodegradation of diesel fuel were found. Nevertheless, both surfactants altered cell surface hydrophobicity of the consortia in similar manner: increased for the hydrophilic and decreased for the hydrophobic cultures. In addition to this, the surfactants exhibited similar influence on diesel fuel biodegradation: Increase was observed for initially slow-degrading cultures and the opposite for fast degraders. This indicates that in the surfactant-mediated biodegradation, effectiveness of surfactants depends on the specification of microorganisms and not on the type of surfactant. In contrary to what was previously reported for pure strains, cell surface hydrophobicity, as determined by MATH, is not a good descriptor of biodegrading potential for mixed cultures.

  10. 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

  11. 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

  12. Roughness gradients on zirconia for rapid screening of cell-surface interactions: Fabrication, characterization and application.

    PubMed

    Flamant, Quentin; Stanciuc, Ana-Maria; Pavailler, Hugo; Sprecher, Christoph Martin; Alini, Mauro; Peroglio, Marianna; Anglada, Marc

    2016-10-01

    Roughness is one of the key parameters for successful osseointegration of dental implants. The understanding of how roughness affects cell response is thus crucial to improve implant performance. Surface gradients, which allow rapid and systematic investigations of cell-surface interactions, have the potential to facilitate this task. In this study, a novel method aiming to produce roughness gradients at the surface of zirconia using hydrofluoric acid etching was implemented. The topography was exhaustively characterized at the microscale and nanoscale by white light interferometry and atomic force microscopy, including the analysis of amplitude, spatial, hybrid, functional, and fractal parameters. A rapid screening of the influence of roughness on human mesenchymal stem cell morphology was conducted and potential correlations between roughness parameters and cell morphology were investigated. The roughness gradient induced significant changes in cell area (p < 0.001), aspect ratio (p = 0.01), and solidity (p = 0.026). Nanoroughness parameters were linearly correlated to cell solidity (p < 0.005), while microroughness parameters appeared nonlinearly correlated to cell area, highlighting the importance of multiscale optimization of implant topography to induce the desired cell response. The gradient method proposed here drastically reduces the efforts and resources necessary to study cell-surface interactions and provides results directly transferable to industry. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2502-2514, 2016. PMID:27227541

  13. 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