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Sample records for cell surface n-linked

  1. Modulation of ecotropic murine retroviruses by N-linked glycosylation of the cell surface receptor/amino acid transporter.

    PubMed Central

    Wang, H; Klamo, E; Kuhmann, S E; Kozak, S L; Kavanaugh, M P; Kabat, D

    1996-01-01

    The cell surface receptor for ecotropic host-range (infection limited to mice or rats) murine leukemia viruses (MuLVs) is the widely expressed system y+ transporter for cationic amino acids (CAT-1). Like other retroviruses, ecotropic MuLV infection eliminates virus-binding sites from cell surfaces and results in complete interference to superinfection. Surprisingly, infection causes only partial (ca 40 to 60%) loss of mouse CAT-1 transporter activity. The NIH/Swiss mouse CAT-1 (mCAT-1) contains 622 amino acids with 14 hydrophobic potential membrane-spanning sequences, and it is known that the third extracellular loop from the amino terminus is required for virus binding. Although loop 3 is hypervariable in different species and mouse strains, consistent with its proposed role in virus-host coevolution, loop 3 sequences of both susceptible and resistant species contain consensus sites for N-linked glycosylation. Both of the consensus sites in loop 3 of mCAT-1 are known to be glycosylated and to contain oligosaccharides with diverse sizes (J. W. Kim and J. M. Cunningham, J. Biol. Chem. 268:16316-16320, 1993). We confirmed by several lines of evidence that N-linked glycosylation occludes a potentially functional virus-binding site in the CAT-1 protein of hamsters, thus contributing to resistance of that species. To study the role of receptor glycosylation in animals susceptible to infection, we eliminated loop 3 glycosylation sites by mutagenesis of an mCAT-1 cDNA clone, and we expressed wild-type and mutant receptors in mink fibroblasts and Xenopus oocytes. These receptors had indistinguishable transport properties, as determined by kinetic and voltage-jump electrophysiological studies of arginine uptake in oocytes and by analyses Of L-[3H]arginine uptake in mink cells. Bindings of ecotropic envelope glycoprotein gp7O to the accessible receptor sites on surfaces of mink cells expressing wild-type or mutant mCAT-1 were not significantly different in kinetics or in

  2. N-linked Glycosylation Enrichment for In-depth Cell Surface Proteomics of Diffuse Large B-cell Lymphoma Subtypes*

    PubMed Central

    Deeb, Sally J.; Cox, Juergen; Schmidt-Supprian, Marc; Mann, Matthias

    2014-01-01

    Global analysis of lymphoma genome integrity and transcriptomes tremendously advanced our understanding of their biology. Technological advances in mass spectrometry-based proteomics promise to complete the picture by allowing the global quantification of proteins and their post-translational modifications. Here we use N-glyco FASP, a recently developed mass spectrometric approach using lectin-enrichment, in conjunction with a super-SILAC approach to quantify N-linked glycoproteins in lymphoma cells. From patient-derived diffuse large B-cell lymphoma cell lines, we mapped 2383 glycosites on 1321 protein groups, which were highly enriched for cell membrane proteins. This N-glyco subproteome alone allowed the segregation of the ABC from the GCB subtypes of diffuse large B-cell lymphoma, which before gene expression studies had been considered one disease entity. Encouragingly, many of the glycopeptides driving the segregation belong to proteins previously characterized as segregators in a deep proteome study of these subtypes (S. J. Deeb et al. MCP 2012 PMID 22442255). This conforms to the high correlation that we observed between the expression level of the glycosites and their corresponding proteins. Detailed examination of glycosites and glycoprotein expression levels uncovered, among other interesting findings, enrichment of transcription factor binding motifs, including known NF-kappa-B related ones. Thus, enrichment of a class of post-translationally modified peptides can classify cancer types as well as reveal cancer specific mechanistic changes. PMID:24190977

  3. Cell surface glycoproteins from Thermoplasma acidophilum are modified with an N-linked glycan containing 6-C-sulfofucose.

    PubMed

    Vinogradov, Evgeny; Deschatelets, Lise; Lamoureux, Marc; Patel, Girishchandra B; Tremblay, Tammy-Lynn; Robotham, Anna; Goneau, Marie-France; Cummings-Lorbetskie, Cathy; Watson, David C; Brisson, Jean-Robert; Kelly, John F; Gilbert, Michel

    2012-09-01

    Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at pH 2 and 59°C. This extremophile is remarkable by the absence of a cell wall or an S-layer. Treating the cells with Triton X-100 at pH 3 allowed the extraction of all of the cell surface glycoproteins while keeping cells intact. The extracted glycoproteins were partially purified by cation-exchange chromatography, and we identified five glycoproteins by N-terminal sequencing and mass spectrometry of in-gel tryptic digests. These glycoproteins are positive for periodic acid-Schiff staining, have a high content of Asn including a large number in the Asn-X-Ser/Thr sequon and have apparent masses that are 34-48% larger than the masses deduced from their amino acid sequences. The pooled glycoproteins were digested with proteinase K and the purified glycopeptides were analyzed by NMR. Structural determination showed that the carbohydrate part was represented by two structures in nearly equal amounts, differing by the presence of one terminal mannose residue. The larger glycan chain consists of eight residues: six hexoses, one heptose and one sugar with an unusual residue mass of 226 Da which was identified as 6-deoxy-6-C-sulfo-D-galactose (6-C-sulfo-D-fucose). Mass spectrometry analyses of the peptides obtained by trypsin and chymotrypsin digestion confirmed the principal structures to be those determined by NMR and identified 14 glycopeptides derived from the main glycoprotein, Ta0280, all containing the Asn-X-Ser/Thr sequons. Thermoplasma acidophilum appears to have a "general" protein N-glycosylation system that targets a number of cell surface proteins.

  4. N-linked glycosylation of platelet P2Y12 ADP receptor is essential for signal transduction but not for ligand binding or cell surface expression.

    PubMed

    Zhong, Xiaotian; Kriz, Ron; Seehra, Jasbir; Kumar, Ravindra

    2004-03-26

    P(2)Y(12) receptor is a G(i)-coupled adenosine diphosphate (ADP) receptor with a critical role in platelet aggregation. It contains two potential N-linked glycosylation sites at its extra cellular amino-terminus, which may modulate its activity. Studies of both tunicamycin treatment and site-directed mutagenesis have revealed a dispensable role of the N-linked glycosylation in the receptor's surface expression and ligand binding activity. However, the non-glycosylated P(2)Y(12) receptor is defective in the P(2)Y(12)-mediated inhibition of the adenylyl cyclase activity. Thus the study uncovers an unexpected vital role of N-linked glycans in receptor's signal transducing step but not in surface expression or ligand binding.

  5. N-linked glycan profiling in neuroblastoma cell lines.

    PubMed

    Hu, Yunli; Mayampurath, Anoop; Khan, Saira; Cohen, Joanna K; Mechref, Yehia; Volchenboum, Samuel L

    2015-05-01

    Although MYCN amplification has been associated with aggressive neuroblastoma, the molecular mechanisms that differentiate low-risk, MYCN-nonamplified neuroblastoma from high-risk, MYCN-amplified disease are largely unknown. Genomic and proteomic studies have been limited in discerning differences in signaling pathways that account for this heterogeneity. N-Linked glycosylation is a common protein modification resulting from the attachment of sugars to protein residues and is important in cell signaling and immune response. Aberrant N-linked glycosylation has been routinely linked to various cancers. In particular, glycomic markers have often proven to be useful in distinguishing cancers from precancerous conditions. Here, we perform a systematic comparison of N-linked glycomic variation between MYCN-nonamplified SY5Y and MYCN-amplified NLF cell lines with the aim of identifying changes in sugar abundance linked to high-risk neuroblastoma. Through a combination of liquid chromatography-mass spectrometry and bioinformatics analysis, we identified 16 glycans that show a statistically significant change in abundance between NLF and SY5Y samples. Closer examination revealed the preference for larger (in terms of total monosaccharide count) and more sialylated glycan structures in the MYCN-amplified samples in comparison to smaller, nonsialylated glycans that are more dominant in the MYCN-nonamplified samples. These results offer clues for deriving marker candidates for accurate neuroblastoma risk diagnosis.

  6. Biosynthesis and Role of N-Linked Glycosylation in Cell Surface Structures of Archaea with a Focus on Flagella and S Layers

    PubMed Central

    Jarrell, Ken F.; Jones, Gareth M.; Nair, Divya B.

    2010-01-01

    The genetics and biochemistry of the N-linked glycosylation system of Archaea have been investigated over the past 5 years using flagellins and S layers as reporter proteins in the model organisms, Methanococcus voltae, Methanococcus maripaludis, and Haloferax volcanii. Structures of archaeal N-linked glycans have indicated a variety of linking sugars as well as unique sugar components. In M. voltae, M. maripaludis, and H. volcanii, a number of archaeal glycosylation genes (agl) have been identified by deletion and complementation studies. These include many of the glycosyltransferases and the oligosaccharyltransferase needed to assemble the glycans as well as some of the genes encoding enzymes required for the biosynthesis of the sugars themselves. The N-linked glycosylation system is not essential for any of M. voltae, M. maripaludis, or H. volcanii, as demonstrated by the successful isolation of mutants carrying deletions in the oligosaccharyltransferase gene aglB (a homologue of the eukaryotic Stt3 subunit of the oligosaccharyltransferase complex). However, mutations that affect the glycan structure have serious effects on both flagellation and S layer function. PMID:20976295

  7. N-Linked Glycosylation Is Required for Vacuolar H(+) -ATPase (V-ATPase) a4 Subunit Stability, Assembly, and Cell Surface Expression.

    PubMed

    Esmail, Sally; Yao, Yeqi; Kartner, Norbert; Li, Jing; Reithmeier, Reinhart A F; Manolson, Morris F

    2016-12-01

    The a subunit is the largest of 14 different subunits that make up the V-ATPase complex. In mammalian species this membrane protein has four paralogous isoforms, a1-a4. Clinically, a subunit isoforms are implicated in diverse diseases; however, little is known about their structure and function. The subunit has conserved, predicted N-glycosylation sites, and the a3 isoform has been directly shown to be N-glycosylated. Here we ask if human a4 (ATP6V0A4) is N-glycosylated at the predicted site, Asn489. We transfected HEK 293 cells, using the pCDNA3.1 expression-vector system, to express cDNA constructs of epitope-tagged human a4 subunit, with or without mutations to eliminate the putative glycosylation site. Glycosylation was characterized also by treatment with endoglycosidases; expression and localization were assessed by immunoblotting and immunofluorescence. Endoglycosidase-treated wild type (WT) a4 showed increased relative mobility on immunoblots, compared with untreated WT a4. This relative mobility was identical to that of unglycosylated mutant a4(N489D) , demonstrating that the a4 subunit is glycosylated. Cycloheximide pulse-chase experiments showed that the unglycosylated subunit degraded at a higher rate than the N-glycosylated form. Unglycosylated a4 was degraded mostly in the proteasomal pathway, but also, in part, through the lysosomal pathway. Immunofluorescence colocalization data showed that unglycosylated a4 was mostly retained in the ER, and that plasma membrane trafficking was defective. Co-immunoprecipitation studies suggested that a4(N489D) does not assemble with the V-ATPase V1 domain. Taken together, these data show that N-glycosylation plays a crucial role in a4 stability, and in V-ATPase assembly and trafficking to the plasma membrane. J. Cell. Biochem. 117: 2757-2768, 2016. © 2016 Wiley Periodicals, Inc.

  8. Recombinant sialidase NanA (rNanA) cleaves α2-3 linked sialic acid of host cell surface N-linked glycoprotein to promote Edwardsiella tarda infection.

    PubMed

    Chigwechokha, Petros Kingstone; Tabata, Mutsumi; Shinyoshi, Sayaka; Oishi, Kazuki; Araki, Kyosuke; Komatsu, Masaharu; Itakura, Takao; Shiozaki, Kazuhiro

    2015-11-01

    Edwardsiella tarda is one of the major pathogenic bacteria affecting both marine and freshwater fish species. Sialidase NanA expressed endogenously in E. tarda is glycosidase removing sialic acids from glycoconjugates. Recently, the relationship of NanA sialidase activity to E. tarda infection has been reported, however, the mechanism with which sialidase NanA aids the pathogenicity of E. tarda remained unclear. Here, we comprehensively determined the biochemical properties of NanA towards various substrates in vitro to provide novel insights on the potential NanA target molecule at the host cell. GAKS cell pretreated with recombinant NanA showed increased susceptibility to E. tarda infection. Moreover, sialidase inhibitor treated E. tarda showed a significantly reduced ability to infect GAKS cells. These results indicate that NanA-induced desialylation of cell surface glycoconjugates is essential for the initial step of E. tarda infection. Among the natural substrates, NanA exhibited the highest activity towards 3-sialyllactose, α2-3 linked sialic acid carrying sialoglycoconjugates. Supporting this finding, intact GAKS cell membrane exposed to recombinant NanA showed changes of glycoconjugates only in α2-3 sialo-linked glycoproteins, but not in glycolipids and α2-6 sialo-linked glycoproteins. Lectin staining of cell surface glycoprotein provided further evidence that α2-3 sialo-linkage of the N-linked glycoproteins was the most plausible target of NanA sialidase. To confirm the significance of α2-3 sialo-linkage desialylation for E. tarda infection, HeLa cells which possessed lower amount of α2-3 sialo-linkage glycoprotein were used for infection experiment along with GAKS cells. As a result, infection of HeLa cells by E. tarda was significantly reduced when compared to GAKS cells. Furthermore, E. tarda infection was significantly inhibited by mannose pretreatment suggesting that the bacterium potentially recognizes and binds to mannose or mannose containing

  9. N-Linked glycans on dengue viruses grown in mammalian and insect cells

    PubMed Central

    Hacker, Kari; White, Laura; de Silva, Aravinda M.

    2009-01-01

    This study compared the ability of mosquito and mammalian cell-derived dengue virus (DENV) to infect human dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN)-expressing cells and characterized the structure of envelope (E) protein N-linked glycans on DENV derived from the two cell types. DENVs derived from both cell types were equally effective at infecting DC-SIGN-expressing human monocytes and dendritic cells. The N-linked glycans on mosquito cell-derived virus were a mix of high-mannose and paucimannose glycans. In virus derived from mammalian cells, the N-linked glycans were a mix of high-mannose and complex glycans. These results indicate that N-linked glycans are incompletely processed during DENV egress from cells, resulting in high-mannose glycans on viruses derived from both cell types. Studies with full-length and truncated E protein demonstrated that incomplete processing was most likely a result of the poor accessibility of glycans on the membrane-anchored protein. PMID:19494052

  10. Natural cell-mediated cytotoxicity: possible role of N-linked glycoproteins

    SciTech Connect

    Oeltmann, T.N.; Chambers, W.H.

    1986-05-01

    The authors have examined the role of N-linked glycoproteins in natural cell-mediated cytotoxicity (NCMC) by treating effector cells or target cells with swainsonine, a specific inhibitor of golgi mannosidase II which is critical for N-linked glycoprotein processing. They have also examined the effects of alpha-mannosidase, an exoglycosidase specific for alpha-linked mannose residues, on both target and effector cells. Pretreatment of nonadherent mononuclear cells with swainsonine (18 hr) resulted in an inhibition of lysis of K-562 target cells as measured by LDH release. Protein synthesis was not inhibited as measured by incorporation of /sup 14/C-amino acids. However, oligosaccharide processing was altered as measured by incorporation of 2(/sup 3/H)-mannose. Similar treatment did not inhibit target cell-effector cell conjugation. Pretreatment of nonadherent mononuclear cells with alpha-mannosidase (1 hr) did not result in a reduction in NK cell function. However, alpha-mannosidase did cause a release of mannose from treated cells. These results suggest that N-linked glycoproteins may play a role in NCMC, but not at the level of recognition and binding.

  11. Natural cell-mediated cytotoxicity: possible role of N-linked glycoproteins

    SciTech Connect

    Chambers, W.H.; Oeltmann, T.N.

    1986-03-01

    The authors have examined the role of N-linked glycoproteins in natural cell-mediated cytotoxicity (NCMC) by treating effector cells or target cells with swainsonine, a specific inhibitor of golgi mannosidase II which is critical for N-linked glycoprotein processing. They have also examined the effects of alpha-mannosidase, an exoglycosidase specific for alpha-linked mannose residues, on both target and effector cells. Pretreatment of nonadherent mononuclear cells with swainsonine (18 hr) resulted in an inhibition of lysis of K-562 target cells as measured by LDH release. Protein synthesis was not inhibited as measured by incorporation of /sup 14/C-amino acids. However, oligosaccharide processing was altered as measured by incorporation of 2(/sup 3/H)-mannose. Similar treatment did not inhibit target cell-effector cell conjugation. Pretreatment of nonadherent mononuclear cells with alpha-mannosidase (1 hr) did not result in a reduction in NK cell function. However, alpha-mannosidase did cause a release of mannose from treated cells. These results suggest that N-linked glycoproteins may play a role in NCMC, but not at the level of recognition and binding.

  12. Archaeal surface appendages: their function and the critical role of N-linked glycosylation in their assembly

    NASA Astrophysics Data System (ADS)

    Jarrell, Ken F.; Nair, Divya B.; Jones, Gareth M.; Aizawa, S.-I.; Chong, James J. P.; Stark, Meg; Logan, Susan M.; Vinogradov, Evgeny; Kelly, John F.

    2011-10-01

    Many cultivated archaea are extremophiles and, as such, various archaea inhabit some of the most inhospitable niches on the planet in terms of temperature, pH, salinity and anaerobiosis. Different archaeal species have been shown to produce a number of unusual and sometimes unique surface structures. The best studied of these are flagella which are fundamentally different from bacterial flagella and instead bear numerous similarities to bacterial type IV pili in their structure and likely assembly. The major structural proteins, flagellins, are made as preproteins with type IV pilin-like signal peptides processed by a specific signal peptidase. In addition, the flagellins are glycoproteins with attached N-linked glycans. Both of these posttranslational modifications have been studied in the anaerobic archaeon, Methanococcus maripaludis, an organism which also possesses other surface appendages, an unusual version of type IV pili, whose major constituents are also glycoproteins. Analysis of mutants unable to make either or both of flagella and pili demonstrated that both are essential for attachment to surfaces. A number of mutants defective in the assembly and biosynthesis of the tetrasaccharide N-linked to the flagellins have been isolated. Investigations of these mutants by electron microscopy, mass spectrometry and motility assays have demonstrated that flagellins possessing no attached glycan or a glycan truncated to a single sugar cannot assemble flagella on their surface. Mutants which can attach a glycan of 2 or 3 sugars to flagellins assemble flagella but they are impaired in their swimming compared with wildtype cells which attach the tetrasaccharide to their flagellins.

  13. N-linked glycosylation is required for plasma membrane localization of D5, but not D1, dopamine receptors in transfected mammalian cells.

    PubMed

    Karpa, K D; Lidow, M S; Pickering, M T; Levenson, R; Bergson, C

    1999-11-01

    We have analyzed the role of N-linked glycosylation in functional cell surface expression of the D1 and D5 dopamine receptor subtypes. Treatment of transfected HEK 293 cells with tunicamycin, an inhibitor of N-linked oligosaccharide addition, was found to prevent localization of D5 receptors in the plasma membrane. In contrast, tunicamycin treatment had no effect on the plasma membrane localization of the D1 receptor. Polymerase chain reaction mutagenesis was used to generate a panel of D5 receptors containing mutations in the three predicted sites of N-linked glycosylation. Expression of mutant receptors indicated that glycosylation of residue N7 was the major determinant of D5 receptor plasma membrane localization. Mutation of a comparable site in the D1 receptor at position N5 had no effect on the delivery of the D1 receptor to the cell surface. Tunicamycin treatment during receptor biosynthesis, but not N-glycosidase F digestion of mature receptors, abrogated binding of the D5 receptor antagonist [(3)H]SCH23390, suggesting that while oligosaccharide moieties play a key role in the cell surface expression of D5 receptors, they do not appear to contribute to the receptor's ligand binding properties. Together, our data indicate a differential requirement for N-linked glycosylation in functional cell surface expression of D1 and D5 dopamine receptors.

  14. Contribution of N-linked glycans on HSV-2 gB to cell–cell fusion and viral entry

    SciTech Connect

    Luo, Sukun; Hu, Kai; He, Siyi; Wang, Ping; Zhang, Mudan; Huang, Xin; Du, Tao; Zheng, Chunfu; Liu, Yalan; Hu, Qinxue

    2015-09-15

    HSV-2 is the major cause of genital herpes and its infection increases the risk of HIV-1 acquisition and transmission. HSV-2 glycoprotein B together with glycoproteins D, H and L are indispensable for viral entry, of which gB, as a class III fusogen, plays an essential role. HSV-2 gB has seven potential N-linked glycosylation (N-CHO) sites, but their significance has yet to be determined. For the first time, we systematically analyzed the contributions of N-linked glycans on gB to cell–cell fusion and viral entry. Our results demonstrated that, of the seven potential N-CHO sites on gB, mutation at N390, N483 or N668 decreased cell–cell fusion and viral entry, while mutation at N133 mainly affected protein expression and the production of infectious virus particles by blocking the transport of gB from the endoplasmic reticulum to Golgi. Our findings highlight the significance of N-linked glycans on HSV-2 gB expression and function. - Highlights: • N-linked glycan at N133 is important for gB intracellular trafficking and maturation. • N-linked glycans at N390, N483 and N668 on gB are necessary for optimal cell–cell fusion. • N-linked glycans at N390, N483 and N668 on gB are necessary for optimal viral entry.

  15. Mapping N-linked Glycosylation Sites in the Secretome and Whole Cells of Aspergillus niger Using Hydrazide Chemistry and Mass Spectrometry

    SciTech Connect

    Wang, Lu; Aryal, Uma K.; Dai, Ziyu; Mason, Alisa C.; Monroe, Matthew E.; Tian, Zhixin; Zhou, Jianying; Su, Dian; Weitz, Karl K.; Liu, Tao; Camp, David G.; Smith, Richard D.; Baker, Scott E.; Qian, Weijun

    2012-01-01

    Protein glycosylation is known to play an essential role in both cellular functions and the secretory pathways; however, little information is available on the dynamics of glycosylated N-linked glycosites of fungi. Herein we present the first extensive mapping of glycosylated N-linked glycosites in industrial strain Aspergillus niger by applying an optimized solid phase enrichment of glycopeptide protocol using hydrazide modified magnetic beads. The enrichment protocol was initially optimized using mouse plasma and A. niger secretome samples, which was then applied to profile N-linked glycosites from both the secretome and whole cell lysates of A. niger. A total of 847 unique N-linked glycosites and 330 N-linked glycoproteins were confidently identified by LC-MS/MS. Based on gene ontology analysis, the identified N-linked glycoproteins in the whole cell lysate were primarily localized in the plasma membrane, endoplasmic reticulum, golgi apparatus, lysosome, and storage vacuoles. The identified N-linked glycoproteins are involved in a wide range of biological processes including gene regulation and signal transduction, protein folding and assembly, protein modification and carbohydrate metabolism. The extensive coverage of glycosylated N-linked glycosites along with identification of partial N-linked glycosylation in those enzymes involving in different biochemical pathways provide useful information for functional studies of N-linked glycosylation and their biotechnological applications in A. niger.

  16. UniPep - a database for human N-linked glycosites: a resource for biomarker discovery

    PubMed Central

    Zhang, Hui; Loriaux, Paul; Eng, Jimmy; Campbell, David; Keller, Andrew; Moss, Pat; Bonneau, Richard; Zhang, Ning; Zhou, Yong; Wollscheid, Bernd; Cooke, Kelly; Yi, Eugene C; Lee, Hookeun; Peskind, Elaine R; Zhang, Jing; Smith, Richard D; Aebersold, Ruedi

    2006-01-01

    There has been considerable recent interest in proteomic analyses of plasma for the purpose of discovering biomarkers. Profiling N-linked glycopeptides is a particularly promising method because the population of N-linked glycosites represents the proteomes of plasma, the cell surface, and secreted proteins at very low redundancy and provides a compelling link between the tissue and plasma proteomes. Here, we describe UniPep - a database of human N-linked glycosites - as a resource for biomarker discovery. PMID:16901351

  17. High molecular weight components containing N-linked oligosaccharides of Ascaris suum extract inhibit the dendritic cells activation through DC-SIGN and MR.

    PubMed

    Favoretto, Bruna C; Casabuono, Adriana A C; Portes-Junior, José A; Jacysyn, Jacqueline F; Couto, Alicia S; Faquim-Mauro, Eliana L

    2017-04-09

    Helminths, as well as their secretory/excretory products, induce a tolerogenic immune microenvironment. High molecular weight components (PI) from Ascaris suum extract down-modulate the immune response against ovalbumin (OVA). The PI exerts direct effect on dendritic cells (DCs) independent of TLR 2, 4 and MyD88 molecule and, thus, decreases the T lymphocytes response. Here, we studied the glycoconjugates in PI and the role of C-type lectin receptors (CLRs), DC-SIGN and MR, in the modulation of DCs activity. Our data showed the presence of glycoconjugates with high mannose- and complex-type N-linked oligosaccharide chains and phosphorylcholine residues on PI. In addition, these N-linked glycoconjugates inhibited the DCs maturation induced by LPS. The binding and internalization of PI-Alexa were decreased on DCs previously incubated with mannan, anti-DC-SIGN and/or anti-MR antibodies. In agreement with this, the incubation of DCs with mannan, anti-DC-SIGN and/or anti-MR antibodies abolished the down-modulatory effect of PI on these cells. It was also observed that the blockage of CLRs, DC-SIGN and MR on DCs reverted the inhibitory effect of PI in in vitro T cells proliferation. Therefore, our data show the involvement of DC-SIGN and MR in the recognition and consequent modulatory effect of N-glycosylated components of PI on DCs.

  18. Perturbation of N-linked oligosaccharide structure results in an altered incorporation of (/sup 3/H)palmitate into specific proteins in Chinese hamster ovary cells

    SciTech Connect

    Wellner, R.B.; Ghosh, P.C.; Roecklein, B.; Wu, H.C.

    1987-09-25

    Increased (/sup 3/H)palmitate incorporation into specific cellular proteins has been reported to occur in Chinese hamster ovary and yeast mutant cells. In this paper we report studies concerning the relationship between N-linked oligosaccharide structure and (/sup 3/H)palmitate incorporation into proteins of Chinese hamster ovary (CHO) cells. We have compared the incorporation of (/sup 3/H)palmitate into proteins of wild-type and four different mutant CHO cell lines defective in various steps of N-linked protein glycosylation. Sodium dodecyl sulfate-gel electrophoretic analysis showed that three of the mutants exhibited increased (/sup 3/H)palmitate incorporation into several CHO cellular proteins (approximately 30,000-38,000 molecular weight) as compared to the wild-type cells. One of the affected mutants which accumulates the Man5Gn2Asn intermediate structure was examined in detail. In agreement with earlier reports, virtually all of the (/sup 3/H) palmitate-labeled proteins of both wild-type and mutant cell lines are membrane-bound. Pretreatment of the mutant cell line with tunicamycin blocked the increased (/sup 3/H)palmitate incorporation into the two specific proteins (both of approximately 30,000 molecular weight) observed in untreated cells; the decreased incorporation of (/sup 3/H)palmitate into the 30,000 molecular weight species was accompanied by a concomitant increase in the incorporation of (/sup 3/H)palmitate into two proteins of approximately 20,000 molecular weight. Pretreatment of wild-type cells with tunicamycin also caused increased (/sup 3/H)palmitate incorporation into the 20,000 molecular weight species.

  19. Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection

    PubMed Central

    Hamm, Melissa; Wang, Yang; Rustandi, Richard R.

    2013-01-01

    The N-linked glycosylation in recombinant monoclonal antibodies (mAb) occurs at Asn297 on the Fc region in the CH2 domain. Glycosylation heterogeneities have been well documented to affect biological activities such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) through their interaction with Fc-receptors. Hence, it is critical to monitor and characterize the N-linked glycosylation profile in a therapeutic protein such as a mAb for product consistency. In one approach, the glycans are first released from the mAb using an enzyme specific digestion, such as Protein N-Glycosidase F (PNGase) and subsequently they are labeled using a fluorophore, for example, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) . Here we have applied this approach and used Capillary Electrophoresis with Laser-Induced Fluorescence detection (CE-LIF) to analyze a recombinant mAb produced in murine myeloma (NS0) cells. The technique provides short analysis times, efficient separations, and high sensitivity. CE-LIF peak identification was done by a combination of glycan standards and treatment with various exoglycosidases. Furthermore, the APTS-labeled glycans were also analyzed using hydrophilic interaction chromatography (HILIC) high performance liquid chromatography (HPLC) to aid identification of minor peaks by sample collection and off-line mass spectrometry (MS) analysis. PMID:24276024

  20. Specificity of the murine IgD receptor on T cells is for N-linked glycans on IgD molecules.

    PubMed Central

    Amin, A R; Tamma, S M; Oppenheim, J D; Finkelman, F D; Kieda, C; Coico, R F; Thorbecke, G J

    1991-01-01

    IgD receptors on murine T cells have been reported in this issue [Tamma, S. M. L., Amin, A. R., Finkelman, F. D., Chen, Y.-W., Thorbecke, G. J. & Coico, R. F. (1991) Proc. Natl. Acad. Sci. USA 88, 9233-9237] to bind either the first or third constant region of the heavy-chain of IgD molecules--findings that could not be satisfactorily explained by IgD amino acid sequences. We now find that boiled IgD molecules or low-Mr fragments from protease-digested IgD still inhibit binding of IgD-coated erythrocytes to IgD receptors. This inhibitory activity can be absorbed with the murine IgD-binding lectin from Griffonia simplicifolia 1 (GS-1) immobilized on Sepharose. N-linked glycans, obtained from N-glycanase-treated IgD and purified by binding to GS-1-Sepharose, also inhibit rosette formation of T-helper cells bearing receptors for IgD with IgD- or mutant IgD-coated erythrocytes. Deglycosylated IgD, produced by treatment with N-glycanase, no longer binds to the lectin and fails to inhibit IgD rosetting. Binding of intact IgD to T cells is also competitively inhibited by N-acetylgalactosamine, galactose, N-acetylglucosamine, and neoglycoproteins containing these sugars. These results clearly show that N-linked glycans, present in both the first and third constant regions of the delta heavy-chain domains, are prerequisites for binding of IgD to IgD receptors. Images PMID:1924387

  1. Absence of an N-Linked Glycosylation Motif in the Glycoprotein of the Live-Attenuated Argentine Hemorrhagic Fever Vaccine, Candid #1, Results in Its Improper Processing, and Reduced Surface Expression

    PubMed Central

    Manning, John T.; Seregin, Alexey V.; Yun, Nadezhda E.; Koma, Takaaki; Huang, Cheng; Barral, José; de la Torre, Juan C.; Paessler, Slobodan

    2017-01-01

    Junin virus (JUNV), a highly pathogenic New World arenavirus, is the causative agent of Argentine hemorrhagic fever (AHF). The live-attenuated Candid #1 (Can) strain currently serves as a vaccine for at-risk populations. We have previously shown that the Can glycoprotein (GPC) gene is the primary gene responsible for attenuation in a guinea pig model of AHF. However, the mechanisms through which the GPC contributes to the attenuation of the Can strain remain unknown. A more complete understanding of the mechanisms underlying the attenuation and immunogenicity of the Can strain will potentially allow for the rational design of additional safe and novel vaccines. Here, we provide a detailed comparison of both RNA and protein expression profiles between both inter- and intra-segment chimeric JUNV recombinant clones expressing combinations of genes from the Can strain and the pathogenic Romero (Rom) strain. The recombinant viruses that express Can GPC, which were shown to be attenuated in guinea pigs, displayed different RNA levels and GPC processing patterns as determined by Northern and Western blot analyses, respectively. Analysis of recombinant viruses containing amino acid substitutions selected at different mouse brain passages during the generation of Can revealed that altered Can GPC processing was primarily due to the T168A substitution within G1, which eliminates an N-linked glycosylation motif. Incorporation of the T168A substitution in the Rom GPC resulted in a Can-like processing pattern of Rom GPC. In addition, JUNV GPCs containing T168A substitution were retained within the endoplasmic reticulum (ER) and displayed significantly lower cell surface expression than wild-type Rom GPC. Interestingly, the reversion A168T in Can GPC significantly increased GPC expression at the cell surface. Our results demonstrate that recombinant JUNV (rJUNV) expressing Can GPC display markedly different protein expression and elevated genomic RNA expression when compared to

  2. Sulphation of proteins secreted by a human hepatoma-derived cell line. Sulphation of N-linked oligosaccharides on alpha 2HS-glycoprotein.

    PubMed Central

    Hortin, G; Green, E D; Baenziger, J U; Strauss, A W

    1986-01-01

    Several human glycoproteins, including alpha 1-antitrypsin, alpha 1-acid glycoprotein, transferrin, caeruloplasmin and alpha 2HS-glycoprotein, synthesized by the hepatoma-derived cell line HepG2 were observed to contain covalently linked sulphate. These proteins were estimated to contain about 0.1 mol of sulphate/mol of protein. The most abundant of the sulphated glycoproteins, alpha 2HS-glycoprotein, was analysed in detail. All of the sulphate on this protein was attached to N-linked oligosaccharides which contained sialic acid and resisted release by endoglycosidase H. Several independent analytical approaches established that approx. 10% of the molecules of alpha 2HS-glycoprotein contained sulphate. Our results suggest that a number of human plasma proteins contain small amounts of sulphate linked to oligosaccharides. Images Fig. 1. Fig. 2. Fig. 3. PMID:3017304

  3. Resveratrol triggers ER stress-mediated apoptosis by disrupting N-linked glycosylation of proteins in ovarian cancer cells.

    PubMed

    Gwak, HyeRan; Kim, Soochi; Dhanasekaran, Danny N; Song, Yong Sang

    2016-02-28

    Malignant tumors have a high glucose demand and alter cellular metabolism to survive. Herein, focusing on the utility of glucose metabolism as a therapeutic target, we found that resveratrol induced endoplasmic reticulum (ER) stress-mediated apoptosis by interrupting protein glycosylation in a cancer-specific manner. Our results indicated that resveratrol suppressed the hexosamine biosynthetic pathway and interrupted protein glycosylation through GSK3β activation. Application of either biochemical intermediates of the hexosamine pathway or small molecular inhibitors of GSK3β reversed the effects of resveratrol on the disruption of protein glycosylation. Additionally, an ER UDPase, ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5), modulated protein glycosylation by Akt attenuation in response to resveratrol. By inhibition or overexpression of Akt functions, we confirmed that the glycosylation activities were dependent on ENTPD5 expression and regulated by the action of Akt in ovarian cancer cells. Resveratrol-mediated disruption of protein glycosylation induced cellular apoptosis as indicated by the up-regulation of GADD153, followed by the activation of ER-stress sensors (PERK and ATF6α). Thus, our results provide novel insight into cancer cell metabolism and protein glycosylation as a therapeutic target for cancers.

  4. Mutation of N-linked glycosylation at Asn548 in CD133 decreases its ability to promote hepatoma cell growth.

    PubMed

    Liu, Ying; Ren, Shifang; Xie, Liqi; Cui, Chunhong; Xing, Yang; Liu, Chanjuan; Cao, Benjin; Yang, Fan; Li, Yinan; Chen, Xiaoning; Wei, Yuanyan; Lu, Haojie; Jiang, Jianhai

    2015-08-21

    The membrane glycoprotein CD133 is a popular marker for cancer stem cells and contributes to cancer initiation and invasion in a number of tumor types. CD133 promotes tumorigenesis partly through an interaction between its phosphorylated Y828 residue and the PI3K regulatory subunit p85, and the interaction with β-catenin. Although CD133 glycosylation is supposed to be associated with its function, the contribution of N-glycosylation to its functions remains unclear. Here we analyzed the exact site(s) of N-glycosylation in CD133 by mass spectrometry and found that all eight potential N-glycosylation sites of CD133 could be indeed occupied by N-glycans. Loss of individual N-glycosylation sites had no effect on the level of expression or membrane localization of CD133. However, mutation at glycosylation site Asn548 significantly decreased the ability of CD133 to promote hepatoma cell growth. Furthermore, mutation of Asn548 reduced the interaction between CD133 and β-catenin and inhibited the activation of β-catenin signaling by CD133 overexpression. Our results identified the characteristics and function of CD133 glycosylation sites. These data could potentially shed light on molecular regulation of CD133 by glycosylation and enhance our understanding of the utility of glycosylated CD133 as a target for cancer therapies.

  5. Mutation of N-linked glycosylation at Asn548 in CD133 decreases its ability to promote hepatoma cell growth

    PubMed Central

    Xie, Liqi; Cui, Chunhong; Xing, Yang; Liu, Chanjuan; Cao, Benjin; Yang, Fan; Li, Yinan; Chen, Xiaoning; Wei, Yuanyan; Lu, Haojie; Jiang, Jianhai

    2015-01-01

    The membrane glycoprotein CD133 is a popular marker for cancer stem cells and contributes to cancer initiation and invasion in a number of tumor types. CD133 promotes tumorigenesis partly through an interaction between its phosphorylated Y828 residue and the PI3K regulatory subunit p85, and the interaction with β-catenin. Although CD133 glycosylation is supposed to be associated with its function, the contribution of N-glycosylation to its functions remains unclear. Here we analyzed the exact site(s) of N-glycosylation in CD133 by mass spectrometry and found that all eight potential N-glycosylation sites of CD133 could be indeed occupied by N-glycans. Loss of individual N-glycosylation sites had no effect on the level of expression or membrane localization of CD133. However, mutation at glycosylation site Asn548 significantly decreased the ability of CD133 to promote hepatoma cell growth. Furthermore, mutation of Asn548 reduced the interaction between CD133 and β-catenin and inhibited the activation of β-catenin signaling by CD133 overexpression. Our results identified the characteristics and function of CD133 glycosylation sites. These data could potentially shed light on molecular regulation of CD133 by glycosylation and enhance our understanding of the utility of glycosylated CD133 as a target for cancer therapies. PMID:26029999

  6. Insertion of N-linked glycosylation sites in the variable regions of the human immunodeficiency virus type 1 surface glycoprotein through AAT triplet reiteration.

    PubMed Central

    Bosch, M L; Andeweg, A C; Schipper, R; Kenter, M

    1994-01-01

    Variable regions with sequence length variation in the human immunodeficiency virus type 1 envelope exhibit an unusual pattern of codon usage with AAT, ACT, and AGT together composing > 70% of all codons used. We postulate that this distribution is caused by insertion of AAT triplets followed by point mutations and selection. Accumulation of the encoded amino acids (asparagine, serine, and threonine) leads to the creation of new N-linked glycosylation sites, which helps the virus to escape from the immune pressure exerted by virus-neutralizing antibodies. PMID:7933144

  7. Characterization of disease-associated N-linked glycoproteins.

    PubMed

    Tian, Yuan; Zhang, Hui

    2013-02-01

    N-linked glycoproteins play important roles in biological processes, including cell-to-cell recognition, growth, differentiation, and programmed cell death. Specific N-linked glycoprotein changes are associated with disease progression and identification of these N-linked glycoproteins has potential for use in disease diagnosis, prognosis, and prediction of treatments. In this review, we summarize common strategies for N-linked glycoprotein characterization and applications of these strategies to identification of glycoprotein changes associated with disease states. We also review the N-linked glycoproteins altered in diseases such as breast cancer, lung cancer, and prostate cancer. Although assays for these glycoproteins have potential clinical utility, research is needed to translate these glycoproteins to clinical biomarkers.

  8. N-Linked Glycosylation of Protease-activated Receptor-1 Second Extracellular Loop

    PubMed Central

    Soto, Antonio G.; Trejo, JoAnn

    2010-01-01

    Protease-activated receptor-1 (PAR1) contains five N-linked glycosylation consensus sites as follows: three residing in the N terminus and two localized on the surface of the second extracellular loop (ECL2). To study the effect of N-linked glycosylation in the regulation of PAR1 signaling and trafficking, we generated mutants in which the critical asparagines of the consensus sites were mutated. Here, we report that both the PAR1 N terminus and ECL2 serve as sites for N-linked glycosylation but have different functions in the regulation of receptor signaling and trafficking. N-Linked glycosylation of the PAR1 N terminus is important for transport to the cell surface, whereas the PAR1 mutant lacking glycosylation at ECL2 (NA ECL2) trafficked to the cell surface like the wild-type receptor. However, activated PAR1 NA ECL2 mutant internalization was impaired compared with wild-type receptor, whereas constitutive internalization of unactivated receptor remained intact. Remarkably, thrombin-activated PAR1 NA ECL2 mutant displayed an enhanced maximal signaling response compared with wild-type receptor. The increased PAR1 NA ECL2 mutant signaling was not due to defects in the ability of thrombin to cleave the receptor or signal termination mechanisms. Rather, the PAR1 NA ECL2 mutant displayed a greater efficacy in thrombin-stimulated G protein signaling. Thus, N-linked glycosylation of the PAR1 extracellular surface likely influences ligand docking interactions and the stability of the active receptor conformation. Together, these studies strongly suggest that N-linked glycosylation of PAR1 at the N terminus versus the surface of ECL2 serves distinct functions critical for proper regulation of receptor trafficking and the fidelity of thrombin signaling. PMID:20368337

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

    PubMed Central

    Thannhauser, Theodore W.; Shen, Miaoqing; Sherwood, Robert; Howe, Kevin; Fish, Tara; Yang, Yong; Chen, Wei; Zhang, Sheng

    2013-01-01

    Glycosylation is a common post-translational modification of plant proteins that impacts a large number of important biological processes. Nevertheless, the impacts of differential site occupancy and the nature of specific glycoforms are obscure. Historically, characterization of glycoproteins has been difficult due to the distinct physicochemical properties of the peptidyl and glycan moieties, the variable and dynamic nature of the glycosylation process, their heterogeneous nature, and the low relative abundance of each glycoform. In this study, we explore a new pipeline developed for large-scale empirical identification of N-linked glycoproteins of tomato fruit as part of our ongoing efforts to characterize the tomato secretome. The workflow presented involves a combination of lectin affinity, tryptic digestion, ion-pairing HILIC and precursor ion-driven data dependent MS/MS analysis with a script to facilitate the identification and characterization of occupied N-linked glycosylation sites. A total of 212 glycoproteins were identified in this study, in which 26 glycopeptides from 24 glycoproteins were successfully characterized in just one HILIC fraction. Further precursor ion discovery (PID)-based MS/MS and deglycosylation followed by high accuracy and resolution MS analysis were used to confirm the glycosylation sites and determine site occupancy rates. The workflow reported is robust and capable of producing large amounts of empirical data involving N-linked glycosylation sites and their associated glycoforms. PMID:23580464

  10. N-Linked glycoengineering for human therapeutic proteins in bacteria.

    PubMed

    Pandhal, Jagroop; Wright, Phillip C

    2010-09-01

    Approx. 70% of human therapeutic proteins are N-linked glycoproteins, and therefore host cells for production must contain the relevant protein modification machinery. The discovery and characterisation of the N-linked glycosylation pathway in the pathogenic bacterium Campylobacter jejuni, and subsequently its functional transfer to Escherichia coli, presents the opportunity of using prokaryotes as cell factories for therapeutic protein production. Not only could bacteria reduce costs and increase yields, but the improved feasibility to genetically control microorganisms means new and improved pharmacokinetics of therapeutics is an exciting possibility. This is a relatively new concept, and progress in bacterial N-glycosylation characterisation is reviewed and metabolic engineering targets revealed.

  11. N-linked protein glycosylation in a bacterial system.

    PubMed

    Nothaft, Harald; Liu, Xin; McNally, David J; Szymanski, Christine M

    2010-01-01

    N-Linked protein glycosylation is conserved throughout the three domains of life and influences protein function, stability, and protein complex formation. N-Linked glycosylation is an essential process in Eukaryotes; however, although N-glycosylation affects multiple cellular processes in Archaea and Bacteria, it is not needed for cell survival. Methods for the analyses of N-glycosylation in eukaryotes are well established, but comparable techniques for the analyses of the pathways in Bacteria and Archaea are needed. In this chapter we describe new methods for the detection and analyses of N-linked, and the recently discovered free oligosaccharides (fOS), from whole cell lysates of Campylobacter jejuni using non-specific pronase E digestion and permethylation followed by mass spectrometry. We also describe the expression and immunodetection of the model N-glycoprotein, AcrA, fused to a hexa-histidine tag to follow protein glycosylation in C. jejuni. This chapter concludes with the recent demonstration that high-resolution magic angle spinning NMR of intact bacterial cells provides a rapid, non-invasive method for analyzing fOS in C. jejuni in vivo. This combination of techniques provides a powerful tool for the exploration, quantification, and structural analyses of N-linked and free oligosaccharides in the bacterial system.

  12. Understanding of decreased sialylation of Fc-fusion protein in hyperosmotic recombinant Chinese hamster ovary cell culture: N-glycosylation gene expression and N-linked glycan antennary profile.

    PubMed

    Lee, Jong Hyun; Jeong, Yeong Ran; Kim, Yeon-Gu; Lee, Gyun Min

    2017-03-07

    To understand the effects of hyperosmolality on protein glycosylation, recombinant Chinese hamster ovary (rCHO) cells producing the Fc-fusion protein were cultivated in hyperosmolar medium resulting from adding NaCl (415 mOsm/kg). The hyperosmotic culture showed increased specific Fc-fusion protein productivity (qFc ) but a decreased proportion of acidic isoforms and sialic acid content of the Fc-fusion protein. The intracellular and extracellular sialidase activities in the hyperosmotic cultures were similar to those in the control culture (314 mOsm/kg), indicating that reduced sialylation of Fc-fusion protein at hyperosmolality was not due to elevated sialidase activity. Expression of 52 N-glycosylation-related genes was assessed by the NanoString nCounter system, which provides a direct digital readout using custom-designed color-coded probes. After three days of hyperosmotic culture, nine genes (ugp, slc35a3, slc35d2, gcs1, manea, mgat2, mgat5b, b4galt3, and b4galt4) were differentially expressed over 1.5-fold of the control, and all these genes were down-regulated. N-linked glycan analysis by anion exchange and hydrophilic interaction HPLC showed that the proportion of highly sialylated (di-, tri-, tetra-) and tetra-antennary N-linked glycans was significantly decreased upon hyperosmotic culture. Addition of betaine, an osmoprotectant, to the hyperosmotic culture significantly increased the proportion of highly sialylated and tetra-antennary N-linked glycans (P ≤ 0.05), while it increased the expression of the N-glycan branching/antennary genes (mgat2 and mgat4b). Thus, decreased expression of the genes with roles in the N-glycan biosynthesis pathway correlated with reduced sialic acid content of Fc-fusion protein caused by hyperosmolar conditions. Taken together, the results obtained in this study provide a better understanding of the detrimental effects of hyperosmolality on N-glycosylation, especially sialylation, in rCHO cells. This article is protected

  13. Cell Wall N-Linked Mannoprotein Biosynthesis Requires Goa1p, a Putative Regulator of Mitochondrial Complex I in Candida albicans

    PubMed Central

    She, Xiaodong; Calderone, Richard; Kruppa, Michael; Lowman, Douglas; Williams, David; Zhang, Lili; Gao, Ying; Khamooshi, Kasra; Liu, Weida; Li, Dongmei

    2016-01-01

    The Goa1p of Candida albicans regulates mitochondrial Complex I (CI) activities in its role as a putative CI accessory protein. Transcriptional profiling of goa1∆ revealed a down regulation of genes encoding β-oligomannosyl transferases. Herein, we present data on cell wall phenotypes of goa1∆ (strain GOA31). We used transmission electron microscopy (TEM), GPC/MALLS, and NMR to compare GOA31 to a gene-reconstituted strain (GOA32) and parental cells. We note by TEM a reduction in outer wall fibrils, increased inner wall transparency, and the loss of a defined wall layer close to the plasma membrane. GPC-MALLS revealed a reduction in high and intermediate Mw mannan by 85% in GOA31. A reduction of β-mannosyl but not α-mannosyl linkages was noted in GOA31 cells. β-(1,6)-linked glucan side chains were branched about twice as often but were shorter in length for GOA31. We conclude that mitochondrial CI energy production is highly integrated with cell wall formation. Our data also suggest that not all cell wall biosynthetic processes are dependent upon Goa1p even though it provides high levels of ATP to cells. The availability of both broadly conserved and fungal-specific mutants lacking CI subunit proteins should be useful in assessing functions of fungal-specific functions subunit proteins. PMID:26809064

  14. A mathematical model of N-linked glycoform biosynthesis.

    PubMed

    Umaña, P; Bailey, J E

    1997-09-20

    Metabolic engineering of N-linked oligosaccharide biosynthesis to produce novel glycoforms or glycoform distributions of a recombinant glycoprotein can potentially lead to an improved therapeutic performance of the glycoprotein product. Effective engineering of this pathway to maximize the fractions of beneficial glycoforms within the glycoform population of a target glycoprotein can be aided by a mathematical model of the N-linked glycosylation process. A mathematical model is presented here, whose main function is to calculate the expected qualitative trends in the N-linked oligosaccharide distribution resulting from changes in the levels of one or more enzymes involved in the network of enzyme-catalyzed reactions that accomplish N-linked oligosaccharide biosynthesis. It consists of mass balances for 33 different oligosaccharide species N-linked to a specified protein that is being transported through the different compartments of the Golgi complex. Values of the model parameters describing Chinese hamster ovary (CHO) cells were estimated from literature information. A basal set of kinetic parameters for the enzyme-catalyzed reactions acting on free oligosaccharide substrates was also obtained from the literature. The solution of the system for this basal set of parameters gave a glycoform distribution consisting mainly of complex-galactosylated oligosaccharides distributed in structures with different numbers of antennae in a fashion similar to that observed for various recombinant proteins produced in CHO cells. Other simulations indicate that changes in the oligosaccharide distribution could easily result from alteration in glycoprotein productivity within the range currently attainable in industry. The overexpression of N-acetylglucosaminyltransferase III in CHO cells was simulated under different conditions to test the main function of the model. These simulations allow a comparison of different strategies, such as simultaneous overexpression of several

  15. N-linked glycans of the human insulin receptor and their distribution over the crystal structure.

    PubMed

    Sparrow, Lindsay G; Lawrence, Michael C; Gorman, Jeffrey J; Strike, Phillip M; Robinson, Christine P; McKern, Neil M; Ward, Colin W

    2008-04-01

    The human insulin receptor (IR) homodimer is heavily glycosylated and contains a total of 19 predicted N-linked glycosylation sites in each monomer. The recent crystal structure of the IR ectodomain shows electron density consistent with N-linked glycosylation at the majority of sites present in the construct. Here, we describe a refined structure of the IR ectodomain that incorporates all of the N-linked glycans and reveals the extent to which the attached glycans mask the surface of the IR dimer from interaction with antibodies or other potential therapeutic binding proteins. The usefulness of Fab complexation in the crystallization of heavily glycosylated proteins is also discussed. The compositions of the glycans on IR expressed in CHO-K1 cells and the glycosylation deficient Lec8 cell line were determined by protease digestion, glycopeptide purification, amino acid sequence analysis, and mass spectrometry. Collectively the data reveal: multiple species of complex glycan at residues 25, 255, 295, 418, 606, 624, 742, 755, and 893 (IR-B numbering); multiple species of high-mannose glycan at residues 111 and 514; a single species of complex glycan at residue 671; and a single species of high-mannose glycan at residue 215. Residue 16 exhibited a mixture of complex, hybrid, and high-mannose glycan species. Of the remaining five predicted N-linked sites, those at residues 397 and 906 were confirmed by amino acid sequencing to be glycosylated, while that at residue 78 and the atypical (NKC) site at residue 282 were not glycosylated. The peptide containing the final site at residue 337 was not recovered but is seen to be glycosylated in the electron density maps of the IR ectodomain. The model of the fully glycosylated IR reveals that the sites carrying high-mannose glycans lie at positions of relatively low steric accessibility.

  16. An N-linked high-mannose type oligosaccharide, expressed at the major outer membrane protein of Chlamydia trachomatis, mediates attachment and infectivity of the microorganism to HeLa cells.

    PubMed

    Kuo, C; Takahashi, N; Swanson, A F; Ozeki, Y; Hakomori, S

    1996-12-15

    The structure of the carbohydrate of the 40-kD major outer membrane component of Chlamydia trachomatis and its role in defining infectivity of the organism were investigated. The oligosaccharides were released from the glycoprotein by N-glycanase digestion, coupled to a 2-aminopyridyl residue, and subjected to two-dimensional sugar mapping technique. The major fractions consisted of "high-mannose type" oligosaccharides containing 8-9 mannose residues. Bi- and tri-antennary "complex type" oligosaccharides having terminal galactose were detected as minor components. These oligosaccharides were N-linked and contained no sialic acid. This structural profile is consistent with our previous characterization based on lectin-binding and glycosidase digestion. Functional specificity of identified chlamydial oligosaccharides was analyzed using glycopeptides fractionated from ovalbumin and structurally defined oligosaccharides from other sources. The glycopeptide fraction having high-mannose type oligosaccharide, as compared to those having complex or hybrid-type, showed a stronger inhibitory effect on attachment and infectivity of chlamydial organisms to HeLa cells. Among high-mannose type oligosaccharides, the strongest inhibition was observed with mannose 8 as compared with mannose 6, 7, or 9. These results indicate that a specific high-mannose type oligosaccharide linked to the major outer membrane protein of C. trachomatis mediates attachment and infectivity of the organism to HeLa cells.

  17. Bioelectrochemistry of cell surfaces

    NASA Astrophysics Data System (ADS)

    Dolowy, Krzysztof

    This paper deals with processes and phenomena of cell surface bioelectrochemistry in which charges do not move across the cell membrane. First, electrochemical properties of the cell membrane and the cell medium interface are described, and different electric potentials present in biological systems are defined. Methods of cell electrophoresis are then discussed. It is shown that none of the simple electrochemical models of the cell membrane can explain the dependence of cell electrophoretic mobility upon ionic strength and other electrochemical properties of the cell membrane, such as the difference in cell membrane charge as determined electrochemically and biochemically, or the effect of neuraminidase, pH, or membrane potential change on cell electrophoretic mobility. Thus, it is apparent that conclusions drawn from electrophoretic mobility data on the basis of simple models are false. The more complex multilayer-electrochemical model of the cell membrane is then described and shown to explain most electrochemical properties of the cell membrane. Next, different electrochemical techniques that were applied to study cell surfaces are described. It is shown that colloid titration, isoelectric focusing, and partition of cells between two immiscible phases is dependent not only on electrical properties of the cell membrane, but also on the energy of adsorption at cell surfaces of organic molecules used in these methods. Powder electrodes, cell polarography, conductometric titration, and Donnan potential methods are described and it is shown that these methods also produce results of doubtful value and are also often misinterpreted. The contact potential difference method produces results difficult to interpret and only electro-osmotic measurements and potential sensitive molecules are valuable methods. The colloid particle interaction theory of Derjaguin, Landau, Verwey, and Overbeek (DLVO) as applied to cell interactions is discussed. It is shown that the

  18. A Single N-Linked Glycosylation Site in the Japanese Encephalitis Virus prM Protein Is Critical for Cell Type-Specific prM Protein Biogenesis, Virus Particle Release, and Pathogenicity in Mice ▿ †

    PubMed Central

    Kim, Jeong-Min; Yun, Sang-Im; Song, Byung-Hak; Hahn, Youn-Soo; Lee, Chan-Hee; Oh, Hyun-Woo; Lee, Young-Min

    2008-01-01

    The prM protein of Japanese encephalitis virus (JEV) contains a single potential N-linked glycosylation site, N15-X16-T17, which is highly conserved among JEV strains and closely related flaviviruses. To investigate the role of this site in JEV replication and pathogenesis, we manipulated the RNA genome by using infectious JEV cDNA to generate three prM mutants (N15A, T17A, and N15A/T17A) with alanine substiting for N15 and/or T17 and one mutant with silent point mutations introduced into the nucleotide sequences corresponding to all three residues in the glycosylation site. An analysis of these mutants in the presence or absence of endoglycosidases confirmed the addition of oligosaccharides to this potential glycosylation site. The loss of prM N glycosylation, without significantly altering the intracellular levels of viral RNA and proteins, led to an ≈20-fold reduction in the production of extracellular virions, which had protein compositions and infectivities nearly identical to those of wild-type virions; this reduction occurred at the stage of virus release, rather than assembly. This release defect was correlated with small-plaque morphology and an N-glycosylation-dependent delay in viral growth. A more conservative mutation, N15Q, had the same effect as N15A. One of the four prM mutants, N15A/T17A, showed an additional defect in virus growth in mosquito C6/36 cells but not human neuroblastoma SH-SY5Y or hamster BHK-21 cells. This cell type dependence was attributed to abnormal N-glycosylation-independent biogenesis of prM. In mice, the elimination of prM N glycosylation resulted in a drastic decrease in virulence after peripheral inoculation. Overall, our findings indicate that this highly conserved N-glycosylation motif in prM is crucial for multiple stages of JEV biology: prM biogenesis, virus release, and pathogenesis. PMID:18524814

  19. A single N-linked glycosylation site in the Japanese encephalitis virus prM protein is critical for cell type-specific prM protein biogenesis, virus particle release, and pathogenicity in mice.

    PubMed

    Kim, Jeong-Min; Yun, Sang-Im; Song, Byung-Hak; Hahn, Youn-Soo; Lee, Chan-Hee; Oh, Hyun-Woo; Lee, Young-Min

    2008-08-01

    The prM protein of Japanese encephalitis virus (JEV) contains a single potential N-linked glycosylation site, N(15)-X(16)-T(17), which is highly conserved among JEV strains and closely related flaviviruses. To investigate the role of this site in JEV replication and pathogenesis, we manipulated the RNA genome by using infectious JEV cDNA to generate three prM mutants (N15A, T17A, and N15A/T17A) with alanine substituting for N(15) and/or T(17) and one mutant with silent point mutations introduced into the nucleotide sequences corresponding to all three residues in the glycosylation site. An analysis of these mutants in the presence or absence of endoglycosidases confirmed the addition of oligosaccharides to this potential glycosylation site. The loss of prM N glycosylation, without significantly altering the intracellular levels of viral RNA and proteins, led to an approximately 20-fold reduction in the production of extracellular virions, which had protein compositions and infectivities nearly identical to those of wild-type virions; this reduction occurred at the stage of virus release, rather than assembly. This release defect was correlated with small-plaque morphology and an N-glycosylation-dependent delay in viral growth. A more conservative mutation, N15Q, had the same effect as N15A. One of the four prM mutants, N15A/T17A, showed an additional defect in virus growth in mosquito C6/36 cells but not human neuroblastoma SH-SY5Y or hamster BHK-21 cells. This cell type dependence was attributed to abnormal N-glycosylation-independent biogenesis of prM. In mice, the elimination of prM N glycosylation resulted in a drastic decrease in virulence after peripheral inoculation. Overall, our findings indicate that this highly conserved N-glycosylation motif in prM is crucial for multiple stages of JEV biology: prM biogenesis, virus release, and pathogenesis.

  20. Evidence that maturation of the N-linked glycans of the respiratory syncytial virus (RSV) glycoproteins is required for virus-mediated cell fusion: The effect of {alpha}-mannosidase inhibitors on RSV infectivity

    SciTech Connect

    McDonald, Terence P.; Jeffree, Chris E.; Li, Ping; Rixon, Helen W. McL.; Brown, Gaie; Aitken, James D.; MacLellan, Kirsty; Sugrue, Richard J. . E-mail: rjsugrue@ntu.edu.sg

    2006-07-05

    Glycan heterogeneity of the respiratory syncytial virus (RSV) fusion (F) protein was demonstrated by proteomics. The effect of maturation of the virus glycoproteins-associated glycans on virus infectivity was therefore examined using the {alpha}-mannosidase inhibitors deoxymannojirimycin (DMJ) and swainsonine (SW). In the presence of SW the N-linked glycans on the F protein appeared in a partially mature form, whereas in the presence of DMJ no maturation of the glycans was observed. Neither inhibitor had a significant effect on G protein processing or on the formation of progeny virus. Although the level of infectious virus and syncytia formation was not significantly affected by SW-treatment, DMJ-treatment correlated with a one hundred-fold reduction in virus infectivity. Our data suggest that glycan maturation of the RSV glycoproteins, in particular those on the F protein, is an important step in virus maturation and is required for virus infectivity.

  1. Cell surface expression of glycosylated, nonglycosylated, and truncated forms of a cytoplasmic protein pyruvate kinase.

    PubMed

    Hiebert, S W; Lamb, R A

    1988-09-01

    The soluble cytoplasmic protein pyruvate kinase (PK) has been expressed at the cell surface in a membrane-anchored form (APK). The hybrid protein contains the NH2-terminal signal/anchor domain of a class II integral membrane protein (hemagglutinin/neuraminidase, of the paramyxovirus SV5) fused to the PK NH2 terminus. APK contains a cryptic site that is used for N-linked glycosylation but elimination of this site by site-specific mutagenesis does not prevent cell surface localization. Truncated forms of the APK molecule, with up to 80% of the PK region of APK removed, can also be expressed at the cell surface. These data suggest that neither the complete PK molecule nor its glycosylation are necessary for intracellular transport of PK to the cell surface, and it is possible that specific signals may not be needed in the ectodomain of this hybrid protein to specify cell surface localization.

  2. Probing the Role of N-Linked Glycans in the Stability and Activity of Fungal Cellobiohydrolases by Mutational Analysis

    SciTech Connect

    Adney, W. S.; Jeoh, T.; Beckham, G. T.; Chou,Y. C.; Baker, J. O.; Michener, W.; Brunecky, R.; Himmel, M. E.

    2009-01-01

    The filamentous fungi Trichoderma reesei and Penicillium funiculosum produce highly effective enzyme mixtures that degrade the cellulose and hemicellulose components of plant cell walls. Many fungal species produce a glycoside hydrolase family 7 (Cel7A) cellobiohydrolase, a class of enzymes that catalytically process from the reducing end of cellulose. A direct amino acid comparison of these two enzymes shows that they not only have high amino acid homology, but also contain analogous N-linked glycosylation sites on the catalytic domain. We have previously shown (Jeoh et al. in Biotechnol Biofuels, 1:10, 2008) that expression of T. reesei cellobiohydrolase I in a commonly used industrial expression host, Aspergillus niger var. awamori, results in an increase in the amount of N-linked glycosylation of the enzyme, which negatively affects crystalline cellulose degradation activity as well as thermal stability. This complementary study examines the significance of individual N-linked glycans on the surface of the catalytic domain of Cel7A cellobiohydrolases from T. reesei and P. funiculosum by genetically adding or removing N-linked glycosylation motifs using site directed mutagenesis. Modified enzymes, expressed in A. niger var. awamori, were tested for activity and thermal stability. It was concluded that N-linked glycans in peptide loops that form part of the active site tunnel have the greatest impact on both thermal stability and enzymatic activity on crystalline cellulose for both the T. reesei and P. funiculosum Cel7A enzymes. Specifically, for the Cel7A T. reesei enzyme expressed in A. niger var. awamori, removal of the N384 glycosylation site yields a mutant with 70% greater activity after 120 h compared to the heterologously expressed wild type T. reesei enzyme. In addition, similar activity improvements were found to be associated with the addition of a new glycosylation motif at N194 in P. funiculosum. This mutant also exhibits 70% greater activity after

  3. cap alpha. -D-Mannopyranosylmethyl-P-nitrophenyltriazene effects on the degradation and biosynthesis of N-linked oligosaccharide chains on. cap alpha. /sub 1/-acid glycoprotein by liver cells

    SciTech Connect

    Docherty, P.A.; Aronson, N.N. Jr.

    1986-05-01

    The effects of ..cap alpha..-D-mannopyranosylmethyl-p-nitrophenyltriazene (..cap alpha..-ManMNT) on the degradation and processing of oligosaccharide chains on ..cap alpha../sub 1/-acid glycoprotein (AGP) were studied. Addition of the triazene to a perfused liver blocked the complete degradation of endocytosed N-acetyl (/sup 14/C)glucosamine-labeled asialo-AGP and caused the accumulation of Man/sub 2/GlcNAc/sub 1/ fragments in the lysosome-enriched fraction of the liver homogenate. This compound also reduced the reincorporation of lysosomally-derived (/sup 14/C)GlcNAc into newly secreted glycoproteins. Cultured hepatocytes treated with the inhibitor synthesized and secreted fully-glycosylated AGP. However, the N-linked oligosaccharide chains on AGP secreted by the ..cap alpha..-ManMNT-treated hepatocytes remained sensitive to digestion with endoglycosidase H, were resistant to neuraminidase, and consisted of Man/sub 9-7/GlcNAc/sub 2/ structures as analyzed by high resolution Bio-Gel P-4 chromatography. As measured by their resistance to cleavage by endoglycosidase H, the normal processing of all six carbohydrate chains on AGP to the complex form did not completely resume until nearly 24 h after triazene treatment. Since ManMNT is likely to irreversibly inactivate ..cap alpha..-D-mannosidases, the return of AGP to secretory forms with complex chains after 24 h probably resulted from synthesis of new processing enzymes.

  4. Furrowing in altered cell surfaces.

    PubMed

    Rappaport, R

    1976-02-01

    Understanding the process which established the cell division mechanism requires analysis of the role of the responding surface as well as that of stimulatory subsurface structures. Cell surface was altered by the expansion which occurs during exovate formation. Exovates appear on the surface of fertilized Arbacia lixula, Paracentrotus lividus and Echinarachnius parma eggs in response to extreme flattening. They result from cytoplasmic outflow initiated in a very restricted portion of the egg surface. Observations of the formation process in pigmented A. lixula eggs revealed that the original surface may be expanded about 100 fold as the exovate swells. When exovates formed 15-30 minutes after fertilization contain the mitotic apparatus, they divide synchronously with flattened controls. If nucleated exovates are established after the beginning of first cleavage, furrows appear in ten minutes. Exovates established after the beginning of second cleavage develop furrows four minutes after the entrance of the the mitsotic apparatus. Cytoplasm beneath damaged exovate surfaces sometimes develops partial constrictions independently of the surface in the plane the furrow would have occupied. These results suggest that normal surface structure is unnecessary for furrow establishment and function.

  5. A Surface-Controlled Solar Cell

    NASA Technical Reports Server (NTRS)

    Daud, T.; Crotty, G. T.

    1987-01-01

    Open-circuit voltage and cell efficiency increased. Proposed technique for controlling recombination velocity on solar-cell surfaces provides cells of increased efficiency and open-circuit voltage. In present cells, uncontrolled surface recombination velocity degrades opencircuit voltage and efficiency. In cell using proposed technique, transparent conducting layer, insulated from cell contacts, biased to enable variable control of surface recombination velocity.

  6. Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment*

    PubMed Central

    Liang, Yaxuan; Eng, William S.; Colquhoun, David R.; Dinglasan, Rhoel R.; Graham, David R.; Mahal, Lara K.

    2014-01-01

    Exosomes, also known as microvesicles (EMVs), are nano-sized membranous particles secreted from nearly all mammalian cell types. These nanoparticles play critical roles in many physiological processes including cell-cell signaling, immune activation, and suppression and are associated with disease states such as tumor progression. The biological functions of EMVs are highly dependent on their protein composition, which can dictate pathogenicity. Although some mechanisms have been proposed for the regulation of EMV protein trafficking, little attention has been paid to N-linked glycosylation as a potential sorting signal. Previous work from our laboratory found a conserved glycan signature for EMVs, which differed from that of the parent cell membranes, suggesting a potential role for glycosylation in EMV biogenesis. In this study, we further explore the role of glycosylation in EMV protein trafficking. We identify EMV glycoproteins and demonstrate alteration of their recruitment as a function of their glycosylation status upon pharmacological manipulation. Furthermore, we show that genetic manipulation of the glycosylation levels of a specific EMV glycoprotein, EWI-2, directly impacts its recruitment as a function of N-linked glycan sites. Taken together, our data provide strong evidence that N-linked glycosylation directs glycoprotein sorting into EMVs. PMID:25261472

  7. Complex N-linked glycans serve as a determinant for exosome/microvesicle cargo recruitment.

    PubMed

    Liang, Yaxuan; Eng, William S; Colquhoun, David R; Dinglasan, Rhoel R; Graham, David R; Mahal, Lara K

    2014-11-21

    Exosomes, also known as microvesicles (EMVs), are nano-sized membranous particles secreted from nearly all mammalian cell types. These nanoparticles play critical roles in many physiological processes including cell-cell signaling, immune activation, and suppression and are associated with disease states such as tumor progression. The biological functions of EMVs are highly dependent on their protein composition, which can dictate pathogenicity. Although some mechanisms have been proposed for the regulation of EMV protein trafficking, little attention has been paid to N-linked glycosylation as a potential sorting signal. Previous work from our laboratory found a conserved glycan signature for EMVs, which differed from that of the parent cell membranes, suggesting a potential role for glycosylation in EMV biogenesis. In this study, we further explore the role of glycosylation in EMV protein trafficking. We identify EMV glycoproteins and demonstrate alteration of their recruitment as a function of their glycosylation status upon pharmacological manipulation. Furthermore, we show that genetic manipulation of the glycosylation levels of a specific EMV glycoprotein, EWI-2, directly impacts its recruitment as a function of N-linked glycan sites. Taken together, our data provide strong evidence that N-linked glycosylation directs glycoprotein sorting into EMVs.

  8. Cell surface expression of v-fms-coded glycoproteins is required for transformation.

    PubMed Central

    Roussel, M F; Rettenmier, C W; Look, A T; Sherr, C J

    1984-01-01

    The viral oncogene v-fms encodes a transforming glycoprotein with in vitro tyrosine-specific protein kinase activity. Although most v-fms-coded molecules remain internally sequestered in transformed cells, a minor population of molecules is transported to the cell surface. An engineered deletion mutant lacking 348 base pairs of the 3.0-kilobase-pair v-fms gene encoded a polypeptide that was 15 kilodaltons smaller than the wild-type v-fms gene product. The in-frame deletion of 116 amino acids was adjacent to the transmembrane anchor peptide located near the middle of the predicted protein sequence and 432 amino acids from the carboxyl terminus. The mutant polypeptide acquired N-linked oligosaccharide chains, was proteolytically processed in a manner similar to the wild-type glycoprotein, and exhibited an associated tyrosine-specific protein kinase activity in vitro. However, the N-linked oligosaccharides of the mutant glycoprotein were not processed to complex carbohydrate chains, and the glycoprotein was not detected at the cell surface. Cells expressing high levels of the mutant glycoprotein did not undergo morphological transformation and did not form colonies in semisolid medium. The transforming activity of the v-fms gene product therefore appears to be mediated through target molecules on the plasma membrane. Images PMID:6390182

  9. Cells behaviors and genotoxicity on topological surface.

    PubMed

    Yang, N; Yang, M K; Bi, S X; Chen, L; Zhu, Z Y; Gao, Y T; Du, Z

    2013-08-01

    To investigate different cells behaviors and genotoxicity, which were driven by specific microenvironments, three patterned surfaces (pillars, wide grooves and narrow grooves) and one smooth surface were prepared by template-based technique. Vinculin is a membrane-cytoskeletal protein in focal adhesion plaques and associates with cell-cell and cell-matrix junctions, which can promote cell adhesion and spreading. The immunofluorescence staining of vinculin revealed that the narrow grooves patterned substrate was favorable for L929 cell adhesion. For cell multiplication, the narrow grooves surface was fitted for the proliferation of L929, L02 and MSC cells, the pillars surface was only in favor of L929 cells to proliferate during 7 days of cell cultivation. Cell genetic toxicity was evaluated by cellular micronuclei test (MNT). The results indicated that topological surfaces were more suitable for L929 cells to proliferate and maintain the stability of genome. On the contrary, the narrow grooves surface induced higher micronuclei ratio of L02 and MSC cells than other surfaces. With the comprehensive results of cell multiplication and MNT, it was concluded that the wide grooves surface was best fitted for L02 cells to proliferate and have less DNA damages, and the smooth surface was optimum for the research of MSC cells in vitro.

  10. Cell surface beta 1,4-galactosyltransferase functions during neural crest cell migration and neurulation in vivo

    PubMed Central

    1992-01-01

    Mesenchymal cell migration and neurite outgrowth are mediated in part by binding of cell surface beta 1,4-galactosyltransferase (GalTase) to N-linked oligosaccharides within the E8 domain of laminin. In this study, we determined whether cell surface GalTase functions during neural crest cell migration and neural development in vivo using antibodies raised against affinity-purified chicken serum GalTase. The antibodies specifically recognized two embryonic proteins of 77 and 67 kD, both of which express GalTase activity. The antibodies also immunoprecipitated and inhibited chick embryo GalTase activity, and inhibited neural crest cell migration on laminin matrices in vitro. Anti-GalTase antibodies were microinjected into the head mesenchyme of stage 7-9 chick embryos or cranial to Henson's node of stage 6 embryos. Anti-avian GalTase IgG decreased cranial neural crest cell migration on the injected side but did not cross the embryonic midline and did not affect neural crest cell migration on the uninjected side. Anti-avian GalTase Fab crossed the embryonic midline and perturbed cranial neural crest cell migration throughout the head. Neural fold elevation and neural tube closure were also disrupted by Fab fragments. Cell surface GalTase was localized to migrating neural crest cells and to the basal surfaces of neural epithelia by indirect immunofluorescence, whereas GalTase was undetectable on neural crest cells prior to migration. These results suggest that, during early embryogenesis, cell surface GalTase participates during neural crest cell migration, perhaps by interacting with laminin, a major component of the basal lamina. Cell surface GalTase also appears to play a role in neural tube formation, possibly by mediating neural epithelial adhesion to the underlying basal lamina. PMID:1560031

  11. Regulation of the Axillary Osmidrosis-Associated ABCC11 Protein Stability by N-Linked Glycosylation: Effect of Glucose Condition.

    PubMed

    Toyoda, Yu; Takada, Tappei; Miyata, Hiroshi; Ishikawa, Toshihisa; Suzuki, Hiroshi

    2016-01-01

    ATP-binding cassette C11 (ABCC11) is a plasma membrane protein involved in the transport of a variety of lipophilic anions. ABCC11 wild-type is responsible for the high-secretion phenotypes in human apocrine glands, such as that of wet-type ear wax, and the risk of axillary osmidrosis. We have previously reported that mature ABCC11 is a glycoprotein containing two N-linked glycans at Asn838 and Asn844. However, little is known about the role of N-linked glycosylation in the regulation of ABCC11 protein. In the current study, we investigated the effects of N-linked glycosylation on the protein level and localization of ABCC11 using polarized Madin-Darby canine kidney II cells. When the N-linked glycosylation in ABCC11-expressing cells was chemically inhibited by tunicamycin treatment, the maturation of ABCC11 was suppressed and its protein level was significantly decreased. Immunoblotting analyses demonstrated that the protein level of the N-linked glycosylation-deficient mutant (N838Q and N844Q: Q838/844) was about half of the ABCC11 wild-type level. Further biochemical studies with the Q838/844 mutant showed that this glycosylation-deficient ABCC11 was degraded faster than wild-type probably due to the enhancement of the MG132-sensitive protein degradation pathway. Moreover, the incubation of ABCC11 wild-type-expressing cells in a low-glucose condition decreased mature, glycosylated ABCC11, compared with the high-glucose condition. On the other hand, the protein level of the Q838/844 mutant was not affected by glucose condition. These results suggest that N-linked glycosylation is important for the protein stability of ABCC11, and physiological alteration in glucose may affect the ABCC11 protein level and ABCC11-related phenotypes in humans, such as axillary osmidrosis.

  12. Regulation of the Axillary Osmidrosis-Associated ABCC11 Protein Stability by N-Linked Glycosylation: Effect of Glucose Condition

    PubMed Central

    Toyoda, Yu; Takada, Tappei; Miyata, Hiroshi; Ishikawa, Toshihisa; Suzuki, Hiroshi

    2016-01-01

    ATP-binding cassette C11 (ABCC11) is a plasma membrane protein involved in the transport of a variety of lipophilic anions. ABCC11 wild-type is responsible for the high-secretion phenotypes in human apocrine glands, such as that of wet-type ear wax, and the risk of axillary osmidrosis. We have previously reported that mature ABCC11 is a glycoprotein containing two N-linked glycans at Asn838 and Asn844. However, little is known about the role of N-linked glycosylation in the regulation of ABCC11 protein. In the current study, we investigated the effects of N-linked glycosylation on the protein level and localization of ABCC11 using polarized Madin-Darby canine kidney II cells. When the N-linked glycosylation in ABCC11-expressing cells was chemically inhibited by tunicamycin treatment, the maturation of ABCC11 was suppressed and its protein level was significantly decreased. Immunoblotting analyses demonstrated that the protein level of the N-linked glycosylation-deficient mutant (N838Q and N844Q: Q838/844) was about half of the ABCC11 wild-type level. Further biochemical studies with the Q838/844 mutant showed that this glycosylation-deficient ABCC11 was degraded faster than wild-type probably due to the enhancement of the MG132-sensitive protein degradation pathway. Moreover, the incubation of ABCC11 wild-type-expressing cells in a low-glucose condition decreased mature, glycosylated ABCC11, compared with the high-glucose condition. On the other hand, the protein level of the Q838/844 mutant was not affected by glucose condition. These results suggest that N-linked glycosylation is important for the protein stability of ABCC11, and physiological alteration in glucose may affect the ABCC11 protein level and ABCC11-related phenotypes in humans, such as axillary osmidrosis. PMID:27281343

  13. Cell attachment on microscopically textured silicon surfaces

    NASA Astrophysics Data System (ADS)

    Turner, Stephen W.; Kam, Lance; Isaacson, Michael; Craighead, Harold G.; Szarowski, Donald H.; Turner, James N.; Shain, W.

    1997-03-01

    To assess the effect of surface topography on cell attachment, central nervous system (astroglial cells) cells were grown on surfaces patterned with two different types of texture. Reactive ion etching (RIE) was used to induce nanometer-scale roughness in silicon wafers. In a subsequent wet etch, photo-patterned resist protected selected areas of the surface, resulting in a pattern of modified and unmodified texture. Scanning electron microscopy (SEM) showed that the RIE-roughened 'primary' surface consists of randomly positioned columnar structures (diameter approximately equals 50 nm, height approximately equals 250 nm). The wet-etched 'secondary' surfaces had shorter and more sparsely distributed projections, controlled to a degree by wet etch duration. Confocal microscopy and SEM demonstrated that transformed astroglial (LRM55) cells preferred secondary surfaces. The morphology of cells on secondary surfaces depended on wet etch duration. with brief wet etch, cells hade stellate or mounded morphology and were not closely adherent to the surface. With long wet etch, cells had an epithelial-like morphology and were closely adherent to substrates. Under all conditions, cells discriminated between primary and secondary surfaces. In contrast to LRM55 cells, astrocytes in primary cell culture preferred primary surfaces. Thus changes in surface topography produce cell-specific selectivity and change cell attachment characteristics.

  14. Core glycan in the yeast multicopper ferroxidase, Fet3p: A case study of N-linked glycosylation, protein maturation, and stability

    PubMed Central

    Ziegler, Lynn; Terzulli, Alaina; Sedlak, Erik; Kosman, Daniel J

    2010-01-01

    Glycosylation is essential to the maintenance of protein quality in the vesicular protein trafficking pathway in eukaryotic cells. Using the yeast multicopper oxidase, Fet3p, the hypothesis is tested that core glycosylation suppresses Fet3p nascent chain aggregation during synthesis into the endoplasmic reticulum (ER). Fet3p has 11 crystallographically mapped N-linked core glycan units. Assembly of four of these units is specifically required for localization of Fet3p to the plasma membrane (PM). Fet3 protein lacking any one of these glycan units is found in an intracellular high-molecular mass species resolvable by blue native gel electrophoresis. Individually, the remaining glycan moieties are not required for ER exit; however, serial deletion of these by N → A substitution correlates with these desglycan species failure to exit the ER. Desglycan Fet3 proteins that localize to the PM are wild type in function indicating that the missing carbohydrate is not required for native structure and biologic activity. This native function includes the interaction with the iron permease, Ftr1p, and wild type high-affinity iron uptake activity. The four essential sequons are found within relatively nonpolar regions located in surface recesses and are strongly conserved among fungal Fet3 proteins. The remaining N-linked sites are found in more surface exposed, less nonpolar environments, and their conservation is weak or absent. The data indicate that in Fet3p the N-linked glycan has little effect on the enzyme's molecular activity but is critical to its cellular activity by maximizing the protein's exit from the ER and assembly into a functional iron uptake complex. PMID:20662012

  15. Ethanol-induced impairment in the biosynthesis of N-linked glycosylation.

    PubMed

    Welti, Michael; Hülsmeier, Andreas J

    2014-04-01

    Deficiency in N-linked protein glycosylation is a long-known characteristic of alcoholic liver disease and congenital disorders of glycosylation. Previous investigations of ethanol-induced glycosylation deficiency demonstrated perturbations in the early steps of substrate synthesis and in the final steps of capping N-linked glycans in the Golgi. The significance of the biosynthesis of N-glycan precursors in the endoplasmic reticulum, however, has not yet been addressed in alcoholic liver disease. Ethanol-metabolizing hepatoma cells were treated with increasing concentrations of ethanol. Transcript analysis of genes involved in the biosynthesis of N-glycans, activity assays of related enzymes, dolichol-phosphate quantification, and analysis of dolichol-linked oligosaccharides were performed. Upon treatment of cells with ethanol, we found a decrease in the final N-glycan precursor Dol-PP-GlcNAc(2) Man(9) Glc(3) and in C95- and C100-dolichol-phosphate levels. Transcript analysis of genes involved in N-glycosylation showed a 17% decrease in expression levels of DPM1, a subunit of the dolichol-phosphate-mannose synthase, and an 8% increase in RPN2, a subunit of the oligosaccharyl transferase. Ethanol treatment decreases the biosynthesis of dolichol-phosphate. Consequently, the formation of N-glycan precursors is affected, resulting in an aberrant precursor assembly. Messenger RNA levels of genes involved in N-glycan biosynthesis are slightly affected by ethanol treatment, indicating that the assembly of N-glycan precursors is not regulated at the transcriptional level. This study confirms that ethanol impairs N-linked glycosylation by affecting dolichol biosynthesis leading to impaired dolichol-linked oligosaccharide assembly. Together our data help to explain the underglycosylation phenotype observed in alcoholic liver disease and congenital disorders of glycosylation.

  16. Surface Glycoproteins of Exosomes Shed by Myeloid-Derived Suppressor Cells Contribute to Function.

    PubMed

    Chauhan, Sitara; Danielson, Steven; Clements, Virginia; Edwards, Nathan; Ostrand-Rosenberg, Suzanne; Fenselau, Catherine

    2017-01-06

    In this report, we use a proteomic strategy to identify glycoproteins on the surface of exosomes derived from myeloid-derived suppressor cells (MDSCs), and then test if selected glycoproteins contribute to exosome-mediated chemotaxis and migration of MDSCs. We report successful modification of a surface chemistry method for use with exosomes and identify 21 surface N-glycoproteins on exosomes released by mouse mammary carcinoma-induced MDSCs. These glycoprotein identities and functionalities are compared with 93 N-linked glycoproteins identified on the surface of the parental cells. As with the lysate proteomes examined previously, the exosome surface N-glycoproteins are primarily a subset of the glycoproteins on the surface of the suppressor cells that released them, with related functions and related potential as therapeutic targets. The "don't eat me" molecule CD47 and its binding partners thrombospondin-1 (TSP1) and signal regulatory protein α (SIRPα) were among the surface N-glycoproteins detected. Functional bioassays using antibodies to these three molecules demonstrated that CD47, TSP1, and to a lesser extent SIRPα facilitate exosome-mediated MDSC chemotaxis and migration.

  17. Standing surface acoustic wave based cell coculture.

    PubMed

    Li, Sixing; Guo, Feng; Chen, Yuchao; Ding, Xiaoyun; Li, Peng; Wang, Lin; Cameron, Craig E; Huang, Tony Jun

    2014-10-07

    Precise reconstruction of heterotypic cell-cell interactions in vitro requires the coculture of different cell types in a highly controlled manner. In this article, we report a standing surface acoustic wave (SSAW)-based cell coculture platform. In our approach, different types of cells are patterned sequentially in the SSAW field to form an organized cell coculture. To validate our platform, we demonstrate a coculture of epithelial cancer cells and endothelial cells. Real-time monitoring of cell migration dynamics reveals increased cancer cell mobility when cancer cells are cocultured with endothelial cells. Our SSAW-based cell coculture platform has the advantages of contactless cell manipulation, high biocompatibility, high controllability, simplicity, and minimal interference of the cellular microenvironment. The SSAW technique demonstrated here can be a valuable analytical tool for various biological studies involving heterotypic cell-cell interactions.

  18. A membrane reservoir at the cell surface

    PubMed Central

    Figard, Lauren; Sokac, Anna Marie

    2014-01-01

    Cell surface expansion is a necessary part of cell shape change. One long-standing hypothesis proposes that membrane for this expansion comes from the flattening out of cell surface projections such as microvilli and membrane folds. Correlative EM data of cells undergoing phagocytosis, cytokinesis, and morphogenesis has hinted at the existence of such an unfolding mechanism for decades; but unfolding has only recently been confirmed using live-cell imaging and biophysical approaches. Considering the wide range of cells in which plasma membrane unfolding has now been reported, it likely represents a fundamental mechanism of cell shape change. PMID:24844289

  19. Standing Surface Acoustic Wave Based Cell Coculture

    PubMed Central

    2015-01-01

    Precise reconstruction of heterotypic cell–cell interactions in vitro requires the coculture of different cell types in a highly controlled manner. In this article, we report a standing surface acoustic wave (SSAW)-based cell coculture platform. In our approach, different types of cells are patterned sequentially in the SSAW field to form an organized cell coculture. To validate our platform, we demonstrate a coculture of epithelial cancer cells and endothelial cells. Real-time monitoring of cell migration dynamics reveals increased cancer cell mobility when cancer cells are cocultured with endothelial cells. Our SSAW-based cell coculture platform has the advantages of contactless cell manipulation, high biocompatibility, high controllability, simplicity, and minimal interference of the cellular microenvironment. The SSAW technique demonstrated here can be a valuable analytical tool for various biological studies involving heterotypic cell–cell interactions. PMID:25232648

  20. Back surface reflectors for solar cells

    NASA Technical Reports Server (NTRS)

    Chai, A. T.

    1980-01-01

    Sample solar cells were fabricated to study the effects of various back surface reflectors on the device performance. They are typical 50 micrometers thick, space quality, silicon solar cells except for variations of the back contact configuration. The back surfaces of the sample cells are polished to a mirror like finish, and have either conventional full contacts or grid finger contacts. Measurements and evaluation of various metallic back surface reflectors, as well as cells with total internal reflection, are presented. Results indicate that back surface reflectors formed using a grid finger back contact are more effective reflectors than cells with full back metallization and that Au, Ag, or Cu are better back surface reflector metals than Al.

  1. Cell behavior on surface modified polydimethylsiloxane (PDMS).

    PubMed

    Stanton, Morgan M; Rankenberg, Johanna M; Park, Byung-Wook; McGimpsey, W Grant; Malcuit, Christopher; Lambert, Christopher R

    2014-07-01

    Designing complex tissue culture systems requires cell alignment and directed extracellular matrix (ECM) and gene expression. Here, a micro-rough, polydimethylsiloxane (PDMS) surface, that also integrates a micro-pattern of 50 µm wide lines of fibronectin (FN) separated by 60 µm wide lines of bovine serum albumin (BSA), is developed. Human fibroblasts cultured on the rough, patterned substrate have aligned growth and a significant change in morphology when compared to cells on a flat, patterned surface. The rough PDMS topography significantly decreases cell area and induces the upregulation of several ECM related genes by two-fold when compared to cells cultured on flat PDMS. This study describes a simple surface engineering procedure for creating surface architecture for scaffolds to design and control the cell-surface interface.

  2. Unconventional N-Linked Glycosylation Promotes Trimeric Autotransporter Function in Kingella kingae and Aggregatibacter aphrophilus

    PubMed Central

    Rempe, Katherine A.; Spruce, Lynn A.; Porsch, Eric A.; Seeholzer, Steven H.; Nørskov-Lauritsen, Niels

    2015-01-01

    ABSTRACT Glycosylation is a widespread mechanism employed by both eukaryotes and bacteria to increase the functional diversity of their proteomes. The nontypeable Haemophilus influenzae glycosyltransferase HMW1C mediates unconventional N-linked glycosylation of the adhesive protein HMW1, which is encoded in a two-partner secretion system gene cluster that also encodes HMW1C. In this system, HMW1 is modified in the cytoplasm by sequential transfer of hexose residues. In the present study, we examined Kingella kingae and Aggregatibacter aphrophilus homologues of HMW1C that are not encoded near a gene encoding an obvious acceptor protein. We found both homologues to be functional glycosyltransferases and identified their substrates as the K. kingae Knh and the A. aphrophilus EmaA trimeric autotransporter proteins. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed multiple sites of N-linked glycosylation on Knh and EmaA. Without glycosylation, Knh and EmaA failed to facilitate wild-type levels of bacterial autoaggregation or adherence to human epithelial cells, establishing that glycosylation is essential for proper protein function. PMID:26307167

  3. A mathematical model of N-linked glycosylation.

    PubMed

    Krambeck, Frederick J; Betenbaugh, Michael J

    2005-12-20

    Metabolic engineering of N-linked oligosaccharide biosynthesis to produce novel glycoforms or glycoform distributions of a recombinant glycoprotein can potentially lead to an improved therapeutic performance of the glycoprotein product. A mathematical model for the initial stages of this process, up to the first galactosylation of an oligosaccharide, was previously developed by Umana and Bailey (1997) (UB1997). Building on this work, an extended model is developed to include further galactosylation, fucosylation, extension of antennae by N-acetyllactosamine repeats, and sialylation. This allows many more structural features to be predicted. A number of simplifying assumptions are also relaxed to incorporate more variables for the control of glycoforms. The full model generates 7565 oligosaccharide structures in a network of 22,871 reactions. Methods for solving the model for the complete product distribution and adjusting the parameters to match experimental data are also developed. A basal set of kinetic parameters for the enzyme-catalyzed reactions acting on free oligosaccharide substrates is obtained from the previous model and existing literature. Enzyme activities are adjusted to match experimental glycoform distributions for Chinese Hamster Ovary (CHO). The model is then used to predict the effect of increasing expression of a target glycoprotein on the product glycoform distribution and evaluate appropriate metabolic engineering strategies to return the glycoform profile to its original distribution pattern. This model may find significant utility in the future to predict glycosylation patterns and direct glycoengineering projects to optimize glycoform distributions.

  4. Bioorthogonal labeling cell-surface proteins expressed in pancreatic cancer cells to identify potential diagnostic/therapeutic biomarkers

    PubMed Central

    Haun, Randy S; Quick, Charles M; Siegel, Eric R; Raju, Ilangovan; Mackintosh, Samuel G; Tackett, Alan J

    2015-01-01

    To develop new diagnostic and therapeutic tools to specifically target pancreatic tumors, it is necessary to identify cell-surface proteins that may serve as potential tumor-specific targets. In this study we used an azido-labeled bioorthogonal chemical reporter to metabolically label N-linked glycoproteins on the surface of pancreatic cancer cell lines to identify potential targets that may be exploited for detection and/or treatment of pancreatic cancer. Labeled glycoproteins were tagged with biotin using click chemistry, purified by streptavidin-coupled magnetic beads, separated by gel electrophoresis, and identified by liquid chromatography-tandem mass spectrometry (MS). MS/MS analysis of peptides from 3 cell lines revealed 954 unique proteins enriched in the azido sugar samples relative to control sugar samples. A comparison of the proteins identified in each sample indicated 20% of these proteins were present in 2 cell lines (193 of 954) and 17 of the proteins were found in all 3 cell lines. Five of the 17 proteins identified in all 3 cell lines have not been previously reported to be expressed in pancreatic cancer; thus indicating that novel cell-surface proteins can be revealed through glycoprotein profiling. Western analysis of one of these glycoproteins, ecto-5′-nucleotidase (NT5E), revealed it is expressed in 8 out of 8 pancreatic cancer cell lines examined. Further, immunohistochemical analysis of human pancreatic tissues indicates NT5E is significantly overexpressed in pancreatic tumors compared to normal pancreas. Thus, we have demonstrated that metabolic labeling with bioorthogonal chemical reporters can be used to selectively enrich and identify novel cell-surface glycoproteins expressed in pancreatic ductal adenocarcinomas. PMID:26176765

  5. High vacuum cells for classical surface techniques

    SciTech Connect

    Martinez, Imee Su; Baldelli, Steven

    2010-04-15

    Novel glass cells were designed and built to be able to perform surface potential and surface tension measurements in a contained environment. The cells can withstand pressures of approximately 1x10{sup -6} Torr, providing a reasonable level of control in terms of the amounts of volatile contaminants during experimentation. The measurements can take several hours; thus the cells help maintain the integrity of the sample in the course of the experiment. To test for the feasibility of the cell design, calibration measurements were performed. For the surface potential cell, the modified TREK 6000B-7C probe exhibited performance comparable to its unmodified counterpart. The correlation measurements between applied potential on the test surface and the measured potential showed R-values very close to 1 as well as standard deviation values of less than 1. Results also demonstrate improved measurement values for experiments performed in vacuum. The surface tension cell, on the other hand, which was used to perform the pendant drop method, was tested on common liquids and showed percentage errors of 0.5% when compared to literature values. The fabricated cells redefine measurements using classical surface techniques, providing unique and novel methods of sample preparation, premeasurement preparation, and sample analysis at highly beneficial expenditure cost.

  6. Functional dynamics of cell surface membrane proteins

    NASA Astrophysics Data System (ADS)

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

    2014-04-01

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

  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. 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.

  9. Detection of Cell Surface Dopamine Receptors

    PubMed Central

    Xiao, Jiping; Bergson, Clare

    2014-01-01

    Dopamine receptors are a class of metabotropic G protein-coupled receptors. Plasma membrane expression is a key determinant of receptor signaling, and one that is regulated both by extra and intracellular cues. Abnormal dopamine receptor signaling is implicated in several neuropsychiatric disorders, including schizophrenia and attention deficit hyperactivity disorder, as well as drug abuse. Here, we describe in detail the application of two complementary applications of protein biotinylation and enzyme-linked immunoabsorbant assay (ELISA) for detecting and quantifying levels of dopamine receptors expressed on the cell surface. In the biotinylation method, cell surface receptors are labeled with Sulfo-NHS-biotin. The charge on the sulfonyl facilitates water solubility of the reactive biotin compound and prevents its diffusion across the plasma membrane. In the ELISA method, cells surface labeling is achieved with antibodies specific to extracellular epitopes on the receptors, and by fixing the cells without detergent such that the plasma membrane remains intact. PMID:23296774

  10. Mechanical guidance through cell-cell and cell-surface contact during multicellular streaming

    NASA Astrophysics Data System (ADS)

    Wang, Chenlu; Driscoll, Meghan; Gupta, Satyandra K.; Parent, Carole; Losert, Wolfgang

    2014-03-01

    During collective cell migration, mechanical forces arise from the extracellular matrix (ECM) through cell-surface contact and from other cells through cell-cell contact. These forces regulate the motion of migrating cell groups. To determine how these mechanical interactions balance during cell migration, we measured the shape dynamics of Dictyostelium discoideum cells at the multicellular streaming stage. We found that cells can coordinate their motion by synchronizing protrusion waves that travel along their membranes when they form proper cell-cell adhesion and cell-surface adhesion. In addition, our experiments on live actin labeled cells show that intracellular actin polymerization actively responds to the change of cell-cell/surface adhesion and helps to stabilize multicellular migration streams. Our finding suggests that the coordination of motion between neighboring cells in collective migration requires a balance between cell-cell adhesion and cell-surface adhesion, and that the cell cytoskeleton plays an important role in this balance.

  11. Dysregulated expression of cell surface glycoprotein CDCP1 in prostate cancer

    PubMed Central

    Yang, Lifang; Dutta, Sucharita M.; Troyer, Dean A.; Lin, Jefferson B.; Lance, Raymond A.; Nyalwidhe, Julius O.; Drake, Richard R; Semmes, O. John

    2015-01-01

    CUB-domain-containing protein 1 (CDCP1) is a trans-membrane protein regulator of cell adhesion with a potent pro-migratory function in tumors. Given that proteolytic cleavage of the ectodomain correlates with outside-in oncogenic signaling, we characterized glycosylation in the context of cellular processing and expression of CDCP1 in prostate cancer. We detected 135 kDa full-length and proteolytic processed 70 kDa species in a panel of PCa cell models. The relative expression of full-length CDCP1 correlated with the metastatic potential of syngeneic cell models and an increase in surface membrane expression of CDCP1 was observed in tumor compared to adjacent normal prostate tissues. We demonstrated that glycosylation of CDCP1 is a prerequisite for protein stability and plasma membrane localization, and that the expression level and extent of N-glycosylation of CDCP1 correlated with metastatic status. Interestingly, complex N-linked glycans with sialic acid chains were restricted to the N-terminal half of the ectodomain and absent in the truncated species. Characterization of the extracellular expression of CDCP1 identified novel circulating forms and revealed that extracellular vesicles provide additional processing pathways. Employing immunoaffinity mass spectrometry, we detected elevated levels of circulating CDCP1 in patient urine with high-risk disease. Our results establish that differential glycosylation, cell surface presentation and extracellular expression of CDCP1 are hallmarks of PCa progression. PMID:26497208

  12. Automated Glycan Sequencing from Tandem Mass Spectra of N-Linked Glycopeptides.

    PubMed

    Yu, Chuan-Yih; Mayampurath, Anoop; Zhu, Rui; Zacharias, Lauren; Song, Ehwang; Wang, Lei; Mechref, Yehia; Tang, Haixu

    2016-06-07

    Mass spectrometry has become a routine experimental tool for proteomic biomarker analysis of human blood samples, partly due to the large availability of informatics tools. As one of the most common protein post-translational modifications (PTMs) in mammals, protein glycosylation has been observed to alter in multiple human diseases and thus may potentially be candidate markers of disease progression. While mass spectrometry instrumentation has seen advancements in capabilities, discovering glycosylation-related markers using existing software is currently not straightforward. Complete characterization of protein glycosylation requires the identification of intact glycopeptides in samples, including identification of the modification site as well as the structure of the attached glycans. In this paper, we present GlycoSeq, an open-source software tool that implements a heuristic iterated glycan sequencing algorithm coupled with prior knowledge for automated elucidation of the glycan structure within a glycopeptide from its collision-induced dissociation tandem mass spectrum. GlycoSeq employs rules of glycosidic linkage as defined by glycan synthetic pathways to eliminate improbable glycan structures and build reasonable glycan trees. We tested the tool on two sets of tandem mass spectra of N-linked glycopeptides cell lines acquired from breast cancer patients. After employing enzymatic specificity within the N-linked glycan synthetic pathway, the sequencing results of GlycoSeq were highly consistent with the manually curated glycan structures. Hence, GlycoSeq is ready to be used for the characterization of glycan structures in glycopeptides from MS/MS analysis. GlycoSeq is released as open source software at https://github.com/chpaul/GlycoSeq/ .

  13. Surface cell immobilization within perfluoroalkoxy microchannels

    NASA Astrophysics Data System (ADS)

    Stojkovič, Gorazd; Krivec, Matic; Vesel, Alenka; Marinšek, Marjan; Žnidaršič-Plazl, Polona

    2014-11-01

    Perfluoroalkoxy (PFA) is one of the most promising materials for the fabrication of cheap, solvent resistant and reusable microfluidic chips, which have been recently recognized as effective tools for biocatalytic process development. The application of biocatalysts significantly depends on efficient immobilization of enzymes or cells within the reactor enabling long-term biocatalyst use. Functionalization of PFA microchannels by 3-aminopropyltriethoxysilane (ATPES) and glutaraldehyde was used for rapid preparation of microbioreactors with surface-immobilized cells. X-ray photoelectron spectroscopy and scanning electron microscopy were used to accurately monitor individual treatment steps and to select conditions for cell immobilization. The optimized protocol for Saccharomyces cerevisiae immobilization on PFA microchannel walls comprised ethanol surface pretreatment, 4 h contacting with 10% APTES aqueous solution, 10 min treatment with 1% glutaraldehyde and 20 min contacting with cells in deionized water. The same protocol enabled also immobilization of Escherichia coli, Pseudomonas putida and Bacillus subtilis cells on PFA surface in high densities. Furthermore, the developed procedure has been proved to be very efficient also for surface immobilization of tested cells on other materials that are used for microreactor fabrication, including glass, polystyrene, poly (methyl methacrylate), polycarbonate, and two olefin-based polymers, namely Zeonor® and Topas®.

  14. GlycoFish: A Database of Zebrafish N-linked Glycoproteins Identified Using SPEG Method Coupled with LC/MS

    PubMed Central

    Baycin-Hizal, Deniz; Tian, Yuan; Akan, Ilhan; Jacobson, Elena; Clark, Dean; Wu, Alexander; Jampol, Russell; Palter, Karen; Betenbaugh, Michael; Zhang, Hui

    2011-01-01

    Zebrafish (Danio rerio) is a model organism to study the mechanisms and pathways of human disorders. Many dysfunctions in neurological, development and neuromuscular systems are due to glycosylation deficiencies, but the glycoproteins involved in zebrafish embryonic development have not been established. In this study, a mass spectrometry-based glycoproteomic characterization of zebrafish embryos was performed to identify the N-linked glycoproteins and N-linked glycosylation sites. To increase the number of glycopeptides, proteins from zebrafish were digested with two different proteases, chymotrypsin and trypsin, into peptides of different length. The N-glycosylated peptides of zebrafish were then captured by the solid phase extraction of N-linked glycopeptides (SPEG) method and the peptides were identified with an LTQ OrbiTrap Velos mass spectrometer. From 265 unique glycopeptides, including 269 consensus NXT/S glycosites, we identified 169 different N-glycosylated proteins. The identified glycoproteins were highly abundant in proteins belonging to the transporter, cell adhesion, and ion channel/ion binding categories which are important to embryonic, organ, and central nervous system development. This proteomics data will expand our knowledge about glycoproteins in zebrafish and may be used to elucidate the role glycosylation plays in cellular processes and disease. The glycoprotein data are available through the GlycoFish database (http://betenbaugh.jhu.edu/GlycoFish) introduced in this paper. PMID:21591763

  15. Detection of cell surface dopamine receptors.

    PubMed

    Xiao, Jiping; Bergson, Clare

    2013-01-01

    Dopamine receptors are a class of metabotropic G protein-coupled receptors. Plasma membrane expression is a key determinant of receptor signaling, and one that is regulated both by extra and intracellular cues. Abnormal dopamine receptor signaling is implicated in several neuropsychiatric disorders, including schizophrenia and attention deficit hyperactivity disorder, as well as drug abuse. Here, we describe in detail the application of two complementary applications of protein biotinylation and enzyme-linked immunoabsorbent assay (ELISA) for detecting and quantifying levels of dopamine receptors expressed on the cell surface. In the biotinylation method, cell surface receptors are labeled with Sulfo-NHS-biotin. The charge on the sulfonyl facilitates water solubility of the reactive biotin compound and prevents its diffusion across the plasma membrane. In the ELISA method, surface labeling is achieved with antibodies specific to extracellular epitopes on the receptors, and by fixing the cells without detergent such that the plasma membrane remains intact.

  16. Vesicle trafficking and cell surface membrane patchiness.

    PubMed Central

    Tang, Q; Edidin, M

    2001-01-01

    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

  17. Cell Surface Glycoside Hydrolases of Streptococcus gordonii Promote Growth in Saliva

    PubMed Central

    Zhou, Yuan; Zhang, Luxia; Shah, Nehal; Palmer, Robert J.; Cisar, John O.

    2016-01-01

    ABSTRACT The growth of the oral commensal Streptococcus gordonii in saliva may depend on a number of glycoside hydrolases (GHs), including three cell wall-anchored proteins that are homologs of pneumococcal β-galactosidase (BgaA), β-N-acetylglucosaminidase (StrH), and endo-β-N-acetylglucosaminidase D (EndoD). In the present study, we introduced unmarked in-frame deletions into the corresponding genes of S. gordonii DL1, verified the presence (or absence) of the encoded proteins on the resulting mutant strains, and compared these strains with wild-type strain DL1 for growth and glycan foraging in saliva. The overnight growth of wild-type DL1 was reduced 3- to 10-fold by the deletion of any one or two genes and approximately 20-fold by the deletion of all three genes. The only notable change in the salivary proteome associated with this reduction of growth was a downward shift in the apparent molecular masses of basic proline-rich glycoproteins (PRG), which was accompanied by the loss of lectin binding sites for galactose-specific Erythrina cristagalli agglutinin (ECA) and mannose-specific Galanthus nivalis agglutinin (GNA). The binding of ECA to PRG was also abolished in saliva cultures of mutants that expressed cell surface BgaA alone or together with either StrH or EndoD. However, the subsequent loss of GNA binding was seen only in saliva cocultures of different mutants that together expressed all three cell surface GHs. The findings indicate that the growth of S. gordonii DL1 in saliva depends to a significant extent on the sequential actions of first BgaA and then StrH and EndoD on N-linked glycans of PRG. IMPORTANCE The ability of oral bacteria to grow on salivary glycoproteins is critical for dental plaque biofilm development. Little is known, however, about how specific salivary components are attacked and utilized by different members of the biofilm community, such as Streptococcus gordonii. Streptococcus gordonii DL1 has three cell wall

  18. Cell surface engineering with edible protein nanoshells.

    PubMed

    Drachuk, Irina; Shchepelina, Olga; Harbaugh, Svetlana; Kelley-Loughnane, Nancy; Stone, Morley; Tsukruk, Vladimir V

    2013-09-23

    Natural protein (silk fibroin) nanoshells are assembled on the surface of Saccharomyces cerevisiae yeast cells without compromising their viability. The nanoshells facilitate initial protection of the cells and allow them to function in encapsulated state for some time period, afterwards being completely biodegraded and consumed by the cells. In contrast to a traditional methanol treatment, the gentle ionic treatment suggested here stabilizes the shell silk fibroin structure but does not compromise the viability of the cells, as indicated by the fast response of the encapsulated cells, with an immediate activation by the inducer molecules. Extremely high viability rates (up to 97%) and preserved activity of encapsulated cells are facilitated by cytocompatibility of the natural proteins and the formation of highly porous shells in contrast to traditional polyelectrolyte-based materials. Moreover, in a high contrast to traditional synthetic shells, the silk proteins are biodegradable and can be consumed by cells at a later stage of growth, thus releasing the cells from their temporary protective capsules. These on-demand encapsulated cells can be considered a valuable platform for biocompatible and biodegradable cell encapsulation, controlled cell protection in a synthetic environment, transfer to a device environment, and cell implantation followed by biodegradation and consumption of protective protein shells.

  19. Fludarabine and cladribine induce changes in surface proteins on human B-lymphoid cell lines involved with apoptosis, cell survival, and antitumor immunity.

    PubMed

    Kohnke, Philippa L; Mactier, Swetlana; Almazi, Juhura G; Crossett, Ben; Christopherson, Richard I

    2012-09-07

    Fludarabine and cladribine are purine analogues used to treat hematological malignancies. Alone or in combination with therapeutic antibodies, they are effective in treating patients with chronic lymphocytic leukemia and non-Hodgkin's lymphoma. However, the mechanisms of action of these drugs are not well understood. Plasma membrane proteins perform a variety of essential functions that can be affected by malignancy and perturbed by chemotherapy. Analysis of surface proteins may contribute to an understanding of the mechanisms of action of purine analogues and identify biomarkers for targeted therapy. The surface of human cells is rich in N-linked glycoproteins, enabling use of a hydrazide-coupling technique to enrich for glycoproteins, with iTRAQ labeling for quantitative comparison. A number of plasma membrane proteins on human leukemia and lymphoma cells were affected by treatment with a purine analogue, including decreases in CD22 (an adhesion and signaling molecule) and increases in CD205 (a "damaged cell marker") and CD80 and CD50 (T-cell interaction molecules). Purine analogues may affect B-cell receptor (BCR) signaling and costimulatory molecules, leading to multiple signals for apoptosis and cell clearance. Fludarabine and cladribine induce differential effects, with some cell survival proteins (ECE-1 and CD100) more abundant after fludarabine treatment. Cell surface proteins induced by fludarabine and cladribine may be targets for therapeutic antibodies.

  20. Glycopeptide capture for cell surface proteomics.

    PubMed

    Lee, M C Gilbert; Sun, Bingyun

    2014-05-09

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

  1. Living Toroids - Cells on Toroidal Surfaces

    NASA Astrophysics Data System (ADS)

    Chang, Ya-Wen; Angelini, Thomas; Marquez, Samantha; Kim, Harold; Fernandez-Nieves, Alberto

    2014-03-01

    Cellular environment influences a multitude of cellular functions by providing chemical and physical signals that modulate cell behavior, dynamics, development, and eventually survival. Substrate mechanics has been recognized as one of the important physical cues that governs cell behavior at single cell level as well as in collective cell motion. Past research has suggested several contact-guided behaviors to be the result of surface curvature. However, studies on the effect of curvature are relatively scarce likely due to the difficulty in generating substrates with well-defined curvature. Here we describe the generation of toroidal droplets, which unlike spherical droplets, have regions of both positive and negative Gaussian curvature. Additionally, the range of curvatures can be controlled by varying the size and aspect ratio of the torus. Cells are either encapsulated inside toroidal droplets or located on toroidal hydrogel surfaces. Preliminary studies use B. Subtilis to study the organization of bacteria biofilms. When confined in droplets surrounded by yield-stress fluid, bacteria self-organize into heterogeneous biofilm at fluid- substrate interface. It is found that the surface curvature in the sub-millimeter scale has little effect on biofilm architecture.

  2. Specialized cell surface structures in cellulolytic bacteria.

    PubMed

    Lamed, R; Naimark, J; Morgenstern, E; Bayer, E A

    1987-08-01

    The cell surface topology of various gram-negative and -positive, anaerobic and aerobic, mesophilic and thermophilic, cellulolytic and noncellulolytic bacteria was investigated by scanning electron microscopic visualization using cationized ferritin. Characteristic protuberant structures were observed on cells of all cellulolytic strains. These structures appeared to be directly related to the previously described exocellular cellulase-containing polycellulosomes of Clostridium thermocellum YS (E. A. Bayer and R. Lamed, J. Bacteriol. 167:828-836, 1986). Immunochemical evidence and lectin-binding studies suggested a further correlation on the molecular level among cellulolytic bacteria. The results indicate that such cell surface cellulase-containing structures may be of general consequence to the bacterial interaction with and degradation of cellulose.

  3. Specialized cell surface structures in cellulolytic bacteria.

    PubMed Central

    Lamed, R; Naimark, J; Morgenstern, E; Bayer, E A

    1987-01-01

    The cell surface topology of various gram-negative and -positive, anaerobic and aerobic, mesophilic and thermophilic, cellulolytic and noncellulolytic bacteria was investigated by scanning electron microscopic visualization using cationized ferritin. Characteristic protuberant structures were observed on cells of all cellulolytic strains. These structures appeared to be directly related to the previously described exocellular cellulase-containing polycellulosomes of Clostridium thermocellum YS (E. A. Bayer and R. Lamed, J. Bacteriol. 167:828-836, 1986). Immunochemical evidence and lectin-binding studies suggested a further correlation on the molecular level among cellulolytic bacteria. The results indicate that such cell surface cellulase-containing structures may be of general consequence to the bacterial interaction with and degradation of cellulose. Images PMID:3301817

  4. Galactosyltransferase 4 is a major control point for glycan branching in N-linked glycosylation

    PubMed Central

    McDonald, Andrew G.; Hayes, Jerrard M.; Bezak, Tania; Głuchowska, Sonia A.; Cosgrave, Eoin F. J.; Struwe, Weston B.; Stroop, Corné J. M.; Kok, Han; van de Laar, Teun; Rudd, Pauline M.; Tipton, Keith F.; Davey, Gavin P.

    2014-01-01

    ABSTRACT Protein N-glycosylation is a common post-translational modification that produces a complex array of branched glycan structures. The levels of branching, or antennarity, give rise to differential biological activities for single glycoproteins. However, the precise mechanism controlling the glycan branching and glycosylation network is unknown. Here, we constructed quantitative mathematical models of N-linked glycosylation that predicted new control points for glycan branching. Galactosyltransferase, which acts on N-acetylglucosamine residues, was unexpectedly found to control metabolic flux through the glycosylation pathway and the level of final antennarity of nascent protein produced in the Golgi network. To further investigate the biological consequences of glycan branching in nascent proteins, we glycoengineered a series of mammalian cells overexpressing human chorionic gonadotropin (hCG). We identified a mechanism in which galactosyltransferase 4 isoform regulated N-glycan branching on the nascent protein, subsequently controlling biological activity in an in vivo model of hCG activity. We found that galactosyltransferase 4 is a major control point for glycan branching decisions taken in the Golgi of the cell, which might ultimately control the biological activity of nascent glycoprotein. PMID:25271059

  5. Reversibility of cell surface label rearrangement

    PubMed Central

    1976-01-01

    Cell surface labeling can cause rearrangements of randomly distributed membrane components. Removal of the label bound to the cell surface allows the membrane components to return to their original random distribution, demonstrating that label is necessary to maintain as well as to induce rearrangements. With scanning electron microscopy, the rearrangement of concanavalin A (con A) and ricin binding sites on LA-9 cells has been followed by means of hemocyanin, a visual label. The removal of con A from its binding sites at the cell surface with alpha- methyl mannoside, and the return of these sites to their original distribution are also followed in this manner. There are labeling differences with con A and ricin. Under some conditions, however, the same rearrangements are seen with both lectins. The disappearance of labeled sites from areas of ruffling activity is a major feature of the rearrangements seen. Both this ruffling activity and the rearrangement of label are sensitive to cytochalasin B, and ruffling activity, perhaps along with other cytochalasin-sensitive structure, may play a role in the rearrangements of labeled sites. PMID:1025154

  6. 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.

  7. Bacterial cell surface structures in Yersinia enterocolitica.

    PubMed

    Białas, Nataniel; Kasperkiewicz, Katarzyna; Radziejewska-Lebrecht, Joanna; Skurnik, Mikael

    2012-06-01

    Yersinia enterocolitica is a widespread member of the family of Enterobacteriaceae that contains both non-virulent and virulent isolates. Pathogenic Y. enterocolitica strains, especially belonging to serotypes O:3, O:5,27, O:8 and O:9 are etiologic agents of yersiniosis in animals and humans. Y. enterocolitica cell surface structures that play a significant role in virulence have been subject to many investigations. These include outer membrane (OM) glycolipids such as lipopolysaccharide (LPS) and enterobacterial common antigen (ECA) and several cell surface adhesion proteins present only in virulent Y. enterocolitica, i.e., Inv, YadA and Ail. While the yadA gene is located on the Yersinia virulence plasmid the Ail, Inv, LPS and ECA are chromosomally encoded. These structures ensure the correct architecture of the OM, provide adhesive properties as well as resistance to antimicrobial peptides and to host innate immune response mechanisms.

  8. Mutation of a Single Envelope N-Linked Glycosylation Site Enhances the Pathogenicity of Bovine Leukemia Virus

    PubMed Central

    Bouzar, Amel Baya; Jacques, Jean-Rock; Cosse, Jean-Philippe; Gillet, Nicolas; Callebaut, Isabelle; Reichert, Michal

    2015-01-01

    ABSTRACT Viruses have coevolved with their host to ensure efficient replication and transmission without inducing excessive pathogenicity that would indirectly impair their persistence. This is exemplified by the bovine leukemia virus (BLV) system in which lymphoproliferative disorders develop in ruminants after latency periods of several years. In principle, the equilibrium reached between the virus and its host could be disrupted by emergence of more pathogenic strains. Intriguingly but fortunately, such a hyperpathogenic BLV strain was never observed in the field or designed in vitro. In this study, we sought to understand the role of envelope N-linked glycosylation with the hypothesis that this posttranslational modification could either favor BLV infection by allowing viral entry or allow immune escape by using glycans as a shield. Using reverse genetics of an infectious molecular provirus, we identified a N-linked envelope glycosylation site (N230) that limits viral replication and pathogenicity. Indeed, mutation N230E unexpectedly leads to enhanced fusogenicity and protein stability. IMPORTANCE Infection by retroviruses requires the interaction of the viral envelope protein (SU) with a membrane-associated receptor allowing fusion and release of the viral genomic RNA into the cell. We show that N-linked glycosylation of the bovine leukemia virus (BLV) SU protein is, as expected, essential for cell infection in vitro. Consistently, mutation of all glycosylation sites of a BLV provirus destroys infectivity in vivo. However, single mutations do not significantly modify replication in vivo. Instead, a particular mutation at SU codon 230 increases replication and accelerates pathogenesis. This unexpected observation has important consequences in terms of disease control and managing. PMID:26085161

  9. Complicated N-linked glycans in simple organisms

    PubMed Central

    Schiller, Birgit; Hykollari, Alba; Yan, Shi; Paschinger, Katharina; Wilson, Iain B. H.

    2013-01-01

    Although countless genomes have now been sequenced, the glycomes of the vast majority of eukaryotes still present a series of unmapped frontiers. However, strides are being made in a few groups of invertebrate and unicellular organisms as regards their N-glycans and N-glycosylation pathways. Thereby, the traditional classification of glycan structures inevitably approaches its boundaries. Indeed, the glycomes of these organisms are rich in surprises including a multitude of modifications of the core regions of N-glycans and unusual antennae. From the actually rather limited glycomic information we have, it is nevertheless obvious that the biotechnological, developmental and immunological relevance of these modifications, especially in insect cell lines, model organisms and parasites means that deciphering unusual glycomes is of more than just academic interest. PMID:22944671

  10. Interactions between N-linked glycosylation and polymerisation of neuroserpin within the endoplasmic reticulum.

    PubMed

    Moriconi, Claudia; Ordoñez, Adriana; Lupo, Giuseppe; Gooptu, Bibek; Irving, James A; Noto, Rosina; Martorana, Vincenzo; Manno, Mauro; Timpano, Valentina; Guadagno, Noemi A; Dalton, Lucy; Marciniak, Stefan J; Lomas, David A; Miranda, Elena

    2015-12-01

    The neuronal serpin neuroserpin undergoes polymerisation as a consequence of point mutations that alter its conformational stability, leading to a neurodegenerative dementia called familial encephalopathy with neuroserpin inclusion bodies (FENIB). Neuroserpin is a glycoprotein with predicted glycosylation sites at asparagines 157, 321 and 401. We used site-directed mutagenesis, transient transfection, western blot, metabolic labelling and ELISA to probe the relationship between glycosylation, folding, polymerisation and degradation of neuroserpin in validated cell models of health and disease. Our data show that glycosylation at N157 and N321 plays an important role in maintaining the monomeric state of neuroserpin, and we propose this is the result of steric hindrance or effects on local conformational dynamics that can contribute to polymerisation. Asparagine residue 401 is not glycosylated in wild type neuroserpin and in several polymerogenic variants that cause FENIB, but partial glycosylation was observed in the G392E mutant of neuroserpin that causes severe, early-onset dementia. Our findings indicate that N401 glycosylation reports lability of the C-terminal end of neuroserpin in its native state. This C-terminal lability is not required for neuroserpin polymerisation in the endoplasmic reticulum, but the additional glycan facilitates degradation of the mutant protein during proteasomal impairment. In summary, our results indicate how normal and variant-specific N-linked glycosylation events relate to intracellular folding, misfolding, degradation and polymerisation of neuroserpin.

  11. N-linked glycoproteome profiling of seedling leaf in Brachypodium distachyon L.

    PubMed

    Zhang, Ming; Chen, Guan-Xing; Lv, Dong-Wen; Li, Xiao-Hui; Yan, Yue-Ming

    2015-04-03

    Brachypodium distachyon L., a model plant for cereal crops, has become important as an alternative and potential biofuel grass. In plants, N-glycosylation is one of the most common and important protein modifications, playing important roles in signal recognition, increase in protein activity, stability of protein structure, and formation of tissues and organs. In this study, we performed the first glycoproteome analysis in the seedling leaves of B. distachyon. Using lectin affinity chromatography enrichment and mass-spectrometry-based analysis, we identified 47 glycosylation sites representing 46 N-linked glycoproteins. Motif-X analysis showed that two conserved motifs, N-X-T/S (X is any amino acid, except Pro), were significantly enriched. Further functional analysis suggested that some of these identified glycoproteins are involved in signal transduction, protein trafficking, and quality control and the modification and remodeling of cell-wall components such as receptor-like kinases, protein disulfide isomerase, and polygalacturonase. Moreover, transmembrane helices and signal peptide prediction showed that most of these glycoproteins could participate in typical protein secretory pathways in eukaryotes. The results provide a general overview of protein N-glycosylation modifications during the early growth of seedling leaves in B. distachyon and supplement the glycoproteome databases of plants.

  12. 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.

  13. 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

  14. N-linked glycosylation of protease-activated receptor-1 at extracellular loop 2 regulates G-protein signaling bias.

    PubMed

    Soto, Antonio G; Smith, Thomas H; Chen, Buxin; Bhattacharya, Supriyo; Cordova, Isabel Canto; Kenakin, Terry; Vaidehi, Nagarajan; Trejo, JoAnn

    2015-07-07

    Protease-activated receptor-1 (PAR1) is a G-protein-coupled receptor (GPCR) for the coagulant protease thrombin. Similar to other GPCRs, PAR1 is promiscuous and couples to multiple heterotrimeric G-protein subtypes in the same cell and promotes diverse cellular responses. The molecular mechanism by which activation of a given GPCR with the same ligand permits coupling to multiple G-protein subtypes is unclear. Here, we report that N-linked glycosylation of PAR1 at extracellular loop 2 (ECL2) controls G12/13 versus Gq coupling specificity in response to thrombin stimulation. A PAR1 mutant deficient in glycosylation at ECL2 was more effective at stimulating Gq-mediated phosphoinositide signaling compared with glycosylated wildtype receptor. In contrast, wildtype PAR1 displayed a greater efficacy at G12/13-dependent RhoA activation compared with mutant receptor lacking glycosylation at ECL2. Endogenous PAR1 rendered deficient in glycosylation using tunicamycin, a glycoprotein synthesis inhibitor, also exhibited increased PI signaling and diminished RhoA activation opposite to native receptor. Remarkably, PAR1 wildtype and glycosylation-deficient mutant were equally effective at coupling to Gi and β-arrestin-1. Consistent with preferential G12/13 coupling, thrombin-stimulated PAR1 wildtype strongly induced RhoA-mediated stress fiber formation compared with mutant receptor. In striking contrast, glycosylation-deficient PAR1 was more effective at increasing cellular proliferation, associated with Gq signaling, than wildtype receptor. These studies suggest that N-linked glycosylation at ECL2 contributes to the stabilization of an active PAR1 state that preferentially couples to G12/13 versus Gq and defines a previously unidentified function for N-linked glycosylation of GPCRs in regulating G-protein signaling bias.

  15. Shape-memory surfaces for cell mechanobiology

    PubMed Central

    Ebara, Mitsuhiro

    2015-01-01

    Shape-memory polymers (SMPs) are a new class of smart materials, which have the capability to change from a temporary shape ‘A’ to a memorized permanent shape ‘B’ upon application of an external stimulus. In recent years, SMPs have attracted much attention from basic and fundamental research to industrial and practical applications due to the cheap and efficient alternative to well-known metallic shape-memory alloys. Since the shape-memory effect in SMPs is not related to a specific material property of single polymers, the control of nanoarchitecture of polymer networks is particularly important for the smart functions of SMPs. Such nanoarchitectonic approaches have enabled us to further create shape-memory surfaces (SMSs) with tunable surface topography at nano scale. The present review aims to bring together the exciting design of SMSs and the ever-expanding range of their uses as tools to control cell functions. The goal for these endeavors is to mimic the surrounding mechanical cues of extracellular environments which have been considered as critical parameters in cell fate determination. The untapped potential of SMSs makes them one of the most exciting interfaces of materials science and cell mechanobiology. PMID:27877747

  16. Shape-memory surfaces for cell mechanobiology

    NASA Astrophysics Data System (ADS)

    Ebara, Mitsuhiro

    2015-02-01

    Shape-memory polymers (SMPs) are a new class of smart materials, which have the capability to change from a temporary shape ‘A’ to a memorized permanent shape ‘B’ upon application of an external stimulus. In recent years, SMPs have attracted much attention from basic and fundamental research to industrial and practical applications due to the cheap and efficient alternative to well-known metallic shape-memory alloys. Since the shape-memory effect in SMPs is not related to a specific material property of single polymers, the control of nanoarchitecture of polymer networks is particularly important for the smart functions of SMPs. Such nanoarchitectonic approaches have enabled us to further create shape-memory surfaces (SMSs) with tunable surface topography at nano scale. The present review aims to bring together the exciting design of SMSs and the ever-expanding range of their uses as tools to control cell functions. The goal for these endeavors is to mimic the surrounding mechanical cues of extracellular environments which have been considered as critical parameters in cell fate determination. The untapped potential of SMSs makes them one of the most exciting interfaces of materials science and cell mechanobiology.

  17. Shape-memory surfaces for cell mechanobiology.

    PubMed

    Ebara, Mitsuhiro

    2015-02-01

    Shape-memory polymers (SMPs) are a new class of smart materials, which have the capability to change from a temporary shape 'A' to a memorized permanent shape 'B' upon application of an external stimulus. In recent years, SMPs have attracted much attention from basic and fundamental research to industrial and practical applications due to the cheap and efficient alternative to well-known metallic shape-memory alloys. Since the shape-memory effect in SMPs is not related to a specific material property of single polymers, the control of nanoarchitecture of polymer networks is particularly important for the smart functions of SMPs. Such nanoarchitectonic approaches have enabled us to further create shape-memory surfaces (SMSs) with tunable surface topography at nano scale. The present review aims to bring together the exciting design of SMSs and the ever-expanding range of their uses as tools to control cell functions. The goal for these endeavors is to mimic the surrounding mechanical cues of extracellular environments which have been considered as critical parameters in cell fate determination. The untapped potential of SMSs makes them one of the most exciting interfaces of materials science and cell mechanobiology.

  18. Production, characterization and pharmacokinetic properties of antibodies with N-linked Mannose-5 glycans

    PubMed Central

    Yu, Marcella; Brown, Darren; Reed, Chae; Chung, Shan; Lutman, Jeff; Stefanich, Eric; Wong, Anne; Stephan, Jean-Philippe; Bayer, Robert

    2012-01-01

    The effector functions of therapeutic antibodies are strongly affected by the specific glycans added to the Fc domain during post-translational processing. Antibodies bearing high levels of N-linked mannose-5 glycan (Man5) have been reported to exhibit enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared with antibodies with fucosylated complex or hybrid glycans. To better understand the relationship between antibodies with high levels of Man5 and their biological activity in vivo, we developed an approach to generate substantially homogeneous antibodies bearing the Man5 glycoform. A mannosidase inhibitor, kifunensine, was first incorporated in the cell culture process to generate antibodies with a distribution of high mannose glycoforms. Antibodies were then purified and treated with a mannosidase for trimming to Man5 in vitro. This 2-step approach can consistently generate antibodies with > 99% Man5 glycan. Antibodies bearing varying levels of Man5 were studied to compare ADCC and Fcγ receptor binding, and they showed enhanced ADCC activity and increased binding affinity to the FcγRIIIA. In addition, the clearance rate of antibodies bearing Man8/9 and Man5 glycans was determined in a pharmacokinetics study in mice. When compared with historical data, the antibodies bearing the high mannose glycoform exhibited faster clearance rate compared with antibodies bearing the fucosylated complex glycoform, while the pharmacokinetic properties of antibodies with Man8/9 and Man5 glycoforms appeared similar. In addition, we identified the presence of a mannosidase in mouse serum that converted most Man8/9 to Man6 after 24 h. PMID:22699308

  19. The criticality of high-resolution N-linked carbohydrate assays and detailed characterization of antibody effector function in the context of biosimilar development.

    PubMed

    Brady, Lowell J; Velayudhan, Jyoti; Visone, Devi B; Daugherty, Ken C; Bartron, Jeff L; Coon, Michael; Cornwall, Cabot; Hinckley, Peter J; Connell-Crowley, Lisa

    2015-01-01

    Accurate measurement and functional characterization of antibody Fc domain N-linked glycans is critical to successful biosimilar development. Here, we describe the application of methods to accurately quantify and characterize the N-linked glycans of 2 IgG1 biosimilars with effector function activity, and show the potential pitfalls of using assays with insufficient resolution. Accurate glycan assessment was combined with glycan enrichment using lectin chromatography or production with glycosylation inhibitors to produce enriched pools of key glycan species for subsequent assessment in cell-based antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity effector function assays. This work highlights the challenges of developing high-quality biosimilar candidates and the need for modern biotechnology capabilities. These results show that high-quality analytics, combined with sensitive cell-based assays to study in vivo mechanisms of action, is an essential part of biosimilar development.

  20. Expanding the glycoengineering toolbox: the rise of bacterial N-linked protein glycosylation.

    PubMed

    Baker, Jenny L; Çelik, Eda; DeLisa, Matthew P

    2013-05-01

    Glycosylation is the most prevalent post-translational modification found on proteins, occurring in all domains of life. Ever since the discovery of asparagine-linked (N-linked) protein glycosylation pathways in bacteria, major efforts have been made to harness these systems for the creation of novel therapeutics, vaccines, and diagnostics. Recent advances such as the ability to produce designer glycans in bacteria, some containing unnatural sugars, and techniques for evolving glycosylation enzymes have spawned an entirely new discipline known as bacterial glycoengineering. In addition to their biotechnological and therapeutic potential, bacteria equipped with recombinant N-linked glycosylation pathways are improving our understanding of the N-glycosylation process. This review discusses the key role played by microorganisms in glycosciences, particularly in the context of N-linked glycosylation.

  1. Knowledge discovery of cell-cell and cell-surface interactions

    NASA Astrophysics Data System (ADS)

    Su, Jing

    High-throughput cell culture is an emerging technology that shows promise as a tool for research in tissue engineering, drug discovery, and medical diagnostics. An important, but overlooked, challenge is the integration of experimental methods with information processing suitable for handling large databases of cell-cell and cell-substrate interactions. In this work the traditional global descriptions of cell behaviors and surface characteristics was shown insufficient for investigating short-distance cell-to-cell and cell-to-surface interactions. Traditional summary metrics cannot distinguish information of cell near neighborhood from the average, global features, thus often is not suitable for studying distance-sensitive cell behaviors. The problem of traditional summary metrics was addressed by introducing individual-cell based local metrics that emphasize cell local environment. An individual-cell based local data analysis method was established. Contact inhibition of cell proliferation was used as a benchmark for the effectiveness of the local metrics and the method. Where global, summary metrics were unsuccessful, the local metrics successfully and quantitatively distinguished the contact inhibition effects of MC3T3-E1 cells on PLGA, PCL, and TCPS surfaces. In order to test the new metrics and analysis method in detail, a model of cell contact inhibition was proposed. Monte Carlo simulation was performed for validating the individual-cell based local data analysis method as well as the cell model itself. The simulation results well matched with the experimental observations. The parameters used in the cell model provided new descriptions of both cell behaviors and surface characteristics. Based on the viewpoint of individual cells, the local metrics and local data analysis method were extended to the investigation of cell-surface interactions, and a new high-throughput screening and knowledge discovery method on combinatorial libraries, local cell

  2. Role of different classes of mammalian cell surface molecules in adherence of coagulase positive and coagulase negative staphylococci.

    PubMed

    Hafez, Mohamed M; Aboulwafa, Mohammad M; Yassien, Mahmoud A; Hassouna, Nadia A

    2008-10-01

    In the present study the role of different mammalian cell receptors in adherence of the coagulase positive pathogen, Staphylococcus aureus and some coagulase negative staphylococci, namely Staphylococcus epidermidis and Staphylococcus saprophyticus was investigated. Upon testing the adherence to Vero and Hep-2 cells, S. aureus isolates showed an adherence to both cell lines while S. epidermidis and S. saprophyticus isolates adhered to Vero cells only. According to the obtained results, both O-linked and N-linked mammalian cell surface glycoproteins are involved in the adherence of S. aureus isolates to Vero and Hep-2 cells, whereas only the O-linked ones serve as receptors for adherence of S. epidermidis and S. saprophyticus isolates to Vero cells. Of the O-linked glycoproteins, GAG-like receptors are involved in adherence of all tested isolates to Vero cells. The coagulase positive staphylococci preferred to adhere to the highly sulphated GAGs (Heparin and chondroitin sulphate B) while the coagulase negative isolates showed higher affinity to the less sulphated ones (Chondroitin sulphate A and C). Mucin like receptors appeared to be important for the adherence of all tested staphylococci. The role exhibited by fibronectin- and fibrinogen-like receptors was detected with S. aureus and S. epidermidis but not with S. saprophyticus isolates. While, collagen and gelatin were found to contribute to the adherence of S. aureus isolates only. Neither carbohydrate moieties of the glycoconjugates nor lipid molecules on the mammalian cell surface played a role in the adherence of the tested staphylococcal isolates. Taken together, the results revealed that both coagulase negative and coagulase positive staphylococcal tested isolates adhere to the same classes of mammalian cell surface receptors such as mucin-like, fibrinogen-like, fibronectin-like and GAG-like receptors. However, the tested isolates exhibited different degrees of affinities to such receptors.

  3. Identification of genes involved in the biosynthesis of the third and fourth sugars of the Methanococcus maripaludis archaellin N-linked tetrasaccharide.

    PubMed

    Ding, Yan; Jones, Gareth M; Uchida, Kaoru; Aizawa, Shin-Ichi; Robotham, Anna; Logan, Susan M; Kelly, John; Jarrell, Ken F

    2013-09-01

    N-glycosylation is a protein posttranslational modification found in all three domains of life. Many surface proteins in Archaea, including S-layer proteins, pilins, and archaellins (archaeal flagellins) are known to contain N-linked glycans. In Methanococcus maripaludis, the archaellins are modified at multiple sites with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is the unique sugar (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-l-erythro-hexos-5-ulo-1,5-pyranose. In this study, four genes--mmp1084, mmp1085, mmp1086, and mmp1087--were targeted to determine their potential involvement of the biosynthesis of the sugar components in the N-glycan, based on bioinformatics analysis and proximity to a number of genes which have been previously demonstrated to be involved in the N-glycosylation pathway. The genes mmp1084 to mmp1087 were shown to be cotranscribed, and in-frame deletions of each gene as well as a Δmmp1086Δmmp1087 double mutant were successfully generated. All mutants were archaellated and motile. Mass spectrometry examination of purified archaella revealed that in Δmmp1084 mutant cells, the threonine linked to the third sugar of the glycan was missing, indicating a putative threonine transferase function of MMP1084. Similar analysis of the archaella of the Δmmp1085 mutant cells demonstrated that the glycan lacked the methyl group at the C-5 position of the terminal sugar, indicating that MMP1085 is a methyltransferase involved in the biosynthesis of this unique sugar. Deletion of the remaining two genes, mmp1086 and mmp1087, either singularly or together, had no effect on the structure of the archaellin N-glycan. Because of their demonstrated involvement in the N-glycosylation pathway, we designated mmp1084 as aglU and mmp1085 as aglV.

  4. Identification of Genes Involved in the Biosynthesis of the Third and Fourth Sugars of the Methanococcus maripaludis Archaellin N-Linked Tetrasaccharide

    PubMed Central

    Ding, Yan; Jones, Gareth M.; Uchida, Kaoru; Aizawa, Shin-Ichi; Robotham, Anna; Logan, Susan M.

    2013-01-01

    N-glycosylation is a protein posttranslational modification found in all three domains of life. Many surface proteins in Archaea, including S-layer proteins, pilins, and archaellins (archaeal flagellins) are known to contain N-linked glycans. In Methanococcus maripaludis, the archaellins are modified at multiple sites with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is the unique sugar (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-l-erythro-hexos-5-ulo-1,5-pyranose. In this study, four genes—mmp1084, mmp1085, mmp1086, and mmp1087—were targeted to determine their potential involvement of the biosynthesis of the sugar components in the N-glycan, based on bioinformatics analysis and proximity to a number of genes which have been previously demonstrated to be involved in the N-glycosylation pathway. The genes mmp1084 to mmp1087 were shown to be cotranscribed, and in-frame deletions of each gene as well as a Δmmp1086Δmmp1087 double mutant were successfully generated. All mutants were archaellated and motile. Mass spectrometry examination of purified archaella revealed that in Δmmp1084 mutant cells, the threonine linked to the third sugar of the glycan was missing, indicating a putative threonine transferase function of MMP1084. Similar analysis of the archaella of the Δmmp1085 mutant cells demonstrated that the glycan lacked the methyl group at the C-5 position of the terminal sugar, indicating that MMP1085 is a methyltransferase involved in the biosynthesis of this unique sugar. Deletion of the remaining two genes, mmp1086 and mmp1087, either singularly or together, had no effect on the structure of the archaellin N-glycan. Because of their demonstrated involvement in the N-glycosylation pathway, we designated mmp1084 as aglU and mmp1085 as aglV. PMID:23836872

  5. Secretion and N-linked glycosylation are required for prostatic acid phosphatase catalytic and antinociceptive activity.

    PubMed

    Hurt, Julie K; Fitzpatrick, Brendan J; Norris-Drouin, Jacqueline; Zylka, Mark J

    2012-01-01

    Secretory human prostatic acid phosphatase (hPAP) is glycosylated at three asparagine residues (N62, N188, N301) and has potent antinociceptive effects when administered to mice. Currently, it is unknown if these N-linked residues are required for hPAP protein stability and activity in vitro or in animal models of chronic pain. Here, we expressed wild-type hPAP and a series of Asn to Gln point mutations in the yeast Pichia pastoris X33 then analyzed protein levels and enzyme activity in cell lysates and in conditioned media. Pichia secreted wild-type recombinant (r)-hPAP into the media (6-7 mg protein/L). This protein was as active as native hPAP in biochemical assays and in mouse models of inflammatory pain and neuropathic pain. In contrast, the N62Q and N188Q single mutants and the N62Q, N188Q double mutant were expressed at lower levels and were less active than wild-type r-hPAP. The purified N62Q, N188Q double mutant protein was also 1.9 fold less active in vivo. The N301Q mutant was not expressed, suggesting a critical role for this residue in protein stability. To explicitly test the importance of secretion, a construct lacking the signal peptide of hPAP was expressed in Pichia and assayed. This "cellular" construct was not expressed at levels detectable by western blotting. Taken together, these data indicate that secretion and post-translational carbohydrate modifications are required for PAP protein stability and catalytic activity. Moreover, our findings indicate that recombinant hPAP can be produced in Pichia--a yeast strain that is used to generate biologics for therapeutic purposes.

  6. Identification of a novel inducible cell-surface ligand of CD5 on activated lymphocytes

    PubMed Central

    1996-01-01

    CD5 is a 67-kD glycoprotein that is expressed on most T lymphocytes and on a subset of mature B cells. Although its physiologic function is unknown, several lines of evidence suggest that CD5 may play a role in the regulation of T cell activation and in T cell-antigen presenting cell interactions. Using a CD5-immunoglobulin fusion protein (CD5Rg, for receptorglobulin) we have uncovered a new CD5 ligand (CD5L) expressed on the surface of activated splenocytes. Stimulation of murine splenocytes with anti-CD3 and anti-CD28 antibodies induce transient expression of CD5L on B lymphocytes that lasts for approximately 72 h. Binding of CD5Rg to activated splenocytes is trypsin resistant and independent of divalent cations. However, it is pronase sensitive and dependent on N-linked glycosylation of CD5, since treatment of CD5Rg with PNGaseF on N-glycanase completely abrogates its ability to bind activated splenocytes. It addition to splenocytes, CD5L is expressed on activated murine T cell clones. Immunoprecipitation, antibody, and recombinant protein blocking studies indicate that CD5L is distinct from CD72, which has been proposed to be a CD5 ligand. To determine whether CD5-CD5L interaction might play a role in vivo, we tested the effect of CD5Rg in a murine model of antibody-mediated membranous glomerulonephritis. Injection of CD5Rg was found to abrogate development of the disease. Taken together, our results help identify a novel ligand of CD5 and propose a role for CD5 in the regulation of immune responses. PMID:9064341

  7. Controlling cell-cell interactions using surface acoustic waves.

    PubMed

    Guo, Feng; Li, Peng; French, Jarrod B; Mao, Zhangming; Zhao, Hong; Li, Sixing; Nama, Nitesh; Fick, James R; Benkovic, Stephen J; Huang, Tony Jun

    2015-01-06

    The interactions between pairs of cells and within multicellular assemblies are critical to many biological processes such as intercellular communication, tissue and organ formation, immunological reactions, and cancer metastasis. The ability to precisely control the position of cells relative to one another and within larger cellular assemblies will enable the investigation and characterization of phenomena not currently accessible by conventional in vitro methods. We present a versatile surface acoustic wave technique that is capable of controlling the intercellular distance and spatial arrangement of cells with micrometer level resolution. This technique is, to our knowledge, among the first of its kind to marry high precision and high throughput into a single extremely versatile and wholly biocompatible technology. We demonstrated the capabilities of the system to precisely control intercellular distance, assemble cells with defined geometries, maintain cellular assemblies in suspension, and translate these suspended assemblies to adherent states, all in a contactless, biocompatible manner. As an example of the power of this system, this technology was used to quantitatively investigate the gap junctional intercellular communication in several homotypic and heterotypic populations by visualizing the transfer of fluorescent dye between cells.

  8. Distribution of cell surface saccharides on pancreatic cells

    PubMed Central

    Maylie-Pfenninger, M; Jamieson, JD

    1979-01-01

    We describe here a simple, general procedure for the purification of a variety of lectins, and for the preparation of lectin-ferritin conjugates of defined molar composition and binding properties to be used as probes for cell surface saccharides. The technique uses a “universal” affinity column for lectins and their conjugates, which consists of hog sulfated gastric mucin glycopeptides covalently coupled to agarose. The procedure involes: (a) purification of lectins by chromatography of aqueous extracts of seeds or other lectin-containing fluids over the affinity column, followed by desorption of the desired lectin with its hapten suge; (b) iodination of the lectin to serve as a marker during subsequent steps; (c) conjugation of lectin to ferritin with glutaraldehyde; (d) collection of active lectin-ferritin conjugates by affinity chromatography; and (e) separation of monomeric lectin-ferritin conjugates from larger aggregates and unconjugated lectin by gel chromatography. Based on radioactivity and absorbancy at 310 nm for lectin and ferritin, respectively, the conjugates consist of one to two molecules of lectin per ferrritin molecule. Binding studies of native lectins and their ferritin conjugates to dispersed pancreatic acinar cells showed that the conjugation procedure does not significantly alter either the affinity constant of the lectin for its receptor on the cell surface or the number of sites detected. PMID:422653

  9. Functional analysis of N-linking oligosaccharyl transferase enzymes encoded by deep-sea vent proteobacteria

    PubMed Central

    Mills, Dominic C.; Jervis, Adrian J.; Abouelhadid, Sherif; Yates, Laura E.; Cuccui, Jon; Linton, Dennis; Wren, Brendan W.

    2016-01-01

    Bacterial N-linking oligosaccharyl transferases (OTase enzymes) transfer lipid-linked glycans to selected proteins in the periplasm and were first described in the intestinal pathogen Campylobacter jejuni, a member of the ε-proteobacteria-subdivision of bacteria. More recently, orthologues from other ε-proteobacterial Campylobacter and Helicobacter species and a δ-proteobacterium, Desulfovibrio desulfuricans, have been described, suggesting that these two subdivisions of bacteria may be a source of further N-linked protein glycosylation systems. Whole-genome sequencing of both ε- and δ-proteobacteria from deep-sea vent habitats, a rich source of species from these subdivisions, revealed putative ORFs encoding OTase enzymes and associated adjacent glycosyltransferases similar to the C. jejuni N-linked glycosylation locus. We expressed putative OTase ORFs from the deep-sea vent species Nitratiruptor tergarcus, Sulfurovum lithotrophicum and Deferribacter desulfuricans in Escherichia coli and showed they were able to functionally complement the C. jejuni OTase, CjPglB . The enzymes were shown to possess relaxed glycan specificity, transferring diverse glycan structures and demonstrated different glycosylation sequon specificities. Additionally a permissive D. desulfuricans acceptor protein was identified, and we provide evidence that the N-linked glycan synthesised by N. tergarcus and S. lithotrophicum contains an acetylated sugar at the reducing end. This work demonstrates that deep-sea vent bacteria encode functional N-glycosylation machineries and are a potential source of biotechnologically important OTase enzymes. PMID:26610891

  10. Functional analysis of N-linking oligosaccharyl transferase enzymes encoded by deep-sea vent proteobacteria.

    PubMed

    Mills, Dominic C; Jervis, Adrian J; Abouelhadid, Sherif; Yates, Laura E; Cuccui, Jon; Linton, Dennis; Wren, Brendan W

    2016-04-01

    Bacterial N-linking oligosaccharyl transferases (OTase enzymes) transfer lipid-linked glycans to selected proteins in the periplasm and were first described in the intestinal pathogen Campylobacter jejuni, a member of the ε-proteobacteria-subdivision of bacteria. More recently, orthologues from other ε-proteobacterial Campylobacter and Helicobacter species and a δ-proteobacterium, Desulfovibrio desulfuricans, have been described, suggesting that these two subdivisions of bacteria may be a source of further N-linked protein glycosylation systems. Whole-genome sequencing of both ε- and δ-proteobacteria from deep-sea vent habitats, a rich source of species from these subdivisions, revealed putative ORFs encoding OTase enzymes and associated adjacent glycosyltransferases similar to the C. jejuni N-linked glycosylation locus. We expressed putative OTase ORFs from the deep-sea vent species Nitratiruptor tergarcus, Sulfurovum lithotrophicum and Deferribacter desulfuricans in Escherichia coli and showed that they were able to functionally complement the C. jejuni OTase, CjPglB. The enzymes were shown to possess relaxed glycan specificity, transferring diverse glycan structures and demonstrated different glycosylation sequon specificities. Additionally, a permissive D. desulfuricans acceptor protein was identified, and we provide evidence that the N-linked glycan synthesized by N. tergarcus and S. lithotrophicum contains an acetylated sugar at the reducing end. This work demonstrates that deep-sea vent bacteria encode functional N-glycosylation machineries and are a potential source of biotechnologically important OTase enzymes.

  11. Functions of red cell surface proteins.

    PubMed

    Daniels, G

    2007-11-01

    The external membrane of the red cell contains numerous proteins that either cross the lipid bilayer one or more times or are anchored to it through a lipid tail. Many of these proteins express blood group activity. The functions of some of these proteins are known; in others their function can only be surmised from the protein structure or from limited experimental evidence. They are loosely divided into four categories based on their functions: membrane transporters; adhesion molecules and receptors; enzymes; and structural proteins that link the membrane with the membrane skeleton. Some of the proteins carry out more than one of these functions. Some proteins may complete their major functions during erythropoiesis or may only be important under adverse physiological conditions. Furthermore, some might be evolutionary relics and may no longer have significant functions. Polymorphisms or rare changes in red cell surface proteins are often responsible for blood groups. The biological significance of these polymorphisms or the selective pressures responsible for their stability within populations are mostly not known, although exploitation of the proteins by pathogenic micro-organisms has probably played a major role.

  12. 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

    Phase objects can become visible by slightly defocusing an optical microscope, a technique seldom used as a useful tool. We revisited the theory of defocusing and apply it to our optical microscope with optics corrected at infinity. In our approximation, we obtain that the image contrast is proportional to the two-dimensional (2D) Laplacian of the phase difference introduced by the phase object. If the index of refraction of the phase object is uniform the image obtained from defocusing microscopy is the image of curvature (Laplacian of the local thickness) of the phase object, while standard phase-contrast microscopy gives information about the thickness of the object. We made artificial phase objects and measured image contrasts with defocusing microscopy. Measured contrasts are in excellent agreement with our theoretical model. We use defocusing microscopy to study curvature fluctuations (ruffles) on the surface of macrophages (cell of the innate immune system), and try to correlate mechanical properties of macrophage surface and phagocytosis. We observe large coherent propagating structures: Their shape, speed, density are measured and curvature energy estimated. Inhomogeneities of cytoskeleton refractive index, curvature modulations due to thermal fluctuations and/or periodic changes in cytoskeleton-membrane interactions cause random fluctuations in image contrast. From the temporal and spatial contrast correlation functions, we obtain the decay time and correlation length of such fluctuations that are related to their size and the viscoelastic properties of the cytoskeleton. In order to associate the dynamics of cytoskeleton with the process of phagocytosis, we use an optical tweezers to grab a zymosan particle and put it into contact with the macrophage. We then measure the time for a single phagocytosis event. We add the drug cytochalasin D that depolymerizes the cytoskeleton F-actin network: It inhibits the large propagating coherent fluctuations on the

  13. Cell surface interaction of annexin A2 and galectin-3 modulates epidermal growth factor receptor signaling in Her-2 negative breast cancer cells.

    PubMed

    Shetty, Praveenkumar; Bargale, Anil; Patil, Basavraj R; Mohan, Rajashekar; Dinesh, U S; Vishwanatha, Jamboor K; Gai, Pramod B; Patil, Vidya S; Amsavardani, T S

    2016-01-01

    Overexpression and activation of tyrosine kinase receptors like EGFR and Src regulate the progression and metastasis of Her-2 negative breast cancer. Recently we have reported the role of cell membrane interaction of phospholipid-binding protein annexin A2 (AnxA2) and EGFR in regulating cellular signaling in the activation of angiogenesis, matrix degradation, invasion, and cancer metastasis. Beta-galactoside-specific animal lectin galectin-3 is an apoptosis inhibitor, and cell surface-associated extracellular galectin-3 also has a role in cell migration, cancer progression, and metastasis. Similar expression pattern and membrane co-localization of these two proteins made us to hypothesize in the current study that galectin-3 and AnxA2 interaction is critical for Her-2 negative breast cancer progression. By various experimental analyses, we confirm that glycosylated AnxA2 at the membrane surface interacts with galectin-3. N-linked glycosylation inhibitor tunicamycin treatment convincingly blocked AnxA2 membrane translocation and its association with galectin-3. To analyze whether this interaction has any functional relevance, we tried to dissociate this interaction with purified plant lectin from chickpea (Cicer arietinum agglutinin). This highly specific 30 kDa plant lectin could dissociate AnxA2 from endogenous lectin galectin-3 interaction at the cell surface. This dissociation could down-regulate Bcl-2 family proteins, cell proliferation, and migration simultaneously triggering cell apoptosis. Targeting this interaction of membrane surface glycoprotein and its animal lectin in Her-2 negative breast cancer may be of therapeutic value.

  14. Dictyostelium cells migrate similarly on surfaces of varying chemical composition.

    PubMed

    McCann, Colin P; Rericha, Erin C; Wang, Chenlu; Losert, Wolfgang; Parent, Carole A

    2014-01-01

    During cell migration, cell-substrate binding is required for pseudopod anchoring to move the cell forward, yet the interactions with the substrate must be sufficiently weak to allow parts of the cell to de-adhere in a controlled manner during typical protrusion/retraction cycles. Mammalian cells actively control cell-substrate binding and respond to extracellular conditions with localized integrin-containing focal adhesions mediating mechanotransduction. We asked whether mechanotransduction also occurs during non-integrin mediated migration by examining the motion of the social amoeba Dictyostelium discoideum, which is thought to bind non-specifically to surfaces. We discovered that Dictyostelium cells are able to regulate forces generated by the actomyosin cortex to maintain optimal cell-surface contact area and adhesion on surfaces of various chemical composition and that individual cells migrate with similar speed and contact area on the different surfaces. In contrast, during collective migration, as observed in wound healing and metastasis, the balance between surface forces and protrusive forces is altered. We found that Dictyostelium collective migration dynamics are strongly affected when cells are plated on different surfaces. These results suggest that the presence of cell-cell contacts, which appear as Dictyostelium cells enter development, alter the mechanism cells use to migrate on surfaces of varying composition.

  15. Toward Cell Selective Surfaces: Cell Adhesion and Proliferation on Breath Figures with Antifouling Surface Chemistry.

    PubMed

    Martínez-Campos, Enrique; Elzein, Tamara; Bejjani, Alice; García-Granda, Maria Jesús; Santos-Coquillat, Ana; Ramos, Viviana; Muñoz-Bonilla, Alexandra; Rodríguez-Hernández, Juan

    2016-03-01

    We report the preparation of microporous functional polymer surfaces that have been proven to be selective surfaces toward eukaryotic cells while maintaining antifouling properties against bacteria. The fabrication of functional porous films has been carried out by the breath figures approach that allowed us to create porous interfaces with either poly(ethylene glycol) methyl ether methacrylate (PEGMA) or 2,3,4,5,6-pentafluorostyrene (5FS). For this purpose, blends of block copolymers in a polystyrene homopolymer matrix have been employed. In contrast to the case of single functional polymer, using blends enables us to vary the chemical distribution of the functional groups inside and outside the formed pores. In particular, fluorinated groups were positioned at the edges while the hydrophilic PEGMA groups were selectively located inside the pores, as demonstrated by TOF-SIMS. More interestingly, studies of cell adhesion, growth, and proliferation on these surfaces confirmed that PEGMA functionalized interfaces are excellent candidates to selectively allow cell growth and proliferation while maintaining antifouling properties.

  16. Cell Surface Markers in Colorectal Cancer Prognosis

    PubMed Central

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

    2011-01-01

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

  17. Calreticulin: Roles in Cell-Surface Protein Expression

    PubMed Central

    Jiang, Yue; Dey, Sandeepa; Matsunami, Hiroaki

    2014-01-01

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

  18. 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

  19. Theory of back-surface-field solar cells

    NASA Technical Reports Server (NTRS)

    Vonroos, O.

    1979-01-01

    Report describes simple concise theory of back-surface-field (BSF) solar cells (npp + junctions) based on Shockley's depletion-layer approximation and cites superiority of two-junction devices over conventional unijunction cells.

  20. Targeting Prostate Cancer Stemlike Cells Through Cell Surface-Expressed GRP78

    DTIC Science & Technology

    2015-10-01

    the cell surface GRP78-expressing subpopulation of cells supports nuclear Akt/GSK-3/ Snail -1 signaling. These findings are important because they are...original tasks outlined in the approved statement of work. 15. SUBJECT TERMS prostate cancer, cell surface GRP78, cancer stem cell, Snail -1 16. SECURITY...associated with cell surface GRP78 (Akt/GSK-3/ Snail -1) were upregulated in GRP78(+) relative to GRP78(-) prostate cancer cells. Our results in this

  1. Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis

    SciTech Connect

    Mahal, L.K.; Yareme, K.J.; Bertozzi, C.R.

    1997-05-16

    Cell surface oligosaccharide can be engineered to display unusual functional groups for the selective chemical remodeling of cell surfaces. An unnatural derivative of N-acetyl-mannosamine, which has a ketone group, was converted to the corresponding sialic acid and incorporated into cell surface oligosaccharide metabolically, resulting in the cell surface display of ketone groups. The ketone group on the cell surface can then be covalently ligated under physiological conditions with molecules carrying a complementary reactive functional group such as the hydrazide. Cell surface reactions of this kind should prove useful in the introduction of new recognition epitopes, such as peptides, oligosaccharide, or small organic molecules, onto cell surfaces and in the subsequent modulation of cell-cell or cell-small molecule binding events. The versatility of this technology was demonstrated by an example of selective drug delivery. Cells were decorated with biotin through selective conjugation to ketone groups, and selectively killed in the presence of a ricin A chain-avidin conjugate. 30 refs., 4 figs.

  2. Glycomic and Proteomic Profiling of Pancreatic Cyst Fluids Identifies Hyperfucosylated Lactosamines on the N-linked Glycans of Overexpressed Glycoproteins*

    PubMed Central

    Mann, Benjamin F.; Goetz, John A.; House, Michael G.; Schmidt, C. Max; Novotny, Milos V.

    2012-01-01

    Pancreatic cancer is now the fourth leading cause of cancer deaths in the United States, and it is associated with an alarmingly low 5-year survival rate of 5%. However, a patient's prognosis is considerably improved when the malignant lesions are identified at an early stage of the disease and removed by surgical resection. Unfortunately, the absence of a practical screening strategy and clinical diagnostic test for identifying premalignant lesions within the pancreas often prevents early detection of pancreatic cancer. To aid in the development of a molecular screening system for early detection of the disease, we have performed glycomic and glycoproteomic profiling experiments on 21 pancreatic cyst fluid samples, including fluids from mucinous cystic neoplasms and intraductal papillary mucinous neoplasms, two types of mucinous cysts that are considered high risk to undergo malignant transformation. A total of 80 asparagine-linked (N-linked) glycans, including high mannose and complex structures, were identified. Of special interest was a series of complex N-linked glycans containing two to six fucose residues, located predominantly as substituents on β-lactosamine extensions. Following the observation of these “hyperfucosylated” glycans, bottom-up proteomics experiments utilizing a label-free quantitative approach were applied to the investigation of two sets of tryptically digested proteins derived from the cyst fluids: 1) all soluble proteins in the raw samples and 2) a subproteome of the soluble cyst fluid proteins that were selectively enriched for fucosylation through the use of surface-immobilized Aleuria aurantia lectin. A comparative analysis of these two proteomic data sets identified glycoproteins that were significantly enriched by lectin affinity. Several candidate glycoproteins that appear hyperfucosylated were identified, including triacylglycerol lipase and pancreatic α-amylase, which were 20- and 22-fold more abundant, respectively

  3. Rapid removal of N-linked oligosaccharides using microwave assisted enzyme catalyzed deglycosylation

    NASA Astrophysics Data System (ADS)

    Sandoval, Wendy N.; Arellano, Fred; Arnott, David; Raab, Helga; Vandlen, Richard; Lill, Jennie R.

    2007-01-01

    The removal of N-linked oligosaccharides from glycoproteins is commonly performed during the preparation of samples for mass spectrometry. A reduction in the protein's structural heterogeneity is sometimes essential to obtain a mass for the intact protein. Alternatively, removal of the sugar may be desired to facilitate oligosaccharide analysis. A typical approach to deglycosylation employs overnight digestion with the enzyme peptide N-glycosidase F (PNGase F). We report a method for the accelerated removal of N-linked oligosaccharides using PNGase F assisted by microwave irradiation. Complete deglycosylation was achieved in less than 30 min for most proteins without compromising the integrity of protein samples. This method was tested on a variety of glycoproteins, including antibodies, at the microgram level.

  4. Single cell profiling of surface carbohydrates on Bacillus cereus

    PubMed Central

    Wang, Congzhou; Ehrhardt, Christopher J.; Yadavalli, Vamsi K.

    2015-01-01

    Cell surface carbohydrates are important to various bacterial activities and functions. It is well known that different types of Bacillus display heterogeneity of surface carbohydrate compositions, but detection of their presence, quantitation and estimation of variation at the single cell level have not been previously solved. Here, using atomic force microscopy (AFM)-based recognition force mapping coupled with lectin probes, the specific carbohydrate distributions of N-acetylglucosamine and mannose/glucose were detected, mapped and quantified on single B. cereus surfaces at the nanoscale across the entire cell. Further, the changes of the surface carbohydrate compositions from the vegetative cell to spore were shown. These results demonstrate AFM-based ‘recognition force mapping’ as a versatile platform to quantitatively detect and spatially map key bacterial surface biomarkers (such as carbohydrate compositions), and monitor in situ changes in surface biochemical properties during intracellular activities at the single cell level. PMID:25505137

  5. Targeting Negative Surface Charges of Cancer Cells by Multifunctional Nanoprobes

    PubMed Central

    Chen, Bingdi; Le, Wenjun; Wang, Yilong; Li, Zhuoquan; Wang, Dong; Ren, Lei; Lin, Ling; Cui, Shaobin; Hu, Jennifer J.; Hu, Yihui; Yang, Pengyuan; Ewing, Rodney C.; Shi, Donglu; Cui, Zheng

    2016-01-01

    A set of electrostatically charged, fluorescent, and superparamagnetic nanoprobes was developed for targeting cancer cells without using any molecular biomarkers. The surface electrostatic properties of the established cancer cell lines and primary normal cells were characterized by using these nanoprobes with various electrostatic signs and amplitudes. All twenty two randomly selected cancer cell lines of different organs, but not normal control cells, bound specifically to the positively charged nanoprobes. The relative surface charges of cancer cells could be quantified by the percentage of cells captured magnetically. The activities of glucose metabolism had a profound impact on the surface charge level of cancer cells. The data indicate that an elevated glycolysis in the cancer cells led to a higher level secretion of lactate. The secreted lactate anions are known to remove the positive ions, leaving behind the negative changes on the cell surfaces. This unique metabolic behavior is responsible for generating negative cancer surface charges in a perpetuating fashion. The metabolically active cancer cells are shown to a unique surface electrostatic pattern that can be used for recovering cancer cells from the circulating blood and other solutions. PMID:27570558

  6. Cell Surface Measurements in Hydrocarbon and Carbohydrate Fermentations

    PubMed Central

    Neufeld, R. J.; Zajic, J. E.; Gerson, D. F.

    1980-01-01

    Acinetobacter calcoaceticus was grown in 11-liter batch fermentations with hexadecane or sodium citrate as the sole source of carbon. Surface and interfacial tension measurements of the microbial broth indicated that surface-active compounds were being produced only during growth on the hydrocarbon substrate. Contact angle measurements of an aqueous drop on a smooth lawn of cells in a hexadecane bath indicated a highly hydrophobic surface of the cells in the initial stages of the hydrocarbon fermentation (120° contact angle). At this stage, the entire cell population was bound to the hydrocarbon-aqueous interface. The contact angle dropped rapidly to approximately 45° after 14 h into the fermentation. This coincided with a shift of the cell population to the aqueous phase. Thus, the cells demonstrated more hydrophilic characteristics in the later stages of the fermentation. Contact angles on cells grown on sodium citrate ranged from 18 to 24° throughout the fermentation. The cells appear to be highly hydrophilic during growth on a soluble substrate. From the contact angle and aqueous-hydrocarbon interfacial tension, the surface free energy of the cells was calculated along with the cell-aqueous and cell-hydrocarbon interfacial tension. The results of these measurements were useful in quantitatively evaluating the hydrophobic nature of the cell surface during growth on hydrocarbons and comparing it with the hydrophilic nature of the cell surface during growth on a soluble substrate. PMID:16345526

  7. Targeting Negative Surface Charges of Cancer Cells by Multifunctional Nanoprobes.

    PubMed

    Chen, Bingdi; Le, Wenjun; Wang, Yilong; Li, Zhuoquan; Wang, Dong; Ren, Lei; Lin, Ling; Cui, Shaobin; Hu, Jennifer J; Hu, Yihui; Yang, Pengyuan; Ewing, Rodney C; Shi, Donglu; Cui, Zheng

    2016-01-01

    A set of electrostatically charged, fluorescent, and superparamagnetic nanoprobes was developed for targeting cancer cells without using any molecular biomarkers. The surface electrostatic properties of the established cancer cell lines and primary normal cells were characterized by using these nanoprobes with various electrostatic signs and amplitudes. All twenty two randomly selected cancer cell lines of different organs, but not normal control cells, bound specifically to the positively charged nanoprobes. The relative surface charges of cancer cells could be quantified by the percentage of cells captured magnetically. The activities of glucose metabolism had a profound impact on the surface charge level of cancer cells. The data indicate that an elevated glycolysis in the cancer cells led to a higher level secretion of lactate. The secreted lactate anions are known to remove the positive ions, leaving behind the negative changes on the cell surfaces. This unique metabolic behavior is responsible for generating negative cancer surface charges in a perpetuating fashion. The metabolically active cancer cells are shown to a unique surface electrostatic pattern that can be used for recovering cancer cells from the circulating blood and other solutions.

  8. Investigation of ovarian cancer associated sialylation changes in N-linked glycopeptides by quantitative proteomics

    PubMed Central

    2012-01-01

    Background In approximately 80% of patients, ovarian cancer is diagnosed when the patient is already in the advanced stages of the disease. CA125 is currently used as the marker for ovarian cancer; however, it lacks specificity and sensitivity for detecting early stage disease. There is a critical unmet need for sensitive and specific routine screening tests for early diagnosis that can reduce ovarian cancer lethality by reliably detecting the disease at its earliest and treatable stages. Results In this study, we investigated the N-linked sialylated glycopeptides in serum samples from healthy and ovarian cancer patients using Lectin-directed Tandem Labeling (LTL) and iTRAQ quantitative proteomics methods. We identified 45 N-linked sialylated glycopeptides containing 46 glycosylation sites. Among those, ten sialylated glycopeptides were significantly up-regulated in ovarian cancer patients’ serum samples. LC-MS/MS analysis of the non-glycosylated peptides from the same samples, western blot data using lectin enriched glycoproteins of various ovarian cancer type samples, and PNGase F (+/−) treatment confirmed the sialylation changes in the ovarian cancer samples. Conclusion Herein, we demonstrated that several proteins are aberrantly sialylated in N-linked glycopeptides in ovarian cancer and detection of glycopeptides with abnormal sialylation changes may have the potential to serve as biomarkers for ovarian cancer. PMID:22856521

  9. A categorification of the quantum sl(N)-link polynomials using foams

    NASA Astrophysics Data System (ADS)

    Vaz, Pedro

    2008-07-01

    In this thesis we define and study a categorification of the sl(N)-link polynomial using foams, for Ngeq 3. For N=3 we define the universal sl(3)-link homology, using foams, which depends on three parameters and show that it is functorial, up to scalars, with respect to link cobordisms. Our theory is integral. We show that tensoring it with Q yields a theory which is equivalent to the rational universal Khovanov-Rozansky sl(3)-link homology. For Ngeq 4 we construct a rational theory categorifying the sl(N)-link polynomial using foams. Our theory is functorial, up to scalars, with respect to link cobordisms. To evaluate closed foams we use the Kapustin-Li formula. We show that for any link our homology is isomorphic to the Khovanov-Rozansky homology. We conjecture that the theory is integral and we compute the conjectured integral sl(N)-link homology for the (2,m)-torus links and show that it has torsion of order N.

  10. Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis

    PubMed Central

    Larkin, Angelyn; Chang, Michelle M.; Whitworth, Garrett E.; Imperiali, Barbara

    2013-01-01

    Asparagine-linked glycosylation is a complex protein modification conserved among all three domains of life. Herein we report the in vitro analysis of N-linked glycosylation from the methanogenic archaeon Methanococcus voltae. Using a suite of synthetic and semisynthetic substrates, we show that AglK initiates N-linked glycosylation in M. voltae through the formation of α-linked dolichyl monophosphate N-acetylglucosamine (Dol-P-GlcNAc), which contrasts with the polyprenyl-diphosphate intermediates that feature in both eukaryotes and bacteria. Intriguingly, AglK exhibits high sequence homology to dolichyl-phosphate β-glucosyltransferases, including Alg5 in eukaryotes, suggesting a common evolutionary origin. The combined action of the first two enzymes, AglK and AglC, afforded an α-linked Dol-P-glycan that serves as a competent substrate for the archaeal oligosaccharyl transferase AglB. These studies provide the first biochemical evidence revealing that despite the apparent similarity of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life. PMID:23624439

  11. AglJ adds the first sugar of the N-linked pentasaccharide decorating the Haloferax volcanii S-layer glycoprotein.

    PubMed

    Kaminski, Lina; Abu-Qarn, Mehtap; Guan, Ziqiang; Naparstek, Shai; Ventura, Valeria V; Raetz, Christian R H; Hitchen, Paul G; Dell, Anne; Eichler, Jerry

    2010-11-01

    Like the Eukarya and Bacteria, the Archaea also perform N glycosylation. Using the haloarchaeon Haloferax volcanii as a model system, a series of Agl proteins involved in the archaeal version of this posttranslational modification has been identified. In the present study, the participation of HVO_1517 in N glycosylation was considered, given its homology to a known component of the eukaryal N-glycosylation pathway and because of the genomic proximity of HVO_1517 to agl genes encoding known elements of the H. volcanii N-glycosylation process. By combining the deletion of HVO_1517 with mass spectrometric analysis of both dolichol phosphate monosaccharide-charged carriers and the S-layer glycoprotein, evidence was obtained showing the participation of HVO_1517, renamed AglJ, in adding the first hexose of the N-linked pentasaccharide decorating this reporter glycoprotein. The deletion of aglJ, however, did not fully prevent the attachment of a hexose residue to the S-layer glycoprotein. Moreover, in the absence of AglJ, the level of only one of the three monosaccharide-charged dolichol phosphate carriers detected in the cell was reduced. Nonetheless, in cells lacking AglJ, no further sugar subunits were added to the remaining monosaccharide-charged dolichol phosphate carriers or to the monosaccharide-modified S-layer glycoprotein, pointing to the importance of the sugar added through the actions of AglJ for proper N glycosylation. Finally, while aglJ can be deleted, H. volcanii surface layer integrity is compromised in the absence of the encoded protein.

  12. Cell surface engineering of yeast for applications in white biotechnology.

    PubMed

    Kuroda, Kouichi; Ueda, Mitsuyoshi

    2011-01-01

    Cell surface engineering is a promising strategy for the molecular breeding of whole-cell biocatalysts. By using this strategy, yeasts can be constructed by the cell surface display of functional proteins; these yeasts are referred to as arming yeasts. Because reactions using arming yeasts as whole-cell biocatalysts occur on the cell surface, materials that cannot enter the cell can be used as reaction substrates. Numerous arming yeasts have therefore been constructed for a wide range of uses such as biofuel production, synthesis of valuable chemicals, adsorption or degradation of environmental pollutants, recovery of rare metal ions, and biosensors. Here, we review the science of yeast cell surface modification as well as current applications and future opportunities.

  13. Molecularly engineered surfaces for cell biology: from static to dynamic surfaces.

    PubMed

    Gooding, J Justin; Parker, Stephen G; Lu, Yong; Gaus, Katharina

    2014-04-01

    Surfaces with a well-defined presentation of ligands for receptors on the cell membrane can serve as models of the extracellular matrix for studying cell adhesion or as model cell surfaces for exploring cell-cell contacts. Because such surfaces can provide exquisite control over, for example, the density of these ligands or when the ligands are presented to the cell, they provide a very precise strategy for understanding the mechanisms by which cells respond to external adhesive cues. In the present feature article, we present an overview of the basic biology of cell adhesion before discussing surfaces that have a static presentation of immobile ligands. We outline the biological information that such surfaces have given us, before progressing to recently developed switchable surfaces and surfaces that mimic the lipid bilayer, having adhesive ligands that can move around the membrane and be remodeled by the cell. Finally, the feature article closes with some of the biological information that these new types of surfaces could provide.

  14. N-linked glycan truncation causes enhanced clearance of plasma-derived von Willebrand factor.

    PubMed

    O'Sullivan, J M; Aguila, S; McRae, E; Ward, S E; Rawley, O; Fallon, P G; Brophy, T M; Preston, R J S; Brady, L; Sheils, O; Chion, A; O'Donnell, J S

    2016-12-01

    Essentials von Willebrands factor (VWF) glycosylation plays a key role in modulating in vivo clearance. VWF glycoforms were used to examine the role of specific glycan moieties in regulating clearance. Reduction in sialylation resulted in enhanced VWF clearance through asialoglycoprotein receptor. Progressive VWF N-linked glycan trimming resulted in increased macrophage-mediated clearance. Click to hear Dr Denis discuss clearance of von Willebrand factor in a free presentation from the ISTH Academy SUMMARY: Background Enhanced von Willebrand factor (VWF) clearance is important in the etiology of both type 1 and type 2 von Willebrand disease (VWD). In addition, previous studies have demonstrated that VWF glycans play a key role in regulating in vivo clearance. However, the molecular mechanisms underlying VWF clearance remain poorly understood. Objective To define the molecular mechanisms through which VWF N-linked glycan structures influence in vivo clearance. Methods By use of a series of exoglycosidases, different plasma-derived VWF (pd-VWF) glycoforms were generated. In vivo clearance of these glycoforms was then assessed in VWF(-/-) mice in the presence or absence of inhibitors of asialoglycoprotein receptor (ASGPR), or following clodronate-induced macrophage depletion. Results Reduced amounts of N-linked and O-linked sialylation resulted in enhanced pd-VWF clearance modulated via ASGPR. In addition to this role of terminal sialylation, we further observed that progressive N-linked glycan trimming also resulted in markedly enhanced VWF clearance. Furthermore, these additional N-linked glycan effects on clearance were ASGPR-independent, and instead involved enhanced macrophage clearance that was mediated, at least in part, through LDL receptor-related protein 1. Conclusion The carbohydrate determinants expressed on VWF regulate susceptibility to proteolysis by ADAMTS-13. In addition, our findings now further demonstrate that non-sialic acid carbohydrate

  15. Characteristics of oligosaccharides from rat parotid (RP) N-linked glycoproteins (GP) after. beta. -adrenoreceptor (. beta. -AR) stimulation

    SciTech Connect

    Baum, B.J.; Yeh, C.K.; Kousvelari, E.E.

    1987-05-01

    The authors have shown that ..beta..-AR stimulation of RP cells leads to marked enhancement of N-linked glycosylation in 4 secretory GP (Mrapprox.220Kd, HMW;approx.32-38Kd, MMW;approx.17Kd, LMW). To characterize oligosaccharides in GP, cells were incubated 60 min +/- isoproterenol (ISO) and analyzed 2 ways. First, cell extracts were subjected to SDS-PAGE and Western blotting with peroxidase-conjugated Con A or wheat germ agglutinin (WGA). Second, double-labeled (/sup 3/H) man//sup 14/C leu) extracts were chromatographed on G200 followed by analysis of GP on Con A-Sepharose. HMW from control (CON) and ISO cells were Endo H insensitive, Endo F sensitive, altered by incubation with deoxynojirimycin (dNM), and bound both Con A and WGA conjugates. Similar findings were observed with LMW while MMW were sensitive to Endo H and Endo F, unaffected by dNM, bound Con A (strongly) and WGA (weakly). MMW and LMW were primarily eluted from Con A-Sepharose with 0.5M ..cap alpha..-methyl mannoside (..cap alpha..-MM) while HMW were eluted sequentially with 10 mM ..cap alpha..-methyl glucoside and ..cap alpha..-MM. HMW, MMW, and LMW had approx.4 fold higher /sup 3/H//sup 14/C ratios after ISO. These results suggest HMW and LMW likely contain biantennary complex and hybrid oligosaccharides while MMW contain only high mannose oligosaccharide types.

  16. Mapping cell surface adhesion by rotation tracking and adhesion footprinting

    NASA Astrophysics Data System (ADS)

    Li, Isaac T. S.; Ha, Taekjip; Chemla, Yann R.

    2017-03-01

    Rolling adhesion, in which cells passively roll along surfaces under shear flow, is a critical process involved in inflammatory responses and cancer metastasis. Surface adhesion properties regulated by adhesion receptors and membrane tethers are critical in understanding cell rolling behavior. Locally, adhesion molecules are distributed at the tips of membrane tethers. However, how functional adhesion properties are globally distributed on the individual cell’s surface is unknown. Here, we developed a label-free technique to determine the spatial distribution of adhesive properties on rolling cell surfaces. Using dark-field imaging and particle tracking, we extract the rotational motion of individual rolling cells. The rotational information allows us to construct an adhesion map along the contact circumference of a single cell. To complement this approach, we also developed a fluorescent adhesion footprint assay to record the molecular adhesion events from cell rolling. Applying the combination of the two methods on human promyelocytic leukemia cells, our results surprisingly reveal that adhesion is non-uniformly distributed in patches on the cell surfaces. Our label-free adhesion mapping methods are applicable to the variety of cell types that undergo rolling adhesion and provide a quantitative picture of cell surface adhesion at the functional and molecular level.

  17. Mapping cell surface adhesion by rotation tracking and adhesion footprinting

    PubMed Central

    Li, Isaac T. S.; Ha, Taekjip; Chemla, Yann R.

    2017-01-01

    Rolling adhesion, in which cells passively roll along surfaces under shear flow, is a critical process involved in inflammatory responses and cancer metastasis. Surface adhesion properties regulated by adhesion receptors and membrane tethers are critical in understanding cell rolling behavior. Locally, adhesion molecules are distributed at the tips of membrane tethers. However, how functional adhesion properties are globally distributed on the individual cell’s surface is unknown. Here, we developed a label-free technique to determine the spatial distribution of adhesive properties on rolling cell surfaces. Using dark-field imaging and particle tracking, we extract the rotational motion of individual rolling cells. The rotational information allows us to construct an adhesion map along the contact circumference of a single cell. To complement this approach, we also developed a fluorescent adhesion footprint assay to record the molecular adhesion events from cell rolling. Applying the combination of the two methods on human promyelocytic leukemia cells, our results surprisingly reveal that adhesion is non-uniformly distributed in patches on the cell surfaces. Our label-free adhesion mapping methods are applicable to the variety of cell types that undergo rolling adhesion and provide a quantitative picture of cell surface adhesion at the functional and molecular level. PMID:28290531

  18. 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.

  19. Growth of connective tissue progenitor cells on microtextured polydimethylsiloxane surfaces.

    PubMed

    Mata, Alvaro; Boehm, Cynthia; Fleischman, Aaron J; Muschler, George; Roy, Shuvo

    2002-12-15

    Growth of human connective tissue progenitor cells (CTPs) was characterized on smooth and microtextured polydimethylsiloxane (PDMS) surfaces. Human bone-marrow-derived cells were cultured for 9 days under conditions promoting osteoblastic differentiation on smooth PDMS surfaces and on PDMS post microtextures that were 6 microm high and 5, 10, 20, and 40 microm in diameter, respectively. Glass tissue-culture dishes were used as controls. The number of viable cells was determined, and an alkaline phosphatase stain was used as a marker for osteoblastic phenotype. CTPs attached, proliferated, and differentiated on all surfaces. Cells on the smooth PDMS and control surfaces spread and proliferated as colonies in proximity to other cells and migrated in random directions, with cell process lengths of up to 80 microm. In contrast, cells on the PDMS post microtextures grew as sparsely distributed networks of cells, with processes, occasionally up to 300 microm, that appeared to interact with the posts. Cell counts revealed that there were fewer (50%) CTPs on the smooth PDMS surface than were on the glass control surfaces. However, there were consistently more (>144%) CTPs on the PDMS post textures than on the controls. In particular, the 10-microm-in-diameter posts (268%) exhibited a significantly (p < 0.05) greater cell number than did the smooth PDMS.

  20. Surface plasmon-based infrared spectroscopy for cell biosensing

    NASA Astrophysics Data System (ADS)

    Yashunsky, Victor; Lirtsman, Vladislav; Zilbershtein, Alexander; Bein, Amir; Schwartz, Betty; Aroeti, Benjamin; Golosovsky, Michael; Davidov, Dan

    2012-08-01

    Cell morphology is often used as a valuable indicator of the physical condition and general status of living cells. We demonstrate a noninvasive method for morphological characterization of adherent cells. We measure infrared reflectivity spectrum at oblique angle from living cells cultured on thin Au film, and utilize the unique properties of the confined infrared waves (i.e., surface plasmon and guided modes) traveling inside the cell layer. The propagation of these waves strongly depends on cell morphology and connectivity. By tracking the resonant wavelength and attenuation of the surface plasmon and guided modes we measure the kinetics of various cellular processes such as (i) cell attachment and spreading on different substrata, (ii) modulation of the outer cell membrane with chlorpromazine, and (iii) formation of intercellular junctions associated with progressive cell polarization. Our method enables monitoring of submicron variations in cell layer morphology in real-time, and in the label-free manner.

  1. Attachment of human primary osteoblast cells to modified polyethylene surfaces.

    PubMed

    Poulsson, Alexandra H C; Mitchell, Stephen A; Davidson, Marcus R; Johnstone, Alan J; Emmison, Neil; Bradley, Robert H

    2009-04-09

    Ultra-high-molecular-weight polyethylene (UHMWPE) has a long history of use in medical devices, primarily for articulating surfaces due to its inherent low surface energy which limits tissue integration. To widen the applications of UHMWPE, the surface energy can be increased. The increase in surface energy would improve the adsorption of proteins and attachment of cells to allow tissue integration, thereby allowing UHMWPE to potentially be used for a wider range of implants. The attachment and function of human primary osteoblast-like (HOB) cells to surfaces of UHMWPE with various levels of incorporated surface oxygen have been investigated. The surface modification of the UHMWPE was produced by exposure to a UV/ozone treatment. The resulting surface chemistry was studied using X-ray photoelectron spectroscopy (XPS), and the topography and surface structure were probed by atomic force microscopy (AFM) and scanning electron microscopy (SEM), which showed an increase in surface oxygen from 11 to 26 atom % with no significant change to the surface topography. The absolute root mean square roughness of both untreated and UV/ozone-treated surfaces was within 350-450 nm, and the water contact angles decreased with increasing oxygen incorporation, i.e., showing an increase in surface hydrophilicity. Cell attachment and functionality were assessed over a 21 day period for each cell-surface combination studied; these were performed using SEM and the alamarBlue assay to study cell attachment and proliferation and energy-dispersive X-ray (EDX) analysis to confirm extracellular mineral deposits, and total protein assay to examine the intra- and extracellular protein expressed by the cells. HOB cells cultured for 21 days on the modified UHMWPE surfaces with 19 and 26 atom % oxygen incorporated showed significantly higher cell densities compared to cells cultured on tissue culture polystyrene (TCPS) from day 3 onward. This indicated that the cells attached and proliferated more

  2. The Assessment of Fuel Cell Power Plants for Surface Combatants.

    DTIC Science & Technology

    1994-09-30

    fuel cell technology on the design and effectiveness of future naval surface combatants. The study involved the collection of data to characterize four different fuel cell technologies; proton exchange membrane, molten carbonate, phosphoric acid, and solid oxide fuel cells. This information was used to expand current computer models to develop specific fuel cell plants that met the power requirements for several applications on a nominal 5000 Lton destroyer and a nominal 200 Lton corvette. Each of the fuel cell

  3. Biomaterial surface proteomic signature determines interaction with epithelial cells.

    PubMed

    Abdallah, Mohamed-Nur; Tran, Simon D; Abughanam, Ghada; Laurenti, Marco; Zuanazzi, David; Mezour, Mohamed A; Xiao, Yizhi; Cerruti, Marta; Siqueira, Walter L; Tamimi, Faleh

    2017-03-01

    Cells interact with biomaterials indirectly through extracellular matrix (ECM) proteins adsorbed onto their surface. Accordingly, it could be hypothesized that the surface proteomic signature of a biomaterial might determine its interaction with cells. Here, we present a surface proteomic approach to test this hypothesis in the specific case of biomaterial-epithelial cell interactions. In particular, we determined the surface proteomic signature of different biomaterials exposed to the ECM of epithelial cells (basal lamina). We revealed that the biomaterial surface chemistry determines the surface proteomic profile, and subsequently the interaction with epithelial cells. In addition, we found that biomaterials with surface chemistries closer to that of percutaneous tissues, such as aminated PMMA and aminated PDLLA, promoted higher selective adsorption of key basal lamina proteins (laminins, nidogen-1) and subsequently improved their interactions with epithelial cells. These findings suggest that mimicking the surface chemistry of natural percutaneous tissues can improve biomaterial-epithelial integration, and thus provide a rationale for the design of improved biomaterial surfaces for skin regeneration and percutaneous medical devices.

  4. Fuel cell technology for lunar surface operations

    NASA Technical Reports Server (NTRS)

    Deronck, Henry J.

    1992-01-01

    Hydrogen-oxygen fuel cells have been shown, in several NASA and contractor studies, to be an enabling technology for providing electrical power for lunar bases, outposts, and vehicles. The fuel cell, in conjunction with similar electrolysis cells, comprises a closed regenerative energy storage system, commonly referred to as a regenerative fuel cell (RFC). For stationary applications, energy densities of 1,000 watt-hours per kilograms an order of magnitude over the best rechargeable batteries, have been projected. In this RFC, the coupled fuel cell and electrolyzer act as an ultra-light battery. Electrical energy from solar arrays 'charges' the system by electrolyzing water into hydrogen and oxygen. When an electrical load is applied, the fuel cell reacts the hydrogen and oxygen to 'discharge' usable power. Several concepts for utilizing RFC's, with varying degrees of integration, have been proposed, including both primary and backup roles. For mobile power needs, such as rovers, an effective configuration may be to have only the fuel cell located on the vehicle, and to use a central electrolysis 'gas station'. Two fuel cell technologies are prime candidates for lunar power system concepts: alkaline electrolyte and proton exchange membrane. Alkaline fuel cells have been developed to a mature production power unit in NASA's Space Shuttle Orbiter. Recent advances in materials offer to significantly improve durability to the level needed for extended lunar operations. Proton exchange membrane fuel cells are receiving considerable support for hydrospace and terrestrial transportation applications. This technology promises durability, simplicity, and flexibility.

  5. Fuel cell technology for lunar surface operations

    NASA Astrophysics Data System (ADS)

    Deronck, Henry J.

    1992-02-01

    Hydrogen-oxygen fuel cells have been shown, in several NASA and contractor studies, to be an enabling technology for providing electrical power for lunar bases, outposts, and vehicles. The fuel cell, in conjunction with similar electrolysis cells, comprises a closed regenerative energy storage system, commonly referred to as a regenerative fuel cell (RFC). For stationary applications, energy densities of 1,000 watt-hours per kilograms an order of magnitude over the best rechargeable batteries, have been projected. In this RFC, the coupled fuel cell and electrolyzer act as an ultra-light battery. Electrical energy from solar arrays 'charges' the system by electrolyzing water into hydrogen and oxygen. When an electrical load is applied, the fuel cell reacts the hydrogen and oxygen to 'discharge' usable power. Several concepts for utilizing RFC's, with varying degrees of integration, have been proposed, including both primary and backup roles. For mobile power needs, such as rovers, an effective configuration may be to have only the fuel cell located on the vehicle, and to use a central electrolysis 'gas station'. Two fuel cell technologies are prime candidates for lunar power system concepts: alkaline electrolyte and proton exchange membrane. Alkaline fuel cells have been developed to a mature production power unit in NASA's Space Shuttle Orbiter. Recent advances in materials offer to significantly improve durability to the level needed for extended lunar operations. Proton exchange membrane fuel cells are receiving considerable support for hydrospace and terrestrial transportation applications. This technology promises durability, simplicity, and flexibility.

  6. Titanium surface topography affects collagen biosynthesis of adherent cells.

    PubMed

    Mendonça, Daniela B S; Miguez, Patrícia A; Mendonça, Gustavo; Yamauchi, Mitsuo; Aragão, Francisco J L; Cooper, Lyndon F

    2011-09-01

    Collagen-dependent microstructure and physicochemical properties of newly formed bone around implant surfaces represent key determinants of implant biomechanics. This study investigated the effects of implant surface topography on collagen biosynthesis of adherent human mesenchymal stem cells (hMSCs). hMSCs were grown for 0 to 42 days on titanium disks (20.0 × 1.0 mm) with smooth or rough surfaces. Cell attachment and spreading were evaluated by incubating cells with Texas-Red-conjugated phalloidin antibody. Quantitative real-time PCR was used to measure the mRNA levels of Col1α1 and collagen modifying genes including prolyl hydroxylases (PHs), lysyl oxidases (LOXs) and lysyl hydroxylases (LHs). Osteogenesis was assessed at the level of osteoblast specific gene expression and alizarin red staining for mineralization. Cell layer-associated matrix and collagen content were determined by amino acid analysis. At 4h, 100% cells were flattened on both surfaces, however the cells on smooth surface had a fibroblast-like shape, while cells on rough surface lacked any defined long axis. PH, LH, and most LOX mRNA levels were greater in hMSCs grown on rough surfaces for 3 days. The mineralized area was greater for rough surface at 28 and 42 days. The collagen content (percent total protein) was also greater at rough surface compared to smooth surface at 28 (36% versus 26%) and 42 days (46% versus 29%), respectively (p<.05). In a cell culture model, rough surface topography positively modulates collagen biosynthesis and accumulation and the expression of genes associated with collagen cross-linking in adherent hMSC. The altered biosynthesis of the collagen-rich ECM adjacent to endosseous implants may influence the biomechanical properties of osseointegrated endosseous implants.

  7. Cells under siege: Viral glycoprotein interactions at the cell surface

    PubMed Central

    Bowden, Thomas A.; Jones, E. Yvonne; Stuart, David I.

    2011-01-01

    As obligate parasites, viruses are required to enter and replicate within their host, a process which employs many of their proteins to hijack natural cellular processes. High resolution X-ray crystallographic analysis has proven to be an ideal method to visualize the mechanisms by which such virus-host interactions occur and has revealed the innovative capacity of viruses to adapt efficiently to their hosts. In this review, we draw upon recently elucidated paramyxovirus-, arenavirus-, and poxvirus-host protein complex crystal structures to reveal both the capacity of viruses to appropriate one component of a physiological protein–protein binding event (often modifying it to out-compete the host-protein), and the ability to utilize novel binding sites on host cell surface receptors. The structures discussed shed light on a number of biological processes ranging from viral entry to virulence and host antagonism. Drawn together they reveal the common strategies which viruses have evolved to interact with their natural host. The structures also support molecular level rationales for how viruses can be transmitted to unrelated organisms and thus pose severe health risks. PMID:21440638

  8. Endothelial cell behavior on vascular prosthetic grafts: effect of polymer chemistry, surface structure, and surface treatment.

    PubMed

    Marois, Y; Sigot-Luizard, M F; Guidoin, R

    1999-01-01

    When implanting any vascular prosthetic grafts, one important goal to ensure long-term patency is achieving complete endothelialization of the luminal surface, a process that has rarely been observed clinically in humans. Seeding vascular grafts with endothelial cells has been seen as an attractive approach but has not been clinically convincing. A determining factor may be the type of polymer and surface structure. Using organotypic culture assays, the present investigation studied the effect of different polymers, surface structures, and surface treatments on endothelial cell behavior. The materials tested were polyester (PET), polytetrafluoroethylene (PTFE), polyesterurethane (PESU), and polyetherurethane (PETU) grafts with different surface structures. The surface treatments on the PET grafts included impregnation with cross-linked albumin, collagen, and gelatin, and treatments with fluoropolymer and electrically conducting polypyrrole polymer. Low density polyethylene (LDPE) and polydimethylsiloxane (PDMS) sheets (smooth surface, plain wall) were used as controls. After incubation for 7 days at 37 degrees C, cell adhesion and migration on the different polymers and structures were as follows: woven and knitted PET (high porosity) > PTFE, PESU, PETU hydrophobic (low porosity) > PETU hydrophilic, LDPE, PDMS (no porosity). Cell density results showed no difference between polymers and porous structures and a higher cell density on smooth nonporous surfaces. Compared with the nonimpregnated PET structures, knitted PET treated with albumin, collagen, or gelatin showed slight decreases of cell adhesion. No differences in cell migration and density were reported between any of the PET grafts, except for one polyester graft with a different chemistry than Dacron, which exhibited greater cell migration and lower cell density. Polyester grafts with a fluoropolymer treatment showed lower cell adhesion and migration and higher cell density than the nontreated PET. Finally

  9. 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

  10. Surface-modified gold nanorods for specific cell targeting

    NASA Astrophysics Data System (ADS)

    Wang, Chan-Ung; Arai, Yoshie; Kim, Insun; Jang, Wonhee; Lee, Seonghyun; Hafner, Jason H.; Jeoung, Eunhee; Jung, Deokho; Kwon, Youngeun

    2012-05-01

    Gold nanoparticles (GNPs) have unique properties that make them highly attractive materials for developing functional reagents for various biomedical applications including photothermal therapy, targeted drug delivery, and molecular imaging. For in vivo applications, GNPs need to be prepared with very little or negligible cytotoxicitiy. Most GNPs are, however, prepared using growth-directing surfactants such as cetyl trimethylammonium bromide (CTAB), which are known to have considerable cytotoxicity. In this paper, we describe an approach to remove CTAB to a non-toxic concentration. We optimized the conditions for surface modification with methoxypolyethylene glycol thiol (mPEG), which replaced CTAB and formed a protective layer on the surface of gold nanorods (GNRs). The cytotoxicities of pristine and surface-modified GNRs were measured in primary human umbilical vein endothelial cells and human cell lines derived from hepatic carcinoma cells, embryonic kidney cells, and thyroid papillary carcinoma cells. Cytotoxicity assays revealed that treating cells with GNRs did not significantly affect cell viability except for thyroid papillary carcinoma cells. Thyroid cancer cells were more susceptible to residual CTAB, so CTAB had to be further removed by dialysis in order to use GNRs for thyroid cell targeting. PEGylated GNRs are further modified to present monoclonal antibodies that recognize a specific surface marker, Na-I symporter, for thyroid cells. Antibody-conjugated GNRs specifically targeted human thyroid cells in vitro.

  11. Effect of Stratification on Surface Properties of Corneal Epithelial Cells

    PubMed Central

    Yáñez-Soto, Bernardo; Leonard, Brian C.; Raghunathan, Vijay Krishna; Abbott, Nicholas L.; Murphy, Christopher J.

    2015-01-01

    Purpose The purpose of this study was to determine the influence of mucin expression in an immortalized human corneal epithelial cell line (hTCEpi) on the surface properties of cells, such as wettability, contact angle, and surface heterogeneity. Methods hTCEpi cells were cultured to confluence in serum-free medium. The medium was then replaced by stratification medium to induce mucin biosynthesis. The mucin expression profile was analyzed using quantitative PCR and Western blotting. Contact angles were measured using a two-immiscible liquid method, and contact angle hysteresis was evaluated by tilting the apparatus and recording advancing and receding contact angles. The spatial distribution of mucins was evaluated with fluorescently labeled lectin. Results hTCEpi cells expressed the three main ocular mucins (MUC1, MUC4, and MUC16) with a maximum between days 1 and 3 of the stratification process. Upon stratification, cells caused a very significant increase in contact angle hysteresis, suggesting the development of spatially discrete and heterogeneously distributed surface features, defined by topography and/or chemical functionality. Although atomic force microscopy measurements showed no formation of appreciable topographic features on the surface of the cells, we observed a significant increase in surface chemical heterogeneity. Conclusions The surface chemical heterogeneity of the corneal epithelium may influence the dynamic behavior of tear film by “pinning” the contact line between the cellular surface and aqueous tear film. Engineering the surface properties of corneal epithelium could potentially lead to novel treatments in dry eye disease. PMID:26747762

  12. Yeast surface display of dehydrogenases in microbial fuel-cells.

    PubMed

    Gal, Idan; Schlesinger, Orr; Amir, Liron; Alfonta, Lital

    2016-12-01

    Two dehydrogenases, cellobiose dehydrogenase from Corynascus thermophilus and pyranose dehydrogenase from Agaricus meleagris, were displayed for the first time on the surface of Saccharomyces cerevisiae using the yeast surface display system. Surface displayed dehydrogenases were used in a microbial fuel cell and generated high power outputs. Surface displayed cellobiose dehydrogenase has demonstrated a midpoint potential of -28mV (vs. Ag/AgCl) at pH=6.5 and was used in a mediator-less anode compartment of a microbial fuel cell producing a power output of 3.3μWcm(-2) using lactose as fuel. Surface-displayed pyranose dehydrogenase was used in a microbial fuel cell and generated high power outputs using different substrates, the highest power output that was achieved was 3.9μWcm(-2) using d-xylose. These results demonstrate that surface displayed cellobiose dehydrogenase and pyranose dehydrogenase may successfully be used in microbial bioelectrochemical systems.

  13. Molecular design of spacer-N-linked sialoglycopolypeptide as polymeric inhibitors against influenza virus infection.

    PubMed

    Ogata, Makoto; Hidari, Kazuya I P J; Kozaki, Wataru; Murata, Takeomi; Hiratake, Jun; Park, Enoch Y; Suzuki, Takashi; Usui, Taichi

    2009-07-13

    A series of spacer-N-linked glycopolymers carrying long/short α2,3/6 sialylated glycan were designed as polymeric inhibitors of influenza virus. Lactose (Lac) and N-acetyllactosamine (LN: Galβ1,4GlcNAc) were first converted to spacer-N-linked disaccharide glycosides, followed by consecutive enzymatic addition of GlcNAc and Gal residues to the glycosides. The resulting spacer-N-linked glycosides with di-, tetra-, and hexasaccharides carrying a Lac, LN, lacto-N-neotetraose (LNnT: Galβ1,4GlcNAcβ1,3Galβ1,4Glc), and LNβ1,3LNnT were coupled to the carboxy group of γ-polyglutamic acid (γ-PGA) and enzymatically converted to glycopolypeptides carrying α2,3/6 sialylated glycans. The interactions of a series of sialoglycopolypeptides with avian and human influenza virus strains were investigated using a hemagglutination inhibition assay. The avian virus A/Duck/HongKong/313/4/78 (H5N3) bound specifically, regardless of the structure of the asialo portion. In contrast, human virus A/Aichi/2/68 (H3N2) bound preferentially to long α2,6sialylated glycans with penta- or heptasaccharides in a glycan length-dependent manner. Furthermore, the Sambucus sieboldiana (SNA) lectin was also useful as a model of human virus hemagglutinin (HA) for understanding the carbohydrate binding properties, because the recognition motifs of the inner sugar in the receptor were very similar.

  14. Multijunction Solar Cell Technology for Mars Surface Applications

    NASA Technical Reports Server (NTRS)

    Stella, Paul M.; Mardesich, Nick; Ewell, Richard C.; Mueller, Robert L.; Endicter, Scott; Aiken, Daniel; Edmondson, Kenneth; Fetze, Chris

    2006-01-01

    Solar cells used for Mars surface applications have been commercial space qualified AM0 optimized devices. Due to the Martian atmosphere, these cells are not optimized for the Mars surface and as a result operate at a reduced efficiency. A multi-year program, MOST (Mars Optimized Solar Cell Technology), managed by JPL and funded by NASA Code S, was initiated in 2004, to develop tools to modify commercial AM0 cells for the Mars surface solar spectrum and to fabricate Mars optimized devices for verification. This effort required defining the surface incident spectrum, developing an appropriate laboratory solar simulator measurement capability, and to develop and test commercial cells modified for the Mars surface spectrum. This paper discusses the program, including results for the initial modified cells. Simulated Mars surface measurements of MER cells and Phoenix Lander cells (2007 launch) are provided to characterize the performance loss for those missions. In addition, the performance of the MER rover solar arrays is updated to reflect their more than two (2) year operation.

  15. Adipogenic differentiation of mesenchymal stem cells on micropatterned polyelectrolyte surfaces.

    PubMed

    Kawazoe, Naoki; Guo, Likun; Wozniak, Michal J; Imaizumi, Yumie; Tateishi, Tetsuya; Zhang, Xingdong; Chen, Guoping

    2009-01-01

    Three kinds of photoreactive polyelectrolytes of polyallylamine (PAAm), poly(acrylic acid) (PAAc), and poly(vinyl alcohol) (PVA) were synthesized by the introduction of azidophenyl groups in the respective polymers. The photoreactive PAAm, PAAc, and PVA were micropatterned on polystyrene surfaces by photolithography. Observation with optical microscopy and scanning probe microscopy demonstrated the formation of a striped pattern of polyelectrolytes with a width of 200 microm. The micropatterned polyelectrolytes swelled in water. The micropatterned surfaces were used for cell culture of mesenchymal stem cells (MSCs) and their effects on adipogenic differentiation were investigated. The MSCs adhered to and proliferated evenly on the PAAm- and PAAc-patterned surfaces while they formed a cell pattern on the PVA-patterned surface. The PAAm-, PAAc-grafted, and polystyrene surfaces supported cell adhesion while the PVA-grafted surface did not. When cultured in adipogenic differentiation medium, the adipogenic differentiation of MSCs on the polyelectrolyte-patterned surfaces was demonstrated by the formation of lipid vacuoles and gene expression analysis. Oil Red-O-positive cells showed an even distribution on the PAAm- and PAAc-patterned surfaces, while they showed a pattern on the PVA-patterned surface. The fraction of Oil RedO-positive cells increased with culture time. The MSCs cultured on the PAAm-, PAAc-grafted, and polystyrene surfaces in adipogenic differentiation medium expressed the adipogenesis marker genes of peroxisome proliferator-activated receptor gamma2 (PPARgamma2), lipoprotein lipase (LPL), and fatty acid binding protein 4 (FABP4). These results indicate that the PAAm-, and PAAc-grafted, and polystyrene surfaces supported the adipogenesis of MSCs while a PVA-grafted surface did not.

  16. Biomechanics of cell rolling: shear flow, cell-surface adhesion, and cell deformability.

    PubMed

    Dong, C; Lei, X X

    2000-01-01

    The mechanics of leukocyte (white blood cell; WBC) deformation and adhesion to endothelial cells (EC) has been investigated using a novel in vitro side-view flow assay. HL-60 cell rolling adhesion to surface-immobilized P-selectin was used to model the WBC-EC adhesion process. Changes in flow shear stress, cell deformability, or substrate ligand strength resulted in significant changes in the characteristic adhesion binding time, cell-surface contact and cell rolling velocity. A 2-D model indicated that cell-substrate contact area under a high wall shear stress (20 dyn/cm2) could be nearly twice of that under a low stress (0.5 dyn/cm2) due to shear flow-induced cell deformation. An increase in contact area resulted in more energy dissipation to both adhesion bonds and viscous cytoplasm, whereas the fluid energy that inputs to a cell decreased due to a flattened cell shape. The model also predicted a plateau of WBC rolling velocity as flow shear stresses further increased. Both experimental and computational studies have described how WBC deformation influences the WBC-EC adhesion process in shear flow.

  17. Heterojunction solar cell with passivated emitter surface

    DOEpatents

    Olson, J.M.; Kurtz, S.R.

    1994-05-31

    A high-efficiency heterojunction solar cell is described wherein a thin emitter layer (preferably Ga[sub 0.52]In[sub 0.48]P) forms a heterojunction with a GaAs absorber layer. A passivating window layer of defined composition is disposed over the emitter layer. The conversion efficiency of the solar cell is at least 25.7%. The solar cell preferably includes a passivating layer between the substrate and the absorber layer. An anti-reflection coating is preferably disposed over the window layer. 1 fig.

  18. Heterojunction solar cell with passivated emitter surface

    DOEpatents

    Olson, Jerry M.; Kurtz, Sarah R.

    1994-01-01

    A high-efficiency heterojunction solar cell wherein a thin emitter layer (preferably Ga.sub.0.52 In.sub.0.48 P) forms a heterojunction with a GaAs absorber layer. A passivating window layer of defined composition is disposed over the emitter layer. The conversion efficiency of the solar cell is at least 25.7%. The solar cell preferably includes a passivating layer between the substrate and the absorber layer. An anti-reflection coating is preferably disposed over the window layer.

  19. Modelling cell motility and chemotaxis with evolving surface finite elements.

    PubMed

    Elliott, Charles M; Stinner, Björn; Venkataraman, Chandrasekhar

    2012-11-07

    We present a mathematical and a computational framework for the modelling of cell motility. The cell membrane is represented by an evolving surface, with the movement of the cell determined by the interaction of various forces that act normal to the surface. We consider external forces such as those that may arise owing to inhomogeneities in the medium and a pressure that constrains the enclosed volume, as well as internal forces that arise from the reaction of the cells' surface to stretching and bending. We also consider a protrusive force associated with a reaction-diffusion system (RDS) posed on the cell membrane, with cell polarization modelled by this surface RDS. The computational method is based on an evolving surface finite-element method. The general method can account for the large deformations that arise in cell motility and allows the simulation of cell migration in three dimensions. We illustrate applications of the proposed modelling framework and numerical method by reporting on numerical simulations of a model for eukaryotic chemotaxis and a model for the persistent movement of keratocytes in two and three space dimensions. Movies of the simulated cells can be obtained from http://homepages.warwick.ac.uk/∼maskae/CV_Warwick/Chemotaxis.html.

  20. The endomembrane requirement for cell surface repair

    NASA Technical Reports Server (NTRS)

    McNeil, Paul L.; Miyake, Katsuya; Vogel, Steven S.

    2003-01-01

    The capacity to reseal a plasma membrane disruption rapidly is required for cell survival in many physiological environments. Intracellular membrane (endomembrane) is thought to play a central role in the rapid resealing response. We here directly compare the resealing response of a cell that lacks endomembrane, the red blood cell, with that of several nucleated cells possessing an abundant endomembrane compartment. RBC membrane disruptions inflicted by a mode-locked Ti:sapphire laser, even those initially smaller than hemoglobin, failed to reseal rapidly. By contrast, much larger laser-induced disruptions made in sea urchin eggs, fibroblasts, and neurons exhibited rapid, Ca(2+)-dependent resealing. We conclude that rapid resealing is not mediated by simple physiochemical mechanisms; endomembrane is required.

  1. Implant Surface Design Regulates Mesenchymal Stem Cell Differentiation and Maturation

    PubMed Central

    Boyan, B.D.; Cheng, A.; Olivares-Navarrete, R.; Schwartz, Z.

    2016-01-01

    Changes in dental implant materials, structural design, and surface properties can all affect biological response. While bulk properties are important for mechanical stability of the implant, surface design ultimately contributes to osseointegration. This article reviews the surface parameters of dental implant materials that contribute to improved cell response and osseointegration. In particular, we focus on how surface design affects mesenchymal cell response and differentiation into the osteoblast lineage. Surface roughness has been largely studied at the microscale, but recent studies have highlighted the importance of hierarchical micron/submicron/nanosurface roughness, as well as surface roughness in combination with surface wettability. Integrins are transmembrane receptors that recognize changes in the surface and mediate downstream signaling pathways. Specifically, the noncanonical Wnt5a pathway has been implicated in osteoblastic differentiation of cells on titanium implant surfaces. However, much remains to be elucidated. Only recently have studies been conducted on the differences in biological response to implants based on sex, age, and clinical factors; these all point toward differences that advocate for patient-specific implant design. Finally, challenges in implant surface characterization must be addressed to optimize and compare data across studies. An understanding of both the science and the biology of the materials is crucial for developing novel dental implant materials and surface modifications for improved osseointegration. PMID:26927483

  2. Regulation of tissue factor coagulant activity on cell surfaces

    PubMed Central

    RAO, L.V.M.; PENDURTHI, U.R.

    2012-01-01

    Summary Tissue factor (TF) is a transmembrane glycoprotein and an essential component of factor VIIa-TF enzymatic complex that triggers activation of the coagulation cascade. Formation of TF-FVIIa complexes on cell surfaces not only trigger the coagulation cascade but also transduce cell signaling via activation of protease-activated receptors. Tissue factor is expressed constitutively on cell surfaces of a variety of extravascular cell types, including fibroblasts and pericytes in and surrounding blood vessel walls and epithelial cells but generally absent on cells that come in contact with blood directly. However, TF expression could be induced in some blood cells, such as monocytes and endothelial cells, following an injury or pathological stimuli. Tissue factor is essential for hemostasis, but aberrant expression of TF leads to thrombosis. Therefore, a proper regulation of TF activity is critical for the maintenance of hemostatic balance and health in general. TF-FVIIa coagulant activity at the cell surface is influenced not only by TF protein expression levels but also independently by a variety of mechanisms, including alterations in membrane phospholipid composition and cholesterol content, thiol-dependent modifications of TF allosteric disulfide bond, and other post-translational modifications of TF. In this article, we critically review key literature on mechanisms by which TF coagulant activity is regulated at the cell surface in the absence of changes in TF protein levels with specific emphasis on recently published data and provide the authors’ perspective on the subject. PMID:23006890

  3. Skin stem cells: rising to the surface.

    PubMed

    Fuchs, Elaine

    2008-01-28

    The skin epidermis and its appendages provide a protective barrier that is impermeable to harmful microbes and also prevents dehydration. To perform their functions while being confronted with the physicochemical traumas of the environment, these tissues undergo continual rejuvenation through homeostasis, and, in addition, they must be primed to undergo wound repair in response to injury. The skin's elixir for maintaining tissue homeostasis, regenerating hair, and repairing the epidermis after injury is its stem cells, which reside in the adult hair follicle, sebaceous gland, and epidermis. Stem cells have the remarkable capacity to both self-perpetuate and also give rise to the differentiating cells that constitute one or more tissues. In recent years, scientists have begun to uncover the properties of skin stem cells and unravel the mysteries underlying their remarkable capacity to perform these feats. In this paper, I outline the basic lineages of the skin epithelia and review some of the major findings about mammalian skin epithelial stem cells that have emerged in the past five years.

  4. 1H NMR characterization of a hen ovalbumin tyrosinamide N-linked oligosaccharide library.

    PubMed

    Corradi Da Silva, M L; Stubbs, H J; Tamura, T; Rice, K G

    1995-04-20

    A library of 15 N-linked oligosaccharide structures was prepared from ovalbumin and characterized using high-field NMR and mass spectrometry. The oligosaccharides were enzymatically released from ovalbumin glycopeptides, and the reducing ends were reacted with ammonium bicarbonate to form oligosaccharide-glycosylamines. These reacted with Boc-tyrosine-N-hydroxysuccinimide ester, resulting in a mixture of tyrosinamide-oligosaccharides. The Boc group was removed to expose an amine terminus which enhanced the resolution of tyrosinamide-oligosaccharides when chromatographed on reverse-phase HPLC. Ten major and five minor oligosaccharides were purified on a micromole scale and characterized using 1H NMR and FAB-MS. The structures include high-mannose, hybrid, and complex oligosaccharides possessing from two to five antenna, providing the most complete definition of ovalbumin N-linked oligosaccharides to date. The resulting library is well suited to biological studies due to the presence of a single terminal tyrosine residue on each oligosaccharide that allows radioiodination or the attachment of additional probes to these glycoconjugates prior to biological studies.

  5. 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

  6. 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.

  7. Elastomers bonded to metal surfaces seal electrochemical cells

    NASA Technical Reports Server (NTRS)

    Sherfey, J. M.

    1964-01-01

    A leakproof seal secondary cell containing alkaline electrolytes was developed by bonding an alkali-resistant elastomer, such as neoprene, to metal contact surfaces. Test results of several different elastomers strongly indicate the feasibility of this sealing method.

  8. 2-DEOXY-GLUCOSE DOWN REGULATES ENDOTHELIAL AKT AND ERK VIA INTERFERENCE WITH N-LINKED GLYCOSYLATION, INDUCTION OF ENDOPLASMIC RETICULUM STRESS AND GSK-3β ACTIVATION

    PubMed Central

    Kovács, Krisztina; Decatur, Christina; Toro, Marcela; Pham, Dien G.; Liu, Huaping; Jing, Yuqi; Murray, Timothy G.; Lampidis, Theodore J.; Merchan, Jaime R.

    2015-01-01

    Interference with endothelial cell metabolism is a promising, yet unexploited strategy for angiogenesis inhibition. We reported that the glucose analog, 2-deoxy-D-Glucose (2-DG) inhibits angiogenesis at significantly lower concentrations than those required for tumor cytotoxicity. Here, we found that hypersensitivity to 2-DG in endothelial cells is not associated with enhanced drug uptake compared to tumor cells, but with time dependent, endothelial selective inhibition of Akt and Erk phosphorylation. Down regulation of these critical survival pathways is shown to be due to 2-DG’s interference with N-linked glycosylation, leading to alterations in VEGFR2 (and downstream signaling) as well as induction of endoplasmic reticulum (ER) stress, GSK-3β activation and apoptosis. In vivo, periocular administration of 2-DG in LHBETATAG mice was associated with significant reduction of newly formed (CD 105 +) tumor capillaries, ER stress (GRP 78 expression), and endothelial apoptosis (TUNEL). These findings uniquely link N-linked glycosylation inhibition, ER stress and Erk/Akt down regulation in endothelial cells, and provide a novel drug development strategy to overcome resistance mechanisms to currently available antiangiogenic agents. PMID:26637370

  9. Contact inhibition of phagocytosis in epithelial sheets: alterations of cell surface properties induced by cell-cell contacts.

    PubMed

    Vasiliev, J M; Gelfand, I M; Domnina, L V; Zacharova, O S; Ljubimov, A V

    1975-02-01

    Contact inhibition of phagocytosis was found to be characteristic for epithelial sheets formed in cultures by several cell types: normal and transformed mouse kidney cells, and differentiated mouse hepatoma cells. In these sheets most central cells surrounded by other cells had very low phagocytic activity. In contrast, marginal cells having a free edge were able to perform an active phagocytosis. Contact inhibition of phagocytosis was absent in dense cultures of mouse embryo fibroblasts and in cultures of anaplastic mouse hepatoma 22a. The upper surface of epithelial sheets was nonadhesive for prelabeled epithelial cells and fibroblasts. In contrast, the upper surface of dense cultures of mouse fibroblasts was adhesive for these cells. These and other data strengthen the suggestion that contact inhibition of phagocytosis is a result of different adhesiveness of the upper cell surface and of the surfaces near the free edge. Agents inhibiting cell surface movements at the free edges of marginal epithelial cells (cytochalasin, azide, sorbitol, low temperature) prevented adhesion of particles to these edges. Possibly, the surface of actively moving cytoplasmic processes is the only cell part that has adhesive properties necessary for the formation of attachments with other cellular and noncellular surfaces. In epithelial sheets, in contrast to fibroblast cultures, Colcemid did not activate movements of immobile contacting cell edges. These results indicate that mechanisms of contact immobilization of cell surface may be different in epithelium and fibroblasts. Firm contacts formed between epithelial cells are sufficient for stable immobilization of the surface; additional stabilization of the surface by microtubules is not essential. Fibroblasts do not form firm contacts and the Colcemid-sensitive stabilization process is essential for maintenance of the immobile state of their surfaces. Differences in the stability of cell surface immobilization produced by cell-cell

  10. Theoretical analysis of cell separation based on cell surface marker density.

    PubMed

    Chalmers, J J; Zborowski, M; Moore, L; Mandal, S; Fang, B B; Sun, L

    1998-07-05

    A theoretical analysis was performed to determine the number of fractions a multidisperse, immunomagnetically labeled cell population can be separated into based on the surface marker (antigen) density. A number of assumptions were made in this analysis: that there is a proportionality between the number of surface markers on the cell surface and the number of immunomagnetic labels bound; that this surface marker density is independent of the cell diameter; and that there is only the presence of magnetic and drag forces acting on the cell. Due to the normal distribution of cell diameters, a "randomizing" effect enters into the analysis, and an analogy between the "theoretical plate" analysis of distillation, adsorption, and chromatography can be made. Using the experimentally determined, normal distribution of cell diameters for human lymphocytes and a breast cancer cell line, and fluorescent activated cell screening data of specific surface marker distributions, examples of theoretical plate calculations were made and discussed.

  11. Cell culture on hydrophilicity-controlled silicon nitride surfaces.

    PubMed

    Masuda, Yuriko; Inami, Wataru; Miyakawa, Atsuo; Kawata, Yoshimasa

    2015-12-01

    Cell culture on silicon nitride membranes is required for atmospheric scanning electron microscopy, electron beam excitation assisted optical microscopy, and various biological sensors. Cell adhesion to silicon nitride membranes is typically weak, and cell proliferation is limited. We increased the adhesion force and proliferation of cultured HeLa cells by controlling the surface hydrophilicity of silicon nitride membranes. We covalently coupled carboxyl groups on silicon nitride membranes, and measured the contact angles of water droplets on the surfaces to evaluate the hydrophilicity. We cultured HeLa cells on the coated membranes and evaluated stretch of the cell. Cell migration and confluence were observed on the coated silicon nitride films. We also demonstrated preliminary observation result with direct electron beam excitation-assisted optical microscope.

  12. Standing surface acoustic wave (SSAW)-based cell washing

    PubMed Central

    Li, Sixing; Ding, Xiaoyun; Mao, Zhangming; Chen, Yuchao; Nama, Nitesh; Guo, Feng; Li, Peng; Wang, Lin; Cameron, Craig E.; Huang, Tony Jun

    2014-01-01

    Cell/bead washing is an indispensable sample preparation procedure used in various cell studies and analytical processes. In this article, we report a standing surface acoustic wave (SSAW)-based microfluidic device for cell and bead washing in a continuous flow. In our approach, the acoustic radiation force generated in a SSAW field is utilized to actively extract cells or beads from their original medium. A unique configuration of tilted-angle standing surface acoustic wave (taSSAW) is employed in our device, enabling us to wash beads with >98% recovery rate and >97% washing efficiency. We also demonstrate the functionality of our device by preparing high-purity (>97%) white blood cells from lysed blood samples through cell washing. Our SSAW-based cell/bead washing device has the advantages of label-free manipulation, simplicity, high biocompatibility, high recovery rate, and high washing efficiency. It can be useful for many lab-on-a-chip applications. PMID:25372273

  13. Surface plasmonic effects on organic solar cells.

    PubMed

    Uddin, Ashraf; Yang, Xiaohan

    2014-02-01

    Most high-performance organic photovoltaic (OPV) devices reported in the literature have been fabricated using the bulk heterojunction (BHJ) concept. Typically, the optimum thickness of the active layer for an OPV device is around 100 nm, or possibly less; such a thin layer can lead to low absorption of light. A thicker layer, however, inevitably increases the device resistance, due to the low carrier mobilities and short exciton diffusion lengths in organic materials. This situation imposes a trade-off between light absorption and charge transport efficiencies in OPV devices, motivating the development of a variety of light-trapping techniques. Metallic nanoparticles (NPs) such as Ag, Au, etc. and other metallic nanostructures are potential candidates for improving the light absorption due to the localized surface plasmon resonance (LSPR). LSPR contributes to the significant enhancement of local electromagnetic fields and improves the optical properties of the nanostructure devices. The excitation of LSPR is achieved when the frequency of the incident light matches its resonance peak, resulting in unique optical properties; selective light extinction as well as local enhancement of electromagnetic fields near the surface of metallic NPs. The resonance peak of LSPR depends strongly on the size, shape, and the dielectric environment of the metallic NPs. In this review article, progress on plasmonic enhanced OPV device performance is examined. The concepts of surface plasmonics for OPV devices, suitable plasmonic materials, location, optimum size and concentration of NP materials within the device are explored.

  14. Multi-scale cell/surface interaction on modified titanium aluminum vanadium surfaces

    NASA Astrophysics Data System (ADS)

    Chen, Jianbo

    This dissertation presents a series of experimental studies of the effects of multi-scale cell/surface interactions on modified Ti-6Al-4V surfaces. These include laser-grooved surfaces; porous structures and RGD-coated laser-grooved surfaces. A nano-second DPSS UV lasers with a Gaussian pulse energy profile was used to introduce the desired micro-groove geometries onto Ti-6Al-4V surfaces. This was done without inducing micro-cracks or significant changes in surface chemistry within the heat affected zones. The desired 8-12 mum groove depths and widths were achieved by the control of pulse frequency, scan speed, and the lens focal length that controls spot size. The interactions between human osteosarcoma (HOS) cells and laser-grooved Ti-6Al-4V surfaces were investigated after 48 hours of cell culture. The cell behavior, including cell spreading, alignment and adhesion, was elucidated using scanning electronic microscopy (SEM), immuno-fluorescence staining and enzymatic detachment. Contact guidance was shown to increase as grooved spacing decreased. For the range of micro-groove geometries studied, micro-grooves with groove spacings of 20 mum provided the best combination of cell orientation and adhesion. Short-term adhesion experiments (15 mins to 1 day) also revealed that there is a positive correlation between cell orientation and cell adhesion. Contact guidance on the micro-grooved surfaces is shown to be enhanced by nano- and micro-scale asperities that provide sites for the attachment of lamellopodia during cell locomotion and spreading. Contact guidance is also promoted by the geometrical confinement provided by laser grooves. An experimental study of initial cell spreading and ingrowth into Ti-6Al-4V porous structures was also carried out on porous structures with different pore sizes and geometries. A combination of SEM, the tetrazolium salt (MTT) colorimetric assay and enzymatic detachment were used to study cell spreading and adhesion. The extent of cell

  15. Microfabricated surface designs for cell culture and diagnosis.

    PubMed

    Matsuda, T; Chung, D J

    1994-01-01

    Grooved and holed surfaces with a well fabricated design may serve as microsubstrates for cell culture and microreactors for diagnosis. In this study, the authors prepared chemically treated, micrometer scale grooved and holed glass surfaces by combined surface modification and ultraviolet (UV) excimer laser ablation techniques, as follows. 1) Microcell-culture substrate: Amino group attached glass surfaces, prepared by the treatment with an aminopropylsilane, were condensed with a carboxylated radical initiator. Subsequently, polyacrylamide was grafted by surface initiated radical polymerization to create a very hydrophilic surface layer. Ultraviolet excimer laser beams (KrF: 248 nm) were irradiated through a microscope onto surfaces to create grooves or holes that were 10 and 50 microns in width or diameter, respectively. The depth, depending on the irradiation light strength, ranged from a few to several tenths of a micrometer. On endothelial cell (EC) seeding, ECs adhered and grew on the bottoms of the grooved or holed surface where glass was exposed on ablation. Little cell adhesion was observed on non ablated, grafted surfaces. Endothelial cells aligned along the groove, resulting in very narrow tube like tissue formation, whereas ECs tended to form a multilayered spherical aggregate in a hole. A single cell resided in a 10 microns square hole. 2) Microreactor for diagnosis: The glass surface, treated with a fluorinated silane, was ablated to create round holes. On addition of a few microliters of water, water could be quantitatively transferred into a hole because of the water repellent characteristics of non ablated, fluorinated glass. As a model of a microreactor, enzyme reactions to affect different levels of glucose were carried out in tiny holed surfaces.

  16. Endothelial cell migration on surfaces modified with immobilized adhesive peptides.

    PubMed

    Kouvroukoglou, S; Dee, K C; Bizios, R; McIntire, L V; Zygourakis, K

    2000-09-01

    Endothelial cell (EC) migration has been studied on aminophase surfaces with covalently bound RGDS and YIGSRG cell adhesion peptides. The fluorescent marker dansyl chloride was used to quantify the spatial distribution of the peptides on the modified surfaces. Peptides appeared to be distributed in uniformly dispersed large clusters separated by areas of lower peptide concentrations. We employed digital time-lapse video microscopy and image analysis to monitor EC migration on the modified surfaces and to reconstruct the cell trajectories. The persistent random walk model was then applied to analyze the cell displacement data and compute the mean root square speed, the persistence time, and the random motility coefficient of EC. We also calculated the time-averaged speed of cell locomotion. No differences in the speed of cell locomotion on the various substrates were noted. Immobilization of the cell adhesion peptides (RGDS and YIGSRG), however, significantly increased the persistence of cell movement and, thus, the random motility coefficient. These results suggest that immobilization of cell adhesion peptides on the surface of implantable biomaterials may lead to enhanced endothelization rates.

  17. Nanometer polymer surface features: the influence on surface energy, protein adsorption and endothelial cell adhesion

    NASA Astrophysics Data System (ADS)

    Carpenter, Joseph; Khang, Dongwoo; Webster, Thomas J.

    2008-12-01

    Current small diameter (<5 mm) synthetic vascular graft materials exhibit poor long-term patency due to thrombosis and intimal hyperplasia. Tissue engineered solutions have yielded functional vascular tissue, but some require an eight-week in vitro culture period prior to implantation—too long for immediate clinical bedside applications. Previous in vitro studies have shown that nanostructured poly(lactic-co-glycolic acid) (PLGA) surfaces elevated endothelial cell adhesion, proliferation, and extracellular matrix synthesis when compared to nanosmooth surfaces. Nonetheless, these studies failed to address the importance of lateral and vertical surface feature dimensionality coupled with surface free energy; nor did such studies elicit an optimum specific surface feature size for promoting endothelial cell adhesion. In this study, a series of highly ordered nanometer to submicron structured PLGA surfaces of identical chemistry were created using a technique employing polystyrene nanobeads and poly(dimethylsiloxane) (PDMS) molds. Results demonstrated increased endothelial cell adhesion on PLGA surfaces with vertical surface features of size less than 18.87 nm but greater than 0 nm due to increased surface energy and subsequently protein (fibronectin and collagen type IV) adsorption. Furthermore, this study provided evidence that the vertical dimension of nanometer surface features, rather than the lateral dimension, is largely responsible for these increases. In this manner, this study provides key design parameters that may promote vascular graft efficacy.

  18. 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

  19. Oxide modified air electrode surface for high temperature electrochemical cells

    DOEpatents

    Singh, Prabhakar; Ruka, Roswell J.

    1992-01-01

    An electrochemical cell is made having a porous cermet electrode (16) and a porous lanthanum manganite electrode (14), with solid oxide electrolyte (15) between them, where the lanthanum manganite surface next to the electrolyte contains a thin discontinuous layer of high surface area cerium oxide and/or praseodymium oxide, preferably as discrete particles (30) in contact with the air electrode and electrolyte.

  20. Shape of red blood cells in contact with artificial surfaces.

    PubMed

    Grzhibovskis, Richards; Krämer, Elisabeth; Bernhardt, Ingolf; Kemper, Björn; Zanden, Carl; Repin, Nikolay V; Tkachuk, Bogdan V; Voinova, Marina V

    2017-03-01

    The phenomenon of physical contact between red blood cells and artificial surfaces is considered. A fully three-dimensional mathematical model of a bilayer membrane in contact with an artificial surface is presented. Numerical results for the different geometries and adhesion intensities are found to be in agreement with experimentally observed geometries obtained by means of digital holographic microscopy.

  1. Synthetically functionalized retroviruses produced from the bioorthogonally engineered cell surface.

    PubMed

    Wong, Shirley; Kwon, Young Jik

    2011-02-16

    Conjugation of desired molecules onto retroviral surfaces through the ease of the bioorthogonal functionalization method was demonstrated. Oxidation of surface sialic acids using periodate and further p-anisidine-catalyzed conjugation with aminooxy-bearing molecules were used to directly label retroviral envelope with a fluorescent dye. The retroviral particles that were produced from a bioorthogonally functionalized virus producing cell surface and further tethered with magnetic nanoparticles were efficiently purified by simple magnetic column separation and capable of magnet-directed transduction.

  2. Cell-surface marker analysis of rat thymic dendritic cells.

    PubMed Central

    Bañuls, M P; Alvarez, A; Ferrero, I; Zapata, A; Ardavin, C

    1993-01-01

    Rat thymic dendritic cells have been isolated by collagenase digestion, separation of the low-density cell fraction by centrifugation on metrizamide, and differential adherence. The resulting dendritic cell preparation had a purity of > 90%, and has been analysed by flow cytometry (FCM) using a large panel of monoclonal antibodies (mAb). Dendritic cells expressed major histocompatibility (MHC) class I and class II molecules, the leucocyte common antigen CD45, the rat leucocyte antigen OX44, the rat macrophage marker ED1, and the adhesion molecules Mac-1, LFA-1 and ICAM-1. They were negative for the T- and B-cell-specific forms of CD45, CD45R and B220, and the B-cell marker OX12. Concerning T-cell marker expression, they were negative for T-cell receptor (TcR) and OX40, but they expressed CD2, CD4 and CD8, and interestingly, 50% of DC were CD5+, 50% expressed the alpha-chain of interleukin-2 receptor (IL-2R), and 80% were positive for the T-cell activation antigen recognized by the mAb OX48. Moreover, 60% of DC expressed high levels of Thy-1, whereas 40% displayed intermediate levels of this T-cell marker. PMID:8102122

  3. Complex oligosaccharides are N-linked to Kv3 voltage-gated K+ channels in rat brain.

    PubMed

    Cartwright, Tara A; Corey, Melissa J; Schwalbe, Ruth A

    2007-04-01

    Neuronal Kv3 voltage-gated K(+) channels have two absolutely conserved N-glycosylation sites. Here, it is shown that Kv3.1, 3.3, and 3.4 channels are N-glycosylated in rat brain. Digestion of total brain membranes with peptide N glycosidase F (PNGase F) produced faster migrating immunobands than those of undigested membranes. Additionally, partial PNGase F digests showed that both sites are occupied by oligosaccharides. Neuraminidase treatment produced a smaller immunoband shift relative to PNGase F treatment. These results indicate that both sites are highly available and occupied by N-linked oligosaccharides for Kv3.1, 3.3, and 3.4 in rat brain, and furthermore that at least one oligosaccharide is of complex type. Additionally, these results point to an extracytoplasmic S1-S2 linker in Kv3 proteins expressed in native membranes. We suggest that N-glycosylation processing of Kv3 channels is critical for the expression of K(+) currents at the surface of neurons, and perhaps contributes to the pathophysiology of congenital disorders of glycosylation.

  4. 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.

  5. Cell surface differentiation of Mycoplasma mobile visualized by surface protein localization.

    PubMed

    Kusumoto, Akiko; Seto, Shintaro; Jaffe, Jacob D; Miyata, Makoto

    2004-12-01

    Mycoplasma mobile has a flask-shaped cell morphology and glides toward its tapered end at a rate of 3-7 cell lengths per s (2.0-4.5 microm s(-1)) by an unknown mechanism. Gliding requires that the surface of the cell is in contact with a solid substrate, such as glass or plastic. In order to characterize the nature of the outer surface of M. mobile, monoclonal antibodies were raised against intact cells and screened for their ability to recognize surface proteins. Four antibodies were identified and their protein targets were determined. One antibody recognized the Gli349 protein, which is known to be involved in glass binding and gliding. This antibody was also able to displace attached M. mobile cells from glass, suggesting that Gli349 is the major adhesion protein in M. mobile. The other three antibodies recognized members of the Mvsp family of proteins, which are presumably the major surface antigens of M. mobile. Immunofluorescence studies were performed to localize these proteins on the surface of M. mobile cells. Gli349 localized to the proximal region of the tapered part of the cell (the 'neck'), while the various Mvsp family members showed several distinct patterns of subcellular localization. MvspN and MvspO localized to the distal end of the tapered part of the cell (the 'head'), MvspK localized to the main part of the cell (the 'body'), and MvspI localized to both the head and body but not the neck. This analysis shows that M. mobile surprisingly expresses multiple versions of its major surface antigen at once but differentiates its surface by differential localization of the various paralogues.

  6. Characterization of N-linked oligosaccharides bearing sialyl lewis x moieties on an alternatively glycosylated form of soluble complement receptor type 1 (sCR1).

    PubMed

    Picard, M D; Pettey, C L; Marsh, H C; Thomas, L J

    2000-02-01

    We sought to produce a complement inhibitory protein possessing oligosaccharides specifically modified to contain the sialyl Lewis x (sLe(x)) moiety. This modified glycoprotein could combine anti-complement activity with the ability to inhibit selectin-mediated interactions and concentrate this activity to sites of activated endothelium where selectins are upregulated. Soluble complement receptor type 1 (sCR1), previously shown to be effective in inhibiting the complement cascade, was produced in a cell line capable of adding fucose to N-linked oligosaccharides in the alpha1-3 linkage, which is necessary for sLe(x) glycosylation. The glycoprotein purified from these cells was designated sCR1sLe(x), and may prove to be more effective than sCR1 in some clinical applications. Detailed analysis and characterization of sCR1sLe(x) was performed to confirm that the N-linked oligosaccharides possessed sLe(x) moieties and also to determine the extent of sLe(x) glycosylation. The glycoproteins were characterized by oligosaccharide profiling, sequencing, linkage analysis and quantified by differential enzymic digestion, using fluorophore-assisted carbohydrate electrophoresis. The major glycans were identified as biantennary oligosaccharides (including sialylated and non-core fucosylated glycans). The linkages of sialic acid and the branched fucose were analysed by digestion with linkage-specific enzymes and subsequent separation by electrophoresis. All data were consistent with the presence of sLe(x) moieties on the N-linked oligosaccharides of sCR1sLe(x). sCR1sLe(x) is a prime example of a recombinant protein expressed with oligosaccharides engineered for a specific biological function, and produced using a commercially viable method.

  7. Amplified effect of surface charge on cell adhesion by nanostructures

    NASA Astrophysics Data System (ADS)

    Xu, Li-Ping; Meng, Jingxin; Zhang, Shuaitao; Ma, Xinlei; Wang, Shutao

    2016-06-01

    Nano-biointerfaces with varied surface charge can be readily fabricated by integrating a template-based process with maleimide-thiol coupling chemistry. Significantly, nanostructures are employed for amplifying the effect of surface charge on cell adhesion, as revealed by the cell-adhesion performance, cell morphology and corresponding cytoskeletal organization. This study may provide a promising strategy for developing new biomedical materials with tailored cell adhesion for tissue implantation and regeneration.Nano-biointerfaces with varied surface charge can be readily fabricated by integrating a template-based process with maleimide-thiol coupling chemistry. Significantly, nanostructures are employed for amplifying the effect of surface charge on cell adhesion, as revealed by the cell-adhesion performance, cell morphology and corresponding cytoskeletal organization. This study may provide a promising strategy for developing new biomedical materials with tailored cell adhesion for tissue implantation and regeneration. Electronic supplementary information (ESI) available: Experimental details, SEM, KFM AFM, chemical modification and characterization. See DOI: 10.1039/c6nr00649c

  8. Surface strategies for control of neuronal cell adhesion: A review

    NASA Astrophysics Data System (ADS)

    Roach, P.; Parker, T.; Gadegaard, N.; Alexander, M. R.

    2010-06-01

    Material engineering methods have been used for many years to develop biomedical devices for use within the body to augment, repair or replace damaged tissues ranging from contact lenses to heart valves. Here we review the findings gathered from the wide and varied surface analytical approaches applied to study the interaction between biology and man-made materials. The key material characteristics identified to be important for biological recognition are surface chemistry, topography and compliance. Model surfaces with controlled chemistry and topography have provided insight into biological response to various types of topographical features over a wide range of length scales from nano to micrometres, along with 3D matrices that have been used as scaffolds to support cells for tissue formation. The cellular response to surfaces with localised areas of patterned chemistry and to those presenting gradually changing chemistry are discussed. Where previous reviews have been structured around specific classes of surface modification, e.g. self-assembly, or have broadly examined the response of various cells to numerous surfaces, we aim in this article to focus in particular on the tissues involved in the nervous system whilst providing a broad overview of key issues from the field of cell and protein surface interactions with surfaces. The goal of repair and treatment of diseases related to the central and peripheral nervous systems rely on understanding the local interfacial environment and controlling responses at the cellular level. The role of the protein layer deposited from serum containing media onto man-made surfaces is discussed. We highlight the particular problems associated with the repair of the nervous system, and review how neuronal attachment and axon guidance can be accomplished using various surface cues when cultured with single and multiple cell types. We include a brief glossary of techniques discussed in the body of this article aimed at the

  9. CD44 is the principal cell surface receptor for hyaluronate.

    PubMed

    Aruffo, A; Stamenkovic, I; Melnick, M; Underhill, C B; Seed, B

    1990-06-29

    CD44 is a broadly distributed cell surface protein thought to mediate cell attachment to extracelular matrix components or specific cell surface ligands. We have created soluble CD44-immunoglobulin fusion proteins and characterized their reactivity with tissue sections and lymph node high endothelial cells in primary culture. The CD44 target on high endothelial cells is sensitive to enzymes that degrade hyaluronate, and binding of soluble CD44 is blocked by low concentrations of hyaluronate or high concentrations of chondroitin 4- and 6-sulfates. A mouse anti-hamster hyaluonate receptor antibody reacts with COS cells expressing hamster CD44 cDNA. In sections of all tissues examined, including lymph nodes and Peyer's patches, predigestion with hyaluronidase eliminated CD44 binding.

  10. Dominant negative mutation in cell surface beta 1,4- galactosyltransferase inhibits cell-cell and cell-matrix interactions

    PubMed Central

    1993-01-01

    In addition to its traditional location within the Golgi complex, beta 1,4-galactosyltransferase (GalTase) is also present on the cell surface, where it is thought to function as a cell adhesion molecule by binding to extracellular oligosaccharide ligands. Recent studies suggest that cells contain two forms of GalTase with distinct cytoplasmic domains. The longer form of GalTase contains a 13-amino acid cytoplasmic extension and is preferentially targeted to the plasma membrane, relative to the shorter GalTase protein that is confined primarily to the Golgi compartment. In this study, we created a dominant negative mutation that interferes with the function of cell surface GalTase by transfecting into cells cDNAs encoding truncated versions of the long form of GalTase containing the complete cytoplasmic and transmembrane domains, but devoid of the catalytic domain. In both F9 embryonal carcinoma cells and Swiss 3T3 fibroblasts, overexpressing the truncated long GalTase (TLGT) protein displaced the endogenous cell surface GalTase from its association with the cytoskeleton, resulting in a loss of intercellular adhesion and cell spreading specifically on matrices that use GalTase as a cell surface receptor. In contrast, overexpressing the analogous truncated short GalTase (TSGT) protein did not affect cell morphology or GalTase activity. In control assays, inducing the TLGT protein had no effect on cell interactions with fibronectin (which is independent of GalTase), or on the cytoskeleton attachment of another matrix receptor (beta 1 integrin), or on overall glycoprotein synthesis, thus eliminating nonspecific effects of the TLGT protein on cellular adhesion and metabolism. These results represent the first molecular manipulation of cell surface GalTase expression and confirm its function as a cell adhesion molecule. These studies further suggest that the cytoskeleton contains a defined, saturable number of binding sites for GalTase, which enables it to function as

  11. Surface modified stainless steels for PEM fuel cell bipolar plates

    DOEpatents

    Brady, Michael P [Oak Ridge, TN; Wang, Heli [Littleton, CO; Turner, John A [Littleton, CO

    2007-07-24

    A nitridation treated stainless steel article (such as a bipolar plate for a proton exchange membrane fuel cell) having lower interfacial contact electrical resistance and better corrosion resistance than an untreated stainless steel article is disclosed. The treated stainless steel article has a surface layer including nitrogen-modified chromium-base oxide and precipitates of chromium nitride formed during nitridation wherein oxygen is present in the surface layer at a greater concentration than nitrogen. The surface layer may further include precipitates of titanium nitride and/or aluminum oxide. The surface layer in the treated article is chemically heterogeneous surface rather than a uniform or semi-uniform surface layer exclusively rich in chromium, titanium or aluminum. The precipitates of titanium nitride and/or aluminum oxide are formed by the nitriding treatment wherein titanium and/or aluminum in the stainless steel are segregated to the surface layer in forms that exhibit a low contact resistance and good corrosion resistance.

  12. The N-linking glycosylation system from Actinobacillus pleuropneumoniae is required for adhesion and has potential use in glycoengineering

    PubMed Central

    Bossé, Janine T.; Abouelhadid, Sherif; Li, Yanwen; Lin, Chia-Wei; Vohra, Prerna; Tucker, Alexander W.; Rycroft, Andrew N.; Maskell, Duncan J.; Aebi, Markus; Langford, Paul R.

    2017-01-01

    Actinobacillus pleuropneumoniae is a mucosal respiratory pathogen causing contagious porcine pleuropneumonia. Pathogenesis studies have demonstrated a major role for the capsule, exotoxins and outer membrane proteins. Actinobacillus pleuropneumoniae can also glycosylate proteins, using a cytoplasmic N-linked glycosylating enzyme designated NGT, but its transcriptional arrangement and role in virulence remains unknown. We investigated the NGT locus and demonstrated that the putative transcriptional unit consists of rimO, ngt and a glycosyltransferase termed agt. From this information we used the A. pleuropneumoniae glycosylation locus to decorate an acceptor protein, within Escherichia coli, with a hexose polymer that reacted with an anti-dextran antibody. Mass spectrometry analysis of a truncated protein revealed that this operon could add up to 29 repeat units to the appropriate sequon. We demonstrated the importance of NGT in virulence, by creating deletion mutants and testing them in a novel respiratory cell line adhesion model. This study demonstrates the importance of the NGT glycosylation system for pathogenesis and its potential biotechnological application for glycoengineering. PMID:28077594

  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. The role of nitric oxide in ocular surface cells.

    PubMed Central

    Kim, Jae Chan; Park, Gun Sic; Kim, Jin Kook; Kim, Young Myeong

    2002-01-01

    The role of nitric oxide (NO) in the ocular surface remains unknown. We investigated the conditions leading to an increase of NO generation in tear and the main sources of NO in ocular surface tissue. We evaluated the dual action (cell survival or cell death) of NO depending on its amount. We measured the concentration of nitrite plus nitrate in the tears of ocular surface diseases and examined the main source of nitric oxide synthase (NOS). When cultured human corneal fibroblast were treated with NO producing donor with or without serum, the viabilities of cells was studied. We found that the main sources of NO in ocular surface tissue were corneal epithelium, fibroblast, endothelium, and inflammatory cells. Three forms of NOS (eNOS, bNOS, and iNOS) were expressed in experimentally induced inflammation. In the fibroblast culture system, the NO donor (SNAP, S-nitroso-N-acetyl-D, L-penicillamine) prevented the death of corneal fibroblast cells caused by serum deprivation in a dose dependent manner up to 500 micrometer SNAP, but a higher dose decreased cell viability. This study suggested that NO might act as a double-edged sword in ocular surface diseases depending on the degree of inflammation related with NO concentration. PMID:12068145

  15. A membrane reservoir at the cell surface: unfolding the plasma membrane to fuel cell shape change.

    PubMed

    Figard, Lauren; Sokac, Anna Marie

    2014-01-01

    Cell surface expansion is a necessary part of cell shape change. One long-standing hypothesis proposes that membrane for this expansion comes from the flattening out of cell surface projections such as microvilli and membrane folds. Correlative EM data of cells undergoing phagocytosis, cytokinesis, and morphogenesis has hinted at the existence of such an unfolding mechanism for decades; but unfolding has only recently been confirmed using live-cell imaging and biophysical approaches. Considering the wide range of cells in which plasma membrane unfolding has now been reported, it likely represents a fundamental mechanism of cell shape change.

  16. Effect of hydroxyapatite surface morphology on cell adhesion.

    PubMed

    Iwamoto, Takashi; Hieda, Yohki; Kogai, Yasumichi

    2016-12-01

    We obtained hydroxyapatite (HAp) materials as a block by mixing HAp nanoparticles and polymer, and then calcining the mixtures. The surface morphology of the HAp materials was tuned by varying heat treatment conditions. After calcining the mixtures at 1200 or 800°C for 4h, the surface morphology of the HAp materials was flat or convexo-concave, respectively. The flat surface morphology, which showed micrometer-ordered grain boundaries, was formed by the aggregation of HAp nanoparticles. On the other hand, the convexo-concave surface morphology resulted from the agglomeration of HAp nanoparticles after heat treatment at 800°C for 4h with nanometer-ordered particle size. We tested cell adhesion to HAp materials with flat or convexo-concave surface morphology and found that cells adhered well to the flat HAp materials but not to the convexo-concave HAp materials. This technique for selectively preparing HAp materials with flat or convexo-concave surface morphology was very easy because we merely mixed commercial HAp nanoparticles with polymer and then calcined the mixtures. As a result, the heat treatment temperature affected the surface morphology of our HAp materials, and their surface morphologies contributed to cell adhesion independently of other material properties.

  17. Surface modification of closed plastic bags for adherent cell cultivation

    NASA Astrophysics Data System (ADS)

    Lachmann, K.; Dohse, A.; Thomas, M.; Pohl, S.; Meyring, W.; Dittmar, K. E. J.; Lindenmeier, W.; Klages, C.-P.

    2011-07-01

    In modern medicine human mesenchymal stem cells are becoming increasingly important. However, a successful cultivation of this type of cells is only possible under very specific conditions. Of great importance, for instance, are the absence of contaminants such as foreign microbiological organisms, i.e., sterility, and the chemical functionalization of the ground on which the cells are grown. As cultivation of these cells makes high demands, a new procedure for cell cultivation has been developed in which closed plastic bags are used. For adherent cell growth chemical functional groups have to be introduced on the inner surface of the plastic bag. This can be achieved by a new, atmospheric-pressure plasma-based method presented in this paper. The method which was developed jointly by the Fraunhofer IST and the Helmholtz HZI can be implemented in automated equipment as is also shown in this contribution. Plasma process gases used include helium or helium-based gas mixtures (He + N2 + H2) and vapors of suitable film-forming agents or precursors such as APTMS, DACH, and TMOS in helium. The effect of plasma treatment is investigated by FTIR-ATR spectroscopy as well as surface tension determination based on contact angle measurements and XPS. Plasma treatment in nominally pure helium increases the surface tension of the polymer foil due to the presence of oxygen traces in the gas and oxygen diffusing through the gas-permeable foil, respectively, reacting with surface radical centers formed during contact with the discharge. Primary amino groups are obtained on the inner surface by treatment in mixtures with nitrogen and hydrogen albeit their amount is comparably small due to diffusion of oxygen through the gas-permeable bag, interfering with the plasma-amination process. Surface modifications introducing amino groups on the inner surface turned out to be most efficient in the promotion of cell growth.

  18. Regulation of the protein glycosylation pathway in yeast: structural control of N-linked oligosaccharide elongation

    SciTech Connect

    Gopal, P.K.; Ballou, C.E.

    1987-12-01

    The yeast Saccharomyces cerevisiae X2180 strain with the mnn1 mnn2 mnn9 mutations, all of which affect mannoprotein glycosylation, synthesizes N-linked oligosaccharides. Membrane fractions from the mnn1 mnn2 and mnn1 mnn2 mnn9 mutants are equally effective in catalyzing transfer from GDP-(/sup 3/H)mannose to add mannose in both ..cap alpha..1 ..-->.. 2 and ..cap alpha..1 ..-->.. 6 linkages to an oligosaccharide. Neither membrane preparation can utilize the homologous mnn1 mnn2 mnn9 oligosaccharide as an acceptor. Thus, addition of the ..cap alpha..1 ..-->.. 2-linked mannose side chain to the terminal ..cap alpha..1 ..-->.. 6-linked mannose in oligosaccharides of the mnn9 mutant inhibits the elongation reaction and may serve as an important structural control of mannoprotein glycosylation. The mnn9 mutation also increases the transit time for invertase secretion, meaning that this mutation could affect the processing machinery in the Golgi apparatus.

  19. Characterization of the O- and N-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni

    SciTech Connect

    Nyame, A.K.

    1987-01-01

    The structures of the O- and N-linked oligosaccharides in glycoproteins synthesized by larval and adult schistosomes of Schistosoma mansoni have been investigated. Mechanically transformed schistosomula or adult schistosomes were incubated in media containing either (/sup 3/H)mannose, (/sup 3/H)glucosamine or (/sup 3/H)galactose for 48 and 24 hr, respectively, to radiolabel metabolically the oligosaccharide moieties of newly synthesized glycoproteins. Analyses of the radiolabeled glycoproteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) and fluorography demonstrated that numerous glycoproteins from 48-hr old schistosomula and adult schistosomes were labeled by both the (/sup 3/H)mannose and (/sup 3/H)glucosamine precursors. The (/sup 3/H)galactose precursor was incorporated into numerous glycoproteins in adult schistosomes; however, few, if any, glycoproteins in schistosomula were labeled by this radioactive sugar precursor.

  20. Engineered microtopographies and surface chemistries direct cell attachment and function

    NASA Astrophysics Data System (ADS)

    Magin, Chelsea Marie

    Harrison, in 1914, first recognized that cells respond to physicochemical cues such as substratum topography when he observed that fibroblasts elongated while cultured on spider silk. Recently, techniques developed in the micro-electronics industry have been used to create molds for producing microscaled topographies with various shapes and spatial arrangements. Although these patterning techniques are well-established, very little is known about the mechanisms underlying cell sensing and response to microtopographies. In this work cellular micro-environments with varying surface topographies and chemistries were evaluated with marine organisms and mammalian cells to investigate cellular sensing and response. Biofouling---the accumulation of micro-organisms, plants, and animals on submerged surfaces---is an environmental and economic concern. Engineered topographies, replicated in polydimethylsiloxane elastomer (PDMSe) and functionalized poly(ethylene glycol)-dimethacrylate (PEGDMA) hydrogels, were evaluated for inhibition of marine fouling organism attachment. Microtopographies replicated in PDMSe inhibited attachment of the marine bacterium, Cobetia marina up to 99% versus smooth. The average normalized attachment densities of cells of C. marina and zoospores of the green algae Ulva on PDMSe topographies scaled inversely with the Engineered Roughness Index (ERIII), a representation of surface energy. Attachment densities of Ulva from four assays and C. marina from two growth phases to PDMSe surfaces scaled inversely with one equation: ERI II multiplied by the Reynolds number of the organism (Re) (R 2 = 0.77). The same microtopographies created in PDMSe reduced the initial attachment density and attachment strength of cells of the diatoms Navicula incerta and Seminavis robusta compared to smooth PDMSe. The average normalized attachment density of Navicula after exposure to shear stress (48 Pa) was correlated with the contact area between the diatom and a

  1. Roles for E-cadherin cell surface regulation in cancer

    PubMed Central

    Petrova, Yuliya I.; Schecterson, Leslayann; Gumbiner, Barry M.

    2016-01-01

    The loss of E-cadherin expression in association with the epithelial–mesenchymal transition (EMT) occurs frequently during tumor metastasis. However, metastases often retain E-cadherin expression, an EMT is not required for metastasis, and metastases can arise from clusters of tumor cells. We demonstrate that the regulation of the adhesive activity of E-cadherin present at the cell surface by an inside-out signaling mechanism is important in cancer. First, we find that the metastasis of an E-cadherin–expressing mammary cell line from the mammary gland to the lung depends on reduced E-cadherin adhesive function. An activating monoclonal antibody to E-cadherin that induces a high adhesive state significantly reduced the number of cells metastasized to the lung without affecting the growth in size of the primary tumor in the mammary gland. Second, we find that many cancer-associated germline missense mutations in the E-cadherin gene in patients with hereditary diffuse gastric cancer selectively affect the mechanism of inside-out cell surface regulation without inhibiting basic E-cadherin adhesion function. This suggests that genetic deficits in E-cadherin cell surface regulation contribute to cancer progression. Analysis of these mutations also provides insights into the molecular mechanisms underlying cadherin regulation at the cell surface. PMID:27582386

  2. Development of exosome surface display technology in living human cells.

    PubMed

    Stickney, Zachary; Losacco, Joseph; McDevitt, Sophie; Zhang, Zhiwen; Lu, Biao

    2016-03-25

    Surface display technology is an emerging key player in presenting functional proteins for targeted drug delivery and therapy. Although a number of technologies exist, a desirable mammalian surface display system is lacking. Exosomes are extracellular vesicles that facilitate cell-cell communication and can be engineered as nano-shuttles for cell-specific delivery. In this study, we report the development of a novel exosome surface display technology by exploiting mammalian cell secreted nano-vesicles and their trans-membrane protein tetraspanins. By constructing a set of fluorescent reporters for both the inner and outer surface display on exosomes at two selected sites of tetraspanins, we demonstrated the successful exosomal display via gene transfection and monitoring fluorescence in vivo. We subsequently validated our system by demonstrating the expected intracellular partitioning of reporter protein into sub-cellular compartments and secretion of exosomes from human HEK293 cells. Lastly, we established the stable engineered cells to harness the ability of this robust system for continuous production, secretion, and uptake of displayed exosomes with minimal impact on human cell biology. In sum, our work paved the way for potential applications of exosome, including exosome tracking and imaging, targeted drug delivery, as well as exosome-mediated vaccine and therapy.

  3. Targeting Prostate Cancer Stemlike Cells through Cell Surface-Expressed GRP78

    DTIC Science & Technology

    2014-10-01

    inhibit sphere growth of GRP78-sorted prostate cancer cells. 15. SUBJECT TERMS prostate cancer, cell surface GRP78, cancer stem cell, Snail -1 16...domains of cell surface GRP78 on Akt/GSK-3/ Snail -1 signaling. RESULTS/DISCUSSION: We are delayed in performing the experiments because of our delay in...addressing Task 6 (originally scheduled for year 1, moved to year 2; see above). Task 7: OBJECTIVE: Using Snail -1 shRNAs, assess the

  4. Subcellular localization of glycosidases and glycosyltransferases involved in the processing of N-linked oligosaccharides

    SciTech Connect

    Sturm, A.; Johnson, K.D.; Szumilo, T.; Elbein, A.D.; Chrispeels, M.J.

    1987-11-01

    Using isopycnic sucrose gradients, we have ascertained the subcellular location of several enzymes involved in the processing of the N-linked oligosaccharides of glycoproteins in developing cotyledons of the common bean, Phaseolus vulgaris. All are localized in the endoplasmic reticulum (ER) or Golgi complex as determined by co-sedimentation with the ER marker, NADH-cytochrome c reductase, or the Golgi marker, glucan synthase I. Glucosidase activity, which removes glucose residues from Glc/sub 3/Man/sub 9/(GlcNAc)/sub 2/, was found exclusively in the ER. All other processing enzymes, which act subsequent to the glucose trimming steps, are associates with Golgi. These include mannosidase I (removes 1-2 mannose residues from Man/sub 6-9/(GlcNAc)/sub 2/), mannosidase II (removes mannose residues from GlcNAcMan/sub 5/(GlcNAc)/sub 2/), and fucosyltransferase (transfers a fucose residue to the Asn-linked GlcNAc of appropriate glycans). The authors have previously reported the localization of two other glycan modifying enzymes (GlcNAc-transferase and xylosyltranferase activities) in the Golgi complex. Attempts at subfractionation of the Golgi fraction on shallow sucrose gradients yielded similar patterns of distribution for all the Golgi processing enzymes. Subfractionation on Percoll gradients resulted in two peaks of the Golgi marker enzyme inosine diphosphatase, whereas the glycan processing enzymes were all enriched in the peak of lower density. These results do not lend support to the hypothesis that N-linked oligosaccharide processing enzymes are associated with Golgi cisternae of different densities.

  5. Analysis of carbohydrate-protein interactions with synthetic N-linked neoglycoconjugate probes.

    PubMed Central

    Wong, S Y; Manger, I D; Guile, G R; Rademacher, T W; Dwek, R A

    1993-01-01

    Recently we have describe a simple efficient chemical method of generating an asparagine side-chain linker with beta-stereochemistry at the anomeric position of neutral oligosaccharides. We now report the 1-N-glycyl beta-derivatization of sialylated saccharides. Several neoglycoconjugates formed using these N-linked inter-mediates were investigated for their usefulness in probing carbohydrate-protein interactions. First, biotinyl derivatives of two xylose/fucose class plant-type oligosaccharides purified from horseradish peroxidase were effective in demonstrating the carbohydrate specificity of polyclonal anti-(horseradish peroxidase) antibodies. Secondly, a fluorescein-labelled asialo- and digalactosylated biantennary complex sugar was synthesized and shown to bind to a Ricinus communis agglutinin column. This galactose-specific recognition was abolished by treating this fluorescein-labelled oligosaccharide with jack bean beta-galactosidase. Finally, two 1-N-glycyl beta-saccharide derivatives were modified with thiophosgene to form their corresponding isothiocyanate derivatives. Coupling of these isothiocyanate derivatives of sugars to BSA, amino-derivatized polystyrene plates and glass-fibre discs resulted in multiple sugar presentation. The binding of an anti-N-acetylglucosamine monoclonal antibody to N,N'-diacetylchitobiose residues presented on BSA and solid supports was shown by e.l.i.s.a. Similarly the binding of concanavalin A to asialo-, agalactosylated biantennary complex oligosaccharide residues attached to BSA was demonstrated by a competitive e.l.i.s.a. Our results demonstrate that N-linked neoglycoconjugates could be made readily available and they are valuable tools for the detailed analyses of carbohydrates and carbohydrate-binding proteins. Images Figure 2 Figure 6 Figure 9 PMID:7506528

  6. 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.

  7. New Monoclonal Antibodies to Defined Cell Surface Proteins on Human Pluripotent Stem Cells.

    PubMed

    O'Brien, Carmel M; Chy, Hun S; Zhou, Qi; Blumenfeld, Shiri; Lambshead, Jack W; Liu, Xiaodong; Kie, Joshua; Capaldo, Bianca D; Chung, Tung-Liang; Adams, Timothy E; Phan, Tram; Bentley, John D; McKinstry, William J; Oliva, Karen; McMurrick, Paul J; Wang, Yu-Chieh; Rossello, Fernando J; Lindeman, Geoffrey J; Chen, Di; Jarde, Thierry; Clark, Amander T; Abud, Helen E; Visvader, Jane E; Nefzger, Christian M; Polo, Jose M; Loring, Jeanne F; Laslett, Andrew L

    2017-03-01

    The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types. Stem Cells 2017;35:626-640.

  8. Hydrodynamics of Sperm Cells near Surfaces

    PubMed Central

    Elgeti, Jens; Kaupp, U. Benjamin; Gompper, Gerhard

    2010-01-01

    Sperm are propelled by an actively beating tail, and display a wide variety of swimming patterns. When confined between two parallel walls, sperm swim either in circles or on curvilinear trajectories close to the walls. We employ mesoscale hydrodynamics simulations in combination with a mechanical sperm model to study the swimming behavior near walls. The simulations show that sperm become captured at the wall due to the hydrodynamic flow fields which are generated by the flagellar beat. The circular trajectories are determined by the chiral asymmetry of the sperm shape. For strong (weak) chirality, sperm swim in tight (wide) circles, with the beating plane of the flagellum oriented perpendicular (parallel) to the wall. For comparison, we also perform simulations based on a local anisotropic friction of the flagellum. In this resistive force approximation, surface adhesion and circular swimming patterns are obtained as well. However, the adhesion mechanism is now due to steric repulsion, and the orientation of the beating plane is different. Our model provides a theoretical framework that explains several distinct swimming behaviors of sperm near and far from a wall. Moreover, the model suggests a mechanism by which sperm navigate in a chemical gradient via a change of their shape. PMID:20712984

  9. Adhesion of platelets to artificial surfaces: effect of red cells.

    PubMed

    Brash, J L; Brophy, J M; Feuerstein, I A

    1976-05-01

    Adhesion of platelets to several polymer- and protein-coated glass surfaces has been studied in vitro. The apparatus consists of a cylindrical probe rotating in a test tube containing the platelet medium and allows close control of fluid shear and mass transport. Suspensions of washed pig platelets constitute the basic platelet medium, and can be modified by adding back red cells and plasma proteins. Adhesion is measured via 51Cr-labeling of platelets. In the absence of red cells, identical low levels of adhesion were seen on all surfaces and saturation was reached within 2 min. In the presence of red cells, adhesion was greater. Saturation on all surfaces except fibrinogen and collagen again occurred within 2 min. The adhesion levels on polymer surfaces and glass were indistinguishable, while those on albumin were lower and those on fibrinogen were higher. Collagen was the most reactive surface. It did not equilibrate within 15 min., and kinetic data indicated a platelet diffusivity strongly dependent on hematocrit. These effects were attributed to rotational and translational motion of the red cells causing increased diffusion and surface-platelet collision energy.

  10. Cell patterning on polylactic acid through surface-tethered oligonucleotides.

    PubMed

    Matsui, Toshiki; Arima, Yusuke; Takemoto, Naohiro; Iwata, Hiroo

    2015-02-01

    Polylactic acid (PLA) is a candidate material to prepare scaffolds for 3-D tissue regeneration. However, cells do not adhere or proliferate well on the surface of PLA because it is hydrophobic. We report a simple and rapid method for inducing cell adhesion to PLA through DNA hybridization. Single-stranded DNA (ssDNA) conjugated to poly(ethylene glycol) (PEG) and to a terminal phospholipid (ssDNA-PEG-lipid) was used for cell surface modification. Through DNA hybridization, modified cells were able to attach to PLA surfaces modified with complementary sequence (ssDNA'). Different cell types can be attached to PLA fibers and films in a spatially controlled manner by using ssDNAs with different sequences. In addition, they proliferate well in a culture medium supplemented with fetal bovine serum. The coexisting modes of cell adhesion through DNA hybridization and natural cytoskeletal adhesion machinery revealed no serious effects on cell growth. The combination of a 3-D scaffold made of PLA and cell immobilization on the PLA scaffold through DNA hybridization will be useful for the preparation of 3-D tissue and organs.

  11. Efficient synthesis of spacer-N-linked double-headed glycosides carrying N-acetylglucosamine and N,N'-diacetylchitobiose and their cross-linking activities with wheat germ agglutinin.

    PubMed

    Misawa, Yoshinori; Masaka, Ryuichi; Maeda, Kayo; Yano, Megumi; Murata, Takeomi; Kawagishi, Hirokazu; Usui, Taichi

    2008-02-25

    We describe here an efficient synthetic route to spacer-N-linked double-headed glycosides via a simple two-step procedure. N-Acetylglucosamine (GlcNAc) and N,N'-diacetylchitobiose [(GlcNAc)(2)] were treated with ammonia and the resulting N-beta-glycosylamines were coupled to a series of dicarboxylic acids. Condensation with each dicarboxylic acid proceeded stereoselectively to give the corresponding beta-N-linked double-headed glycoside without the need for any protection/deprotection steps. Interaction of the resulting N-linked double-headed glycosides with wheat germ agglutinin (WGA) were then investigated using a precipitation assay and an optical biosensor based on surface plasmon resonance (SPR). Spacer-N-linked double-headed glycosides bearing GlcNAc and (GlcNAc)(2) were found to be capable of binding and precipitating WGA as divalent ligands. However, the length of the spacer groups between the two terminal sugar residues was found to greatly influence the cross-linking activities with the lectin.

  12. Quantum Efficiency Loss after PID Stress: Wavelength Dependence on Cell Surface and Cell Edge

    SciTech Connect

    Oh, Jaewon; Bowden, Stuart; TamizhMani, GovindaSamy; Hacke, Peter

    2015-06-14

    It is known that the potential induced degradation (PID) stress of conventional p-base solar cells affects power, shunt resistance, junction recombination, and quantum efficiency (QE). One of the primary solutions to address the PID issue is a modification of chemical and physical properties of antireflection coating (ARC) on the cell surface. Depending on the edge isolation method used during cell processing, the ARC layer near the edges may be uniformly or non-uniformly damaged. Therefore, the pathway for sodium migration from glass to the cell junction could be either through all of the ARC surface if surface and edge ARC have low quality or through the cell edge if surface ARC has high quality but edge ARC is defective due to certain edge isolation process. In this study, two PID susceptible cells from two different manufacturers have been investigated. The QE measurements of these cells before and after PID stress were performed at both surface and edge. We observed the wavelength dependent QE loss only in the first manufacturer's cell but not in the second manufacturer's cell. The first manufacturer's cell appeared to have low quality ARC whereas the second manufacturer's cell appeared to have high quality ARC with defective edge. To rapidly screen a large number of cells for PID stress testing, a new but simple test setup that does not require laminated cell coupon has been developed and is used in this investigation.

  13. Effects of surface viscoelasticity on cellular responses of endothelial cells

    PubMed Central

    Hosseini, Motahare-Sadat; Katbab, Ali Asghar

    2014-01-01

    Background: One area of nanoscience deals with nanoscopic interactions between nanostructured materials and biological systems. To elucidate the effects of the substrate surface morphology and viscoelasticity on cell proliferation, fractal analysis was performed on endothelial cells cultured on nanocomposite samples based on silicone rubber (SR) and various concentrations of organomodified nanoclay (OC). Methods: The nanoclay/SR ratio was tailored to enhance cell behavior via changes in sample substrate surface roughness and viscoelasticity. Results: Surface roughness of the cured SR filled with negatively-charged nanosilicate layers had a greater effect than elasticity on cell growth. The surface roughness of SR nanocomposite samples increased with increasing the OC content, leading to enhanced cell growth and extracellular matrix (ECM) remodeling. This was consistent with the decrease in SR segmental motions and damping factor as the primary viscoelastic parameters by the nanosilicate layers with increasing clay concentrations. Conclusions: The inclusion of clay nanolayers affected the growth and behavior of endothelial cells on microtextured SR. PMID:26989733

  14. Biological surface engineering: a simple system for cell pattern formation.

    PubMed

    Zhang, S; Yan, L; Altman, M; Lässle, M; Nugent, H; Frankel, F; Lauffenburger, D A; Whitesides, G M; Rich, A

    1999-07-01

    Biological surface engineering using synthetic biological materials has a great potential for advances in our understanding of complex biological phenomena. We developed a simple system to engineer biologically relevant surfaces using a combination of self-assembling oligopeptide monolayers and microcontact printing (muCP). We designed and synthesized two oligopeptides containing a cell adhesion motif (RADS)n (n = 2 and 3) at the N-terminus, followed by an oligo(alanine) linker and a cysteine residue at the C-terminus. The thiol group of cysteine allows the oligopeptides to attach covalently onto a gold-coated surface to form monolayers. We then microfabricated a variety of surface patterns using the cell adhesion peptides in combination with hexa-ethylene glycol thiolate which resist non-specific adsorption of proteins and cells. The resulting patterns consist of areas either supporting or inhibiting cell adhesion, thus they are capable of aligning cells in a well-defined manner, leading to specific cell array and pattern formations.

  15. Origin of subdiffusion of water molecules on cell membrane surfaces

    PubMed Central

    Yamamoto, Eiji; Akimoto, Takuma; Yasui, Masato; Yasuoka, Kenji

    2014-01-01

    Water molecules play an important role in providing unique environments for biological reactions on cell membranes. It is widely believed that water molecules form bridges that connect lipid molecules and stabilize cell membranes. Using all-atom molecular dynamics simulations, we show that translational and rotational diffusion of water molecules on lipid membrane surfaces exhibit subdiffusion and aging. Moreover, we provide evidence that both divergent mean trapping time (continuous-time random walk) and long-correlated noise (fractional Brownian motion) contribute to this subdiffusion. These results suggest that subdiffusion on cell membranes causes the water retardation, an enhancement of cell membrane stability, and a higher reaction efficiency. PMID:24739933

  16. Regulation of Cell Surface CB2 Receptor during Human B Cell Activation and Differentiation.

    PubMed

    Castaneda, Julie T; Harui, Airi; Roth, Michael D

    2017-03-31

    Cannabinoid receptor type 2 (CB2) is the primary receptor pathway mediating the immunologic consequences of cannabinoids. We recently reported that human peripheral blood B cells express CB2 on both the extracellular membrane and at intracellular sites, where-as monocytes and T cells only express intracellular CB2. To better understand the pattern of CB2 expression by human B cells, we examined CD20(+) B cells from three tissue sources. Both surface and intracellular expression were present and uniform in cord blood B cells, where all cells exhibited a naïve mature phenotype (IgD(+)/CD38(Dim)). While naïve mature and quiescent memory B cells (IgD(-)/CD38(-)) from tonsils and peripheral blood exhibited a similar pattern, tonsillar activated B cells (IgD(-)/CD38(+)) expressed little to no surface CB2. We hypothesized that regulation of the surface CB2 receptor may occur during B cell activation. Consistent with this, a B cell lymphoma cell line known to exhibit an activated phenotype (SUDHL-4) was found to lack cell surface CB2 but express intracellular CB2. Furthermore, in vitro activation of human cord blood resulted in a down-regulation of surface CB2 on those B cells acquiring the activated phenotype but not on those retaining IgD expression. Using a CB2 expressing cell line (293 T/CB2-GFP), confocal microscopy confirmed the presence of both cell surface expression and multifocal intracellular expression, the latter of which co-localized with endoplasmic reticulum but not with mitochondria, lysosomes, or nucleus. Our findings suggest a dynamic multi-compartment expression pattern for CB2 in B cells that is specifically modulated during the course of B cell activation.

  17. 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.

  18. Surface-Enhanced Raman Scattering Nanoparticles as Optical Labels for Imaging Cell Surface Proteins

    NASA Astrophysics Data System (ADS)

    MacLaughlin, Christina M.

    Assaying the expression of cell surface proteins has widespread application for characterizing cell type, developmental stage, and monitoring disease transformation. Immunophenotyping is conducted by treating cells with labelled targeting moieties that have high affinity for relevant surface protein(s). The sensitivity and specificity of immunophenotyping is defined by the choice of contrast agent and therefore, the number of resolvable signals that can be used to simultaneously label cells. Narrow band width surface-enhanced Raman scattering (SERS) nanoparticles are proposed as optical labels for multiplexed immunophenotying. Two types of surface coatings were investigated to passivate the gold nanoparticles, incorporate SERS functionality, and to facilitate attachment of targeting antibodies. Thiolated poly(ethylene glycol) forms dative bonds with the gold surface and is compatible with multiple physisorbed Raman-active reporter molecules. Ternary lipid bilayers are used to encapsulate the gold nanoparticles particles, and incorporate three different classes of Raman reporters. TEM, UV-Visible absorbance spectroscopy, DLS, and electrophoretic light scattering were used characterize the particle coating. Colourimetric protein assay, and secondary antibody labelling were used to quantify the antibody conjugation. Three different in vitromodels were used to investigate the binding efficacy and specificity of SERS labels for their biomarker targets. Primary human CLL cells, LY10 B lymphoma, and A549 adenocarcinoma lines were targeted. Dark field imaging was used to visualize the colocalization of SERS labels with cells, and evidence of receptor clustering was obtained based on colour shifts of the particles' Rayleigh scattering. Widefield, and spatially-resolved Raman spectra were used to detect labels singly, and in combination from labelled cells. Fluorescence flow cytometry was used to test the particles' binding specificity, and SERS from labelled cells was also

  19. 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

  20. Surface free energy activated high-throughput cell sorting.

    PubMed

    Zhang, Xinru; Zhang, Qian; Yan, Tao; Jiang, Zeyi; Zhang, Xinxin; Zuo, Yi Y

    2014-09-16

    Cell sorting is an important screening process in microbiology, biotechnology, and clinical research. Existing methods are mainly based on single-cell analysis as in flow cytometric and microfluidic cell sorters. Here we report a label-free bulk method for sorting cells by differentiating their characteristic surface free energies (SFEs). We demonstrated the feasibility of this method by sorting model binary cell mixtures of various bacterial species, including Pseudomonas putida KT2440, Enterococcus faecalis ATCC 29212, Salmonella Typhimurium ATCC 14028, and Escherichia coli DH5α. This method can effectively separate 10(10) bacterial cells within 30 min. Individual bacterial species can be sorted with up to 96% efficiency, and the cell viability ratio can be as high as 99%. In addition to its capacity of sorting evenly mixed bacterial cells, we demonstrated the feasibility of this method in selecting and enriching cells of minor populations in the mixture (presenting at only 1% in quantity) to a purity as high as 99%. This SFE-activated method may be used as a stand-alone method for quickly sorting a large quantity of bacterial cells or as a prescreening tool for microbial discrimination. Given its advantages of label-free, high-throughput, low cost, and simplicity, this SFE-activated cell sorting method has potential in various applications of sorting cells and abiotic particles.

  1. Leukocyte cell surface proteinases: regulation of expression, functions, and mechanisms of surface localization.

    PubMed

    Owen, Caroline A

    2008-01-01

    A number of proteinases are expressed on the surface of leukocytes including members of the serine, metallo-, and cysteine proteinase superfamilies. Some proteinases are anchored to the plasma membrane of leukocytes by a transmembrane domain or a glycosyl phosphatidyl inositol (GPI) anchor. Other proteinases bind with high affinity to classical receptors, or with lower affinity to integrins, proteoglycans, or other leukocyte surface molecules. Leukocyte surface levels of proteinases are regulated by: (1) cytokines, chemokines, bacterial products, and growth factors which stimulate synthesis and/or release of proteinases by cells; (2) the availability of surface binding sites for proteinases; and/or (3) internalization or shedding of surface-bound proteinases. The binding of proteinases to leukocyte surfaces serves many functions including: (1) concentrating the activity of proteinases to the immediate pericellular environment; (2) facilitating pro-enzyme activation; (3) increasing proteinase stability and retention in the extracellular space; (4) regulating leukocyte function by proteinases signaling through cell surface binding sites or other surface proteins; and (5) protecting proteinases from inhibition by extracellular proteinase inhibitors. There is strong evidence that membrane-associated proteinases on leukocytes play critical roles in wound healing, inflammation, extracellular matrix remodeling, fibrinolysis, and coagulation. This review will outline the biology of membrane-associated proteinases expressed by leukocytes and their roles in physiologic and pathologic processes.

  2. Role of the N-linked glycans of the prM and E envelope proteins in tick-borne encephalitis virus particle secretion.

    PubMed

    Goto, Akiko; Yoshii, Kentarou; Obara, Mayumi; Ueki, Tomotaka; Mizutani, Tetsuya; Kariwa, Hiroaki; Takashima, Ikuo

    2005-04-27

    The tick-borne encephalitis (TBE) virus has two membrane glycoproteins (prM and E), which each has one N-linked glycan. Constructs that express prM and E proteins of TBE virus have been shown to produce virus-like particles (VLPs), which have surface properties that are similar to those of infectious viruses. To reveal the function of glycosylation of the TBE virus prM and E proteins in the secretion of VLPs, we expressed glycosylation-mutated prM and E proteins and compared the secretion levels and biological properties of the VLPs. In the prM protein glycosylation-deficient mutant, the level of secreted E protein was reduced to 60% of the wild-type level. On the other hand, in the E or prM-E protein glycosylation-deficient mutant, the level of secreted E protein was reduced to 10% of the wild-type level. Furthermore, the mutant which was glycosylated at positions 66 and 154 in protein E, the level of secreted E protein was four-fold higher than that of the wild-type. However, in the mutant which was glycosylated at position 66 only, E protein secretion was reduced to only 10% of the wild-type level. These data suggest that the glycan associated with the N-linked glycosylation site at position 154 in protein E plays an important role in VLP secretion.

  3. 3D Surface Topology Guides Stem Cell Adhesion and Differentiation

    PubMed Central

    Viswanathan, Priyalakshmi; Ondeck, Matthew G.; Chirasatitsin, Somyot; Nghamkham, Kamolchanok; Reilly, Gwendolen C.; Engler, Adam J.; Battaglia, Giuseppe

    2015-01-01

    Polymerized high internal phase emulsion (polyHIPE) foams are extremely versatile materials for investigating cell-substrate interactions in vitro. Foam morphologies can be controlled by polymerization conditions to result in either open or closed pore structures with different levels of connectivity, consequently enabling the comparison between 2D and 3D matrices using the same substrate with identical surface chemistry conditions. Additionally, here we achieve the control of pore surface topology (i.e. how different ligands are clustered together) using amphiphilic block copolymers as emulsion stabilisers. We demonstrate that adhesion of human mesenchymal progenitor (hES-MP) cells cultured on polyHIPE foams is dependent on foam surface topology and chemistry but is independent of porosity and interconnectivity. We also demonstrate that the interconnectivity, architecture and surface topology of the foams has an effect on the osteogenic differentiation potential of hES-MP cells. Together these data demonstrate that the adhesive heterogeneity of a 3D scaffold could regulate not only mesenchymal stem cell attachment but also cell behavior in the absence of soluble growth factors. PMID:25818420

  4. 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.

  5. Dynamic and reversible surface topography influences cell morphology.

    PubMed

    Kiang, Jennifer D; Wen, Jessica H; del Álamo, Juan C; Engler, Adam J

    2013-08-01

    Microscale and nanoscale surface topography changes can influence cell functions, including morphology. Although in vitro responses to static topography are novel, cells in vivo constantly remodel topography. To better understand how cells respond to changes in topography over time, we developed a soft polyacrylamide hydrogel with magnetic nickel microwires randomly oriented in the surface of the material. Varying the magnetic field around the microwires reversibly induced their alignment with the direction of the field, causing the smooth hydrogel surface to develop small wrinkles; changes in surface roughness, ΔRRMS , ranged from 0.05 to 0.70 μm and could be oscillated without hydrogel creep. Vascular smooth muscle cell morphology was assessed when exposed to acute and dynamic topography changes. Area and shape changes occurred when an acute topographical change was imposed for substrates exceeding roughness of 0.2 μm, but longer-term oscillating topography did not produce significant changes in morphology irrespective of wire stiffness. These data imply that cells may be able to use topography changes to transmit signals as they respond immediately to changes in roughness.

  6. Fabrication of cell container arrays with overlaid surface topographies.

    PubMed

    Truckenmüller, Roman; Giselbrecht, Stefan; Escalante-Marun, Maryana; Groenendijk, Max; Papenburg, Bernke; Rivron, Nicolas; Unadkat, Hemant; Saile, Volker; Subramaniam, Vinod; van den Berg, Albert; van Blitterswijk, Clemens; Wessling, Matthias; de Boer, Jan; Stamatialis, Dimitrios

    2012-02-01

    This paper presents cell culture substrates in the form of microcontainer arrays with overlaid surface topographies, and a technology for their fabrication. The new fabrication technology is based on microscale thermoforming of thin polymer films whose surfaces are topographically prepatterned on a micro- or nanoscale. For microthermoforming, we apply a new process on the basis of temporary back moulding of polymer films and use the novel concept of a perforated-sheet-like mould. Thermal micro- or nanoimprinting is applied for prepatterning. The novel cell container arrays are fabricated from polylactic acid (PLA) films. The thin-walled microcontainer structures have the shape of a spherical calotte merging into a hexagonal shape at their upper circumferential edges. In the arrays, the cell containers are arranged densely packed in honeycomb fashion. The inner surfaces of the highly curved container walls are provided with various topographical micro- and nanopatterns. For a first validation of the microcontainer arrays as in vitro cell culture substrates, C2C12 mouse premyoblasts are cultured in containers with microgrooved surfaces and shown to align along the grooves in the three-dimensional film substrates. In future stem-cell-biological and tissue engineering applications, microcontainers fabricated using the proposed technology may act as geometrically defined artificial microenvironments or niches.

  7. Cell surface markers of cancer stem cells: diagnostic macromolecules and targets for drug delivery.

    PubMed

    Andrews, Timothy E; Wang, Dan; Harki, Daniel A

    2013-04-01

    The recognition that the persistence of cancer stem cells (CSCs) in patients following chemotherapy can result in disease relapse underscores the necessity to develop therapeutics against those cells. CSCs display a unique repertoire of cell surface macromolecules, which have proven essential for their characterization and isolation. Additionally, CSC-specific cell surface macromolecules or markers provide targets for the development of specific agents to destroy them. In this review, we compiled those cell surface molecules that have been validated as CSC markers for many common blood and solid tumors. We describe the unique chemical and structural features of the most common cell surface markers, as well as recent efforts to deliver chemotherapeutic agents into CSCs by targeting those macromolecules.

  8. Autonomous Molecular Cascades for Evaluation of Cell Surfaces

    PubMed Central

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

    2013-01-01

    Molecular automata are mixtures of molecules that undergo precisely defined structural changes in response to sequential interactions with inputs1–4. Previously studied nucleic acid-based-automata include game-playing molecular devices (MAYA automata3,5) and finite-state automata for analysis of nucleic acids6 with the latter inspiring circuits for the analysis of RNA species inside cells7,8. Here, we describe automata based on strand-displacement9,10 cascades directed by antibodies that can analyze cells by using their surface markers as inputs. The final output of a molecular automaton that successfully completes its analysis is the presence of a unique molecular tag on the cell surface of a specific subpopulation of lymphocytes within human blood cells. PMID:23892986

  9. Human NK cells: From surface receptors to clinical applications.

    PubMed

    Moretta, Lorenzo; Pietra, Gabriella; Vacca, Paola; Pende, Daniela; Moretta, Francesca; Bertaina, Alice; Mingari, Maria Cristina; Locatelli, Franco; Moretta, Alessandro

    2016-10-01

    Natural killer (NK) cells play a major role in innate defenses against pathogens, primarily viruses, and are also thought to be part of the immunosurveillance against tumors. They express an array of surface receptors that mediate NK cell function. The human leukocytes antigen (HLA) class I-specific inhibitory receptors allow NK cells to detect and kill cells that have lost or under-express HLA class I antigens, a typical feature of tumor or virally infected cells. However, NK cell activation and induction of cytolytic activity and cytokine production depends on another important checkpoint, namely the expression on target cells of ligands recognized by activating NK receptors. Despite their potent cytolytic activity, NK cells frequently fail to eliminate tumors. This is due to mechanisms of tumor escape, determined by the tumor cells themselves or by tumor-associated cells (i.e. the tumor microenvironment) via the release of soluble suppressive factors or the induction of inhibitory loops involving induction of regulatory T cells, M2-polarized macrophages and myeloid-derived suppressor cells. The most important clinical application involving NK cells is the cure of high-risk leukemias in the haplo-identical hematopoietic stem cell transplant (HSCT) setting. NK cells originated from hematopoietic stem cells (HSC) of HLA-haploidentical donors may express Killer Immunoglobulin-like receptors (KIRs) that are mismatched with the HLA class I alleles of the recipient. This allows NK cells to kill leukemia blasts residual after the conditioning regimen, while sparing normal cells (that do not express ligands for activating NK receptors). More recent approaches based on the specific removal of TCR α/β(+) T cells and of CD19(+) B cells, allow the infusion, together with CD34(+) HSC, of mature KIR(+) NK cells and of TCR γ/δ(+) T cells, both characterized by a potent anti-leukemia activity. This greatly reduces the time interval necessary to obtain alloreactive, KIR(+) NK

  10. Acid base properties of cyanobacterial surfaces. II: Silica as a chemical stressor influencing cell surface reactivity

    NASA Astrophysics Data System (ADS)

    Lalonde, S. V.; Smith, D. S.; Owttrim, G. W.; Konhauser, K. O.

    2008-03-01

    Bacteria grow in complex solutions where the adsorption of aqueous species and nucleation of mineral phases on the cell surface may interfere with membrane-dependent homeostatic functions. While previous investigations have provided evidence that bacteria may alter their surface chemical properties in response to environmental stimuli, to our knowledge no effort has been made to evaluate surface compositional changes resulting from non-nutritional chemical stresses within a quantitative framework applicable to surface complexation modeling. We consider here the influence of exposure to silica on cyanobacterial surface chemistry, particularly in light of the propensity for cyanobacteria to become silicified in geothermal environments. Using data modeled from over 50 potentiometric titrations of the unsheathed cyanobacterium Anabaena sp. strain PCC 7120, we find that both abiotic geochemical and biotic biochemical-assimilatory factors have important and different effects on cell surface chemistry. Changes in functional group distribution that resulted from growth by different nitrogen assimilation pathways were greatest in the absence of dissolved silica and less important in its presence. Furthermore, out of the three nitrogen assimilation pathways investigated, in terms of surface functional group distribution, nitrate-reducing cultures were least sensitive, and ammonium-assimilating cultures were most sensitive, to changes in media silica concentration. When functional group distributions were plotted as a function of silica concentration, it appears that, with higher silica concentrations, basic groups (p Ka > 7) increase in concentration relative to acidic groups (p Ka < 7), and the total ligand densities (on a per-weight basis) decreased. The results imply a decrease in both the magnitude and density of surface charge as the net result of growth at high silica concentrations. Thus, Anabaena sp. appears to actively respond to growth in silicifying solutions by

  11. 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.

  12. Entry Kinetics and Cell-Cell Transmission of Surface-Bound Retroviral Vector Particles

    PubMed Central

    O’Neill, Lee S.; Skinner, Amy M.; Woodward, Josha A.; Kurre, Peter

    2010-01-01

    Background Transduction with recombinant Human Immunodeficiency Virus (HIV) -1 derived lentivirus vectors is a multi-step process initiated by surface attachment and subsequent receptor-directed uptake into the target cell. We previously reported the retention of vesicular stomatitis virus G protein (VSV-G) pseudotyped particles on murine progenitor cells and their delayed cell-cell transfer. Methods To examine the underlying mechanism in more detail we used a combination of approaches focused on investigating the role of receptor-independent factors in modulating attachment. Results Studies of synchronized transduction herein reveal cell-type specific rates of vector particle clearance with substantial delays during particle entry into murine hematopoietic progenitor cells. The observed uptake kinetics from the surface of the 1° cell correlate inversely with the magnitude of transfer to 2° targets, corresponding with our initial observation of preferential cell-cell transfer in the context of brief vector exposures. We further demonstrate that vector particle entry into cells is associated with the cell–type specific abundance of extracellular matrix fibronectin. Residual particle – ECM binding and 2° transfer can be competitively disrupted by heparin exposure without affecting murine progenitor homing and repopulation. Conclusions While cellular attachment factors, including fibronectin, aid gene transfer by colocalizing particles to cells and disfavoring early dissociation from targets, they also appear to stabilize particles on the cell surface. Our study highlights the inadvertent consequences for cell entry and cell-cell transfer. PMID:20440757

  13. Expression of cell surface antigens on mast cells: mast cell phenotyping.

    PubMed

    Hauswirth, Alexander W; Florian, Stefan; Schernthaner, Gerit-Holger; Krauth, Maria-Theresa; Sonneck, Karoline; Sperr, Wolfgang R; Valent, Peter

    2006-01-01

    During the past few decades, a number of functionally important cell surface antigens have been detected on human mast cells (MCs). These antigens include the stem cell factor receptor (SCFR/CD117), the high-affinity immunoglobulin E receptor, adhesion molecules, and activation-linked membrane determinants. Several of these antigens (CD2, CD25, CD35, CD88, CD203c) appear to be upregulated on MCs in patients with systemic mastocytosis and therefore are used as diagnostic markers. Quantitative measurement of these markers on MCs is thus of diagnostic value and is usually performed by multicolor-based flow cytometry techniques utilizing a PE- or APC-labeled antibody against CD117 for MCs detection. This chapter gives an overview about the methods of staining of MC in various tissues with special reference to novel diagnostic markers applied in patients with suspected systemic mastocytosis.

  14. Convergent Synthesis of N-Linked Glycopeptides via Aminolysis of ω-Asp p-Nitrophenyl Thioesters in Solution.

    PubMed

    Du, Jing-Jing; Gao, Xiao-Fei; Xin, Ling-Ming; Lei, Ze; Liu, Zheng; Guo, Jun

    2016-10-07

    An efficient N-linked glycosylation reaction between glycosylamines and p-nitrophenyl thioester peptides has been developed. The reaction conditions are mild and compatible with the C-terminal free carboxylic acid group and the unprotected N-linked sialyloligosaccharide. By means of this convergent strategy, a versatile N-glycopeptide fragment containing an N-terminal Thz and a C-terminal thioester was readily prepared, which is available for the synthesis of long glycopeptides and glycoproteins using the protocol of native chemical ligation.

  15. Simplified fabrication of back surface electric field silicon cells and novel characteristics of such cells

    NASA Technical Reports Server (NTRS)

    Mandelkorn, J.; Lamneck, J. H., Jr.

    1972-01-01

    An investigation of the characteristics and behavior of 10 ohm-cm silicon cells having abnormally high open-circuit voltages was made. The cells studied were made by a new, highly simplified, contact fabrication process which creates both a contact and a thin electric field region at the cell back surface without the need for phosphorus layer removal. These cells had open-circuit voltages of about 0.58 V and their performance as a function of thickness, temperature, and 1 MeV electron irradiation is detailed. The study showed that 10 ohm-cm back-surface-field cells can have the high initial efficiencies and desirable temperature behavior of low resistivity cells. Thin back-surface-field cells were made and showed, in addition, much greater radiation damage resistance. A mechanism is proposed to explain the results.

  16. Simplified fabrication of back surface electric field silicon cells and novel characteristics of such cells.

    NASA Technical Reports Server (NTRS)

    Mandelkorn, J.; Lamneck, J. H., Jr.

    1972-01-01

    An investigation of the characteristics and behavior of 10 ohm-cm silicon cells having abnormally high open-circuit voltages was made. The cells studied were made by a new, highly simplified, contact fabrication process which creates both a contact and a thin electric field region at the cell back surface without the need for phosphorus layer removal. These cells had open-circuit voltages of about 0.58 V and their performance as a function of thickness, temperature, and 1 MeV electron irradiation is detailed. The study showed that 10 ohm-cm back-surface-field cells can have the high initial efficiencies and desirable temperature behavior of low resistivity cells. Thin back-surface-field cells were made and showed, in addition, much greater radiation damage resistance. A mechanism is proposed to explain the results.

  17. Distinct glycan-charged phosphodolichol carriers are required for the assembly of the pentasaccharide N-linked to the Haloferax volcanii S-layer glycoprotein.

    PubMed

    Guan, Ziqiang; Naparstek, Shai; Kaminski, Lina; Konrad, Zvia; Eichler, Jerry

    2010-12-01

    In Archaea, dolichol phosphates have been implicated as glycan carriers in the N-glycosylation pathway, much like their eukaryal counterparts. To clarify this relation, highly sensitive liquid chromatography/mass spectrometry was employed to detect and characterize glycan-charged phosphodolichols in the haloarchaeon Haloferax volcanii. It is reported that Hfx. volcanii contains a series of C(55) and C(60) dolichol phosphates presenting saturated isoprene subunits at the α and ω positions and sequentially modified with the first, second, third and methylated fourth sugar subunits comprising the first four subunits of the pentasaccharide N-linked to the S-layer glycoprotein, a reporter of N-glycosylation. Moreover, when this glycan-charged phosphodolichol pool was examined in cells deleted of agl genes encoding glycosyltransferases participating in N-glycosylation and previously assigned roles in adding pentasaccharide residues one to four, the composition of the lipid-linked glycans was perturbed in the identical manner as was S-layer glycoprotein N-glycosylation in these mutants. In contrast, the fifth sugar of the pentasaccharide, identified as mannose in this study, is added to a distinct dolichol phosphate carrier. This represents the first evidence that in Archaea, as in Eukarya, the oligosaccharides N-linked to glycoproteins are sequentially assembled from glycans originating from distinct phosphodolichol carriers.

  18. Surface plasmon resonance imaging of cells and surface-associated fibronectin

    PubMed Central

    Peterson, Alexander W; Halter, Michael; Tona, Alessandro; Bhadriraju, Kiran; Plant, Anne L

    2009-01-01

    Background A critical challenge in cell biology is quantifying the interactions of cells with their extracellular matrix (ECM) environment and the active remodeling by cells of their ECM. Fluorescence microscopy is a commonly employed technique for examining cell-matrix interactions. A label-free imaging method would provide an alternative that would eliminate the requirement of transfected cells and modified biological molecules, and if collected nondestructively, would allow long term observation and analysis of live cells. Results Using surface plasmon resonance imaging (SPRI), the deposition of protein by vascular smooth muscle cells (vSMC) cultured on fibronectin was quantified as a function of cell density and distance from the cell periphery. We observed that as much as 120 ng/cm2 of protein was deposited by cells in 24 h. Conclusion SPRI is a real-time, low-light-level, label-free imaging technique that allows the simultaneous observation and quantification of protein layers and cellular features. This technique is compatible with live cells such that it is possible to monitor cellular modifications to the extracellular matrix in real-time. PMID:19245706

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

    PubMed

    Gerson, D F; Akit, J

    1980-11-04

    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.

  20. 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...

  1. Multijunction Solar Cells Optimized for the Mars Surface Solar Spectrum

    NASA Technical Reports Server (NTRS)

    Edmondson, Kenneth M.; Fetzer, Chris; Karam, Nasser H.; Stella, Paul; Mardesich, Nick; Mueller, Robert

    2007-01-01

    This paper gives an update on the performance of the Mars Exploration Rovers (MER) which have been continually performing for more than 3 years beyond their original 90-day missions. The paper also gives the latest results on the optimization of a multijunction solar cell that is optimized to give more power on the surface of Mars.

  2. Diffusion-limited reactions on the cell surface

    NASA Astrophysics Data System (ADS)

    Gopalakrishnan, Manoj; Tauber, Uwe; Forsten-Williams, Kimberly

    2003-03-01

    Fibroblast growth factors (FGF) stimulates proliferation of many cell types, and are crucial in such processes as eg. wound healing. Cells have specific receptor (R) protein molecules on their surface which bind FGF for this purpose. FGF is also bound by Heparan Sulfate Proteoglycan (HSPG) molecules which are present on the cell surface. In isolation, both these complexes are unstable, with half-life of the order of 10-20 minutes, wheras in intact cells, the half-life of FGF-R complex is nearly 5 hours! To account for this increased stability, it has been proposed that R-FGF complex combines with HSPG via surface diffusion and forms the triad R-FGF-HSPG. We examine the feasibility of this reaction using the well-known Smoluchowski theory and Monte Carlo simulations. Our results support the triad formation theory, and are in qualitative agreement with experimental results. We also discuss the effects of slowing down of surface diffusion of these molecules by such factors as eg. the cytosekeletal network and anchored proteins.

  3. Mapping the N-linked glycosites of rice (Oryza sativa L.) germinating embryos

    PubMed Central

    Hou, Yuxuan; Wang, Yifeng; Qiu, Jiehua; Li, Zhiyong; Tong, Xiaohong; Shi, Zhaomei; Zhu, Jun

    2017-01-01

    Germination is a key event in the angiosperm life cycle. N-glycosylation of proteins is one of the most common post-translational modifications, and has been recognized to be an important regulator of the proteome of the germinating embryo. Here, we report the first N-linked glycosites mapping of rice embryos during germination by using a hydrophilic interaction chromatography (HILIC) glycopeptides enrichment strategy associated with high accuracy mass spectrometry identification. A total of 242 glycosites from 191 unique proteins was discovered. Inspection of the motifs and sequence structures involved suggested that all the glycosites were concentrated within [NxS/T] motif, while 82.3% of them were in a coil structure. N-glycosylation preferentially occurred on proteins with glycoside hydrolase activities, which were significantly enriched in the starch and sucrose metabolism pathway, suggesting that N-glycosylation is involved in embryo germination by regulating carbohydrate metabolism. Notably, protein-protein interaction analysis revealed a network with several Brassinosteroids signaling proteins, including XIAO and other BR-responsive proteins, implying that glycosylation-mediated Brassinosteroids signaling may be a key mechanism regulating rice embryo germination. In summary, this study expanded our knowledge of protein glycosylation in rice, and provided novel insight into the PTM regulation in rice seed germination. PMID:28328971

  4. N-linked glycosylation in Archaea: a structural, functional, and genetic analysis.

    PubMed

    Jarrell, Ken F; Ding, Yan; Meyer, Benjamin H; Albers, Sonja-Verena; Kaminski, Lina; Eichler, Jerry

    2014-06-01

    N-glycosylation of proteins is one of the most prevalent posttranslational modifications in nature. Accordingly, a pathway with shared commonalities is found in all three domains of life. While excellent model systems have been developed for studying N-glycosylation in both Eukarya and Bacteria, an understanding of this process in Archaea was hampered until recently by a lack of effective molecular tools. However, within the last decade, impressive advances in the study of the archaeal version of this important pathway have been made for halophiles, methanogens, and thermoacidophiles, combining glycan structural information obtained by mass spectrometry with bioinformatic, genetic, biochemical, and enzymatic data. These studies reveal both features shared with the eukaryal and bacterial domains and novel archaeon-specific aspects. Unique features of N-glycosylation in Archaea include the presence of unusual dolichol lipid carriers, the use of a variety of linking sugars that connect the glycan to proteins, the presence of novel sugars as glycan constituents, the presence of two very different N-linked glycans attached to the same protein, and the ability to vary the N-glycan composition under different growth conditions. These advances are the focus of this review, with an emphasis on N-glycosylation pathways in Haloferax, Methanococcus, and Sulfolobus.

  5. Diversity in prokaryotic glycosylation: an archaeal-derived N-linked glycan contains legionaminic acid.

    PubMed

    Kandiba, Lina; Aitio, Olli; Helin, Jari; Guan, Ziqiang; Permi, Perttu; Bamford, Dennis H; Eichler, Jerry; Roine, Elina

    2012-05-01

    VP4, the major structural protein of the haloarchaeal pleomorphic virus, HRPV-1, is glycosylated. To define the glycan structure attached to this protein, oligosaccharides released by β-elimination were analysed by mass spectrometry and nuclear magnetic resonance spectroscopy. Such analyses showed that the major VP4-derived glycan is a pentasaccharide comprising glucose, glucuronic acid, mannose, sulphated glucuronic acid and a terminal 5-N-formyl-legionaminic acid residue. This is the first observation of legionaminic acid, a sialic acid-like sugar, in an archaeal-derived glycan structure. The importance of this residue for viral infection was demonstrated upon incubation with N-acetylneuraminic acid, a similar monosaccharide. Such treatment reduced progeny virus production by half 4 h post infection. LC-ESI/MS analysis confirmed the presence of pentasaccharide precursors on two different VP4-derived peptides bearing the N-glycosylation signal, NTT. The same sites modified by the native host, Halorubrum sp. strain PV6, were also recognized by the Haloferax volcanii N-glycosylation apparatus, as determined by LC-ESI/MS of heterologously expressed VP4. Here, however, the N-linked pentasaccharide was the same as shown to decorate the S-layer glycoprotein in this species. Hence, N-glycosylation of the haloarchaeal viral protein, VP4, is host-specific. These results thus present additional examples of archaeal N-glycosylation diversity and show the ability of Archaea to modify heterologously expressed proteins.

  6. N-Linked Glycosylation in Archaea: a Structural, Functional, and Genetic Analysis

    PubMed Central

    Ding, Yan; Meyer, Benjamin H.; Albers, Sonja-Verena; Kaminski, Lina; Eichler, Jerry

    2014-01-01

    SUMMARY N-glycosylation of proteins is one of the most prevalent posttranslational modifications in nature. Accordingly, a pathway with shared commonalities is found in all three domains of life. While excellent model systems have been developed for studying N-glycosylation in both Eukarya and Bacteria, an understanding of this process in Archaea was hampered until recently by a lack of effective molecular tools. However, within the last decade, impressive advances in the study of the archaeal version of this important pathway have been made for halophiles, methanogens, and thermoacidophiles, combining glycan structural information obtained by mass spectrometry with bioinformatic, genetic, biochemical, and enzymatic data. These studies reveal both features shared with the eukaryal and bacterial domains and novel archaeon-specific aspects. Unique features of N-glycosylation in Archaea include the presence of unusual dolichol lipid carriers, the use of a variety of linking sugars that connect the glycan to proteins, the presence of novel sugars as glycan constituents, the presence of two very different N-linked glycans attached to the same protein, and the ability to vary the N-glycan composition under different growth conditions. These advances are the focus of this review, with an emphasis on N-glycosylation pathways in Haloferax, Methanococcus, and Sulfolobus. PMID:24847024

  7. MAGIC-web: a platform for untargeted and targeted N-linked glycoprotein identification.

    PubMed

    Lih, T Mamie; Choong, Wai-Kok; Chen, Chen-Chun; Cheng, Cheng-Wei; Lin, Hsin-Nan; Chen, Ching-Tai; Chang, Hui-Yin; Hsu, Wen-Lian; Sung, Ting-Yi

    2016-07-08

    MAGIC-web is the first web server, to the best of our knowledge, that performs both untargeted and targeted analyses of mass spectrometry-based glycoproteomics data for site-specific N-linked glycoprotein identification. The first two modules, MAGIC and MAGIC+, are designed for untargeted and targeted analysis, respectively. MAGIC is implemented with our previously proposed novel Y1-ion pattern matching method, which adequately detects Y1- and Y0-ion without prior information of proteins and glycans, and then generates in silico MS(2) spectra that serve as input to a database search engine (e.g. Mascot) to search against a large-scale protein sequence database. On top of that, the newly implemented MAGIC+ allows users to determine glycopeptide sequences using their own protein sequence file. The third module, Reports Integrator, provides the service of combining protein identification results from Mascot and glycan-related information from MAGIC-web to generate a complete site-specific protein-glycan summary report. The last module, Glycan Search, is designed for the users who are interested in finding possible glycan structures with specific numbers and types of monosaccharides. The results from MAGIC, MAGIC+ and Reports Integrator can be downloaded via provided links whereas the annotated spectra and glycan structures can be visualized in the browser. MAGIC-web is accessible from http://ms.iis.sinica.edu.tw/MAGIC-web/index.html.

  8. Xylose Migration During Tandem Mass Spectrometry of N-Linked Glycans

    NASA Astrophysics Data System (ADS)

    Hecht, Elizabeth S.; Loziuk, Philip L.; Muddiman, David C.

    2017-01-01

    Understanding the rearrangement of gas-phase ions via tandem mass spectrometry is critical to improving manual and automated interpretation of complex datasets. N-glycan analysis may be carried out under collision induced (CID) or higher energy collision dissociation (HCD), which favors cleavage at the glycosidic bond. However, fucose migration has been observed in tandem MS, leading to the formation of new bonds over four saccharide units away. In the following work, we report the second instance of saccharide migration ever to occur for N-glycans. Using horseradish peroxidase as a standard, the beta-1,2 xylose was observed to migrate from a hexose to a glucosamine residue on the (Xyl)Man3GlcNac2 glycan. This investigation was followed up in a complex N-linked glycan mixture derived from stem differentiating xylem tissue, and the rearranged product ion was observed for 75% of the glycans. Rearrangement was not favored in isomeric glycans with a core or antennae fucose and unobserved in glycans predicted to have a permanent core-fucose modification. As the first empirical observation of this rearrangement, this work warrants dissemination so it may be searched in de novo sequencing glycan workflows.

  9. Mutational and functional analysis of N-linked glycosylation of envelope fusion protein F of Helicoverpa armigera nucleopolyhedrovirus.

    PubMed

    Shen, Shu; Wang, Manli; Li, Xin; Li, Shufen; van Oers, Monique M; Vlak, Just M; Braakman, Ineke; Hu, Zhihong; Deng, Fei; Wang, Hualin

    2016-04-01

    The envelope fusion (F) protein of baculoviruses is a heavily N-glycosylated protein that plays a significant role in the virus infection cycle. N-Linked glycosylation of virus envelope glycoprotein is important for virus envelope glycoprotein folding and its function in general. There are six predicted N-glycosylation sites in the F (HaF) protein of Helicoverpa armigera nucleopolyhedrovirus (HearNPV). The N-glycosylation site located in the F(2) subunit (N104) of HaF has been identified and functionally characterized previously (Long et al., 2007). In this study, the other five potential N-glycosylation sites located in the HaF1 subunit, namely, N293, N361, N526, N571 and N595, were analysed extensively to examine their N-glycosylation and relative importance to the function of HaF. The results showed that four of these five potential glycosylation sites in the F(1) subunit, N293, N361, N526 and N571, were N-glycosylated in F proteins of mature HearNPV budded viruses (BVs) but that N595 was not. In general, the conserved site N526 was critical to the functioning of HaF, as absence of N-glycosylation of N526 reduced the efficiency of HaF folding and trafficking, consequently decreased fusogenicity and modified the subcellular localization of HaF proteins, and thus impaired virus production and infectivity. The absence of N-glycosylation at other individual sites was found to have different effects on the fusogenicity and subcelluar distribution of HaF proteins in HzAM1 cells. In summary, N-glycosylation plays comprehensive roles in HaF function and virus infectivity, which is further discussed.

  10. Comparative proteomics and glycoproteomics reveal increased N-linked glycosylation and relaxed sequon specificity in Campylobacter jejuni NCTC11168 O.

    PubMed

    Scott, Nichollas E; Marzook, N Bishara; Cain, Joel A; Solis, Nestor; Thaysen-Andersen, Morten; Djordjevic, Steven P; Packer, Nicolle H; Larsen, Martin R; Cordwell, Stuart J

    2014-11-07

    Campylobacter jejuni is a major cause of bacterial gastroenteritis. C. jejuni encodes a protein glycosylation (Pgl) locus responsible for the N-glycosylation of membrane-associated proteins. We examined two variants of the genome sequenced strain NCTC11168: O, a representative of the original clinical isolate, and GS, a laboratory-adapted relative of O. Comparative proteomics by iTRAQ and two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS) allowed the confident identification of 1214 proteins (73.9% of the predicted C. jejuni proteome), of which 187 were present at statistically significant altered levels of abundance between variants. Proteins associated with the O variant included adhesins (CadF and FlpA), proteases, capsule biosynthesis, and cell shape determinants as well as six proteins encoded by the Pgl system, including the PglK flippase and PglB oligosaccharyltransferase. Lectin blotting highlighted specific glycoproteins more abundant in NCTC11168 O, whereas others remained unaltered. Hydrophilic interaction liquid chromatography (HILIC) and LC-MS/MS identified 30 completely novel glycosites from 15 proteins. A novel glycopeptide from a 14 kDa membrane protein (Cj0455c) was identified that did not contain the C. jejuni N-linked sequon D/E-X-N-X-S/T (X ≠ Pro) but that instead contained a sequon with leucine at the -2 position. Occupied atypical sequons were also observed in Cj0958c (OxaA; Gln at the -2 position) and Cj0152c (Ala at the +2 position). The relative O and GS abundances of 30 glycopeptides were determined by label-free quantitation, which revealed a >100-fold increase in the atypical glycopeptide from Cj0455c in isolate O. Our data provide further evidence for the importance of the Pgl system in C. jejuni.

  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. Diverse specificity of cellulosome attachment to the bacterial cell surface

    PubMed Central

    Brás, Joana L. A.; Pinheiro, Benedita A.; Cameron, Kate; Cuskin, Fiona; Viegas, Aldino; Najmudin, Shabir; Bule, Pedro; Pires, Virginia M. R.; Romão, Maria João; Bayer, Edward A.; Spencer, Holly L.; Smith, Steven; Gilbert, Harry J.; Alves, Victor D.; Carvalho, Ana Luísa; Fontes, Carlos M. G. A.

    2016-01-01

    During the course of evolution, the cellulosome, one of Nature’s most intricate multi-enzyme complexes, has been continuously fine-tuned to efficiently deconstruct recalcitrant carbohydrates. To facilitate the uptake of released sugars, anaerobic bacteria use highly ordered protein-protein interactions to recruit these nanomachines to the cell surface. Dockerin modules located within a non-catalytic macromolecular scaffold, whose primary role is to assemble cellulosomal enzymatic subunits, bind cohesin modules of cell envelope proteins, thereby anchoring the cellulosome onto the bacterial cell. Here we have elucidated the unique molecular mechanisms used by anaerobic bacteria for cellulosome cellular attachment. The structure and biochemical analysis of five cohesin-dockerin complexes revealed that cell surface dockerins contain two cohesin-binding interfaces, which can present different or identical specificities. In contrast to the current static model, we propose that dockerins utilize multivalent modes of cohesin recognition to recruit cellulosomes to the cell surface, a mechanism that maximises substrate access while facilitating complex assembly. PMID:27924829

  13. 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

  14. Cell-surface markers for colon adenoma and adenocarcinoma.

    PubMed

    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

    2016-04-05

    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.

  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. Science and Art of Cell-Based Ocular Surface Regeneration.

    PubMed

    Singh, Vivek; Shukla, Sachin; Ramachandran, Charanya; Mishra, Dilip Kumar; Katikireddy, Kishore R; Lal, Ikeda; Chauhan, Sunil K; Sangwan, Virender S

    2015-01-01

    The potential cause of blindness worldwide includes diseases of the cornea, ocular surface (limbal stem cell deficiency, allergic conjunctivitis, dry eye diseases), and retinal diseases. The presence of stem cells (limbal stem cells) in the basal region of the limbus makes it an important tool for the ocular regeneration and also in maintaining the transparency of eye by replacing the corneal epithelium continuously. Various surgical modalities have been developed like cultured limbal epithelial transplantation, cultured oral mucosal epithelial transplantation, simple limbal epithelial transplantation, etc., utilizing the cell-based regenerative properties to treat limbal disorder. Cell-based therapies for ocular repair and regeneration comprise a major hope by therapies involving the mesenchymal stem cells, embryonic stem cells, and limbal stem cells for the restoration of vision in individuals whose ocular tissue has been irreversibly damaged by disease or trauma. This review explores critical needs in human disease mainly the ocular problem where cell-based therapeutics is exceptionally well suited and also the use of animal models, various artificial scaffolds, as well as advancement in clinical technique to challenge the current demand to overcome corneal blindness.

  17. Proteomics and glycoproteomics of pluripotent stem-cell surface proteins.

    PubMed

    Sun, Bingyun

    2015-03-01

    Pluripotent stem cells are a unique cell type with promising potential in regenerative and personalized medicine. Yet the difficulty to understand and coax their seemingly stochastic differentiation and spontaneous self-renewal have largely limited their clinical applications. A call has been made by numerous researchers for a better characterization of surface proteins on these cells, in search of biomarkers that can dictate developmental stages and lineage specifications, and can help formulate mechanistic insight of stem-cell fate choices. In the past two decades, proteomics has gained significant recognition in profiling surface proteins at high throughput. This review will summarize the impact of these studies on stem-cell biology, and discuss the used proteomic techniques. A systematic comparison of all the techniques and their results is also attempted here to help reveal pros, cons, and the complementarity of the existing methods. This awareness should assist in selecting suitable strategies for stem-cell related research, and shed light on technical improvements that can be explored in the future.

  18. Sorption of heavy metals by prepared bacterial cell surfaces

    SciTech Connect

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

    1995-10-01

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

  19. Development of a novel mammalian cell surface antibody display platform.

    PubMed

    Zhou, Chen; Jacobsen, Frederick W; Cai, Ling; Chen, Qing; Shen, Weyen David

    2010-01-01

    Antibody display systems have been successfully applied to screen, select and characterize antibody fragments. These systems typically use prokaryotic organisms such as phage and bacteria or lower eukaryotic organisms, such as yeast. These organisms possess either no or different post-translational modification functions from mammalian cells and prefer to display small antibody fragments instead of full-length IgGs. We report here a novel mammalian cell-based antibody display platform that displays full-length functional antibodies on the surface of mammalian cells. Through recombinase-mediated DNA integration, each host cell contains one copy of the gene of interest in the genome. Utilizing a hot-spot integration site, the expression levels of the gene of interest are high and comparable between clones, ensuring a high signal to noise ratio. Coupled with fluorescence-activated cell sorting (FACS) technology, our platform is high throughput and can distinguish antibodies with very high antigen binding affinities directly on the cell surface. Single-round FACS can enrich high affinity antibodies by more than 500 fold. Antibodies with significantly improved neutralizing activity have been identified from a randomly mutagenized library, demonstrating the power of this platform in screening and selecting antibody therapeutics.

  20. N-linked Glycosylation of Classical Swine Fever Virus Strain Brescia Erns Glycoprotein Alters Virulence in Swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Erns is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). We recently reported the influence of glycosylation of E2 in the virulence of CSFV strain Brescia. Here, we studied the effect of Erns N-linked glycosylation pattern on virulence of CSFV strain Brescia in swine. ...

  1. N-Linked Glycosyl Auxiliary-Mediated Native Chemical Ligation on Aspartic Acid: Application towards N-Glycopeptide Synthesis.

    PubMed

    Chai, Hua; Le Mai Hoang, Kim; Vu, Minh Duy; Pasunooti, Kalyan; Liu, Chuan-Fa; Liu, Xue-Wei

    2016-08-22

    A practical approach towards N-glycopeptide synthesis using an auxiliary-mediated dual native chemical ligation (NCL) has been developed. The first NCL connects an N-linked glycosyl auxiliary to the thioester side chain of an N-terminal aspartate oligopeptide. This intermediate undergoes a second NCL with a C-terminal thioester oligopeptide. Mild cleavage provides the desired N-glycopeptide.

  2. Fixation and stabilization of Escherichia coli cells displaying genetically engineered cell surface proteins.

    PubMed

    Freeman, A; Abramov, S; Georgiou, G

    1996-12-05

    A large biotechnological potential is inherent in the display of proteins (e.g., enzymes, single-chain antibodies, on the surface of bacterial cells) (Georgiou et al., 1993). Applications such as immobilized whole-cell biocatalysts or cellular adsorbents require cell fixation to prevent disintegration, stabilization of the anchored protein from leakage, denaturation or proteolysis, and total loss of cell viability, preventing medium and potential product contamination with cells. In this article we describe the adaptation of a simple two-stage chemical crosslinking procedure based on "bi-layer encagement" (Tor et al., 1989) for stabilizing Escherichia coli cells expressing an Lpp-OmpA (46-159)-beta-lactamase fusion that displays beta-lactamase on the cell surface. Bilayer crosslinking and coating the bacteria with a polymeric matrix is accomplished by treating the cells first with either glutaraldehyde or polyglutaraldehyde, followed by secondary crosslinking with polyacrylamide hydrazide. These treatments resulted in a 5- to 25-fold reduction of the thermal inactivation rate constant at 55 degrees C of surface anchored beta-lactamase and completely prevented the deterioration of the cells for at least a week of storage at 4 degrees C. The stabilization procedure developed paves the way to scalable biotechnological applications of E. coli displaying surface anchored proteins as whole-cell biocatalysts and adsorbents.

  3. Surface code—biophysical signals for apoptotic cell clearance

    NASA Astrophysics Data System (ADS)

    Biermann, Mona; Maueröder, Christian; Brauner, Jan M.; Chaurio, Ricardo; Janko, Christina; Herrmann, Martin; Muñoz, Luis E.

    2013-12-01

    Apoptotic cell death and the clearance of dying cells play an important and physiological role in embryonic development and normal tissue turnover. In contrast to necrosis, apoptosis proceeds in an anti-inflammatory manner. It is orchestrated by the timed release and/or exposure of so-called ‘find-me’, ‘eat me’ and ‘tolerate me’ signals. Mononuclear phagocytes are attracted by various ‘find-me’ signals, including proteins, nucleotides, and phospholipids released by the dying cell, whereas the involvement of granulocytes is prevented via ‘stay away’ signals. The exposure of anionic phospholipids like phosphatidylserine (PS) by apoptotic cells on the outer leaflet of the plasma membrane is one of the main ‘eat me’ signals. PS is recognized by a number of innate receptors as well as by soluble bridging molecules on the surface of phagocytes. Importantly, phagocytes are able to discriminate between viable and apoptotic cells both exposing PS. Due to cytoskeleton remodeling PS has a higher lateral mobility on the surfaces of apoptotic cells thereby promoting receptor clustering on the phagocyte. PS not only plays an important role in the engulfment process, but also acts as ‘tolerate me’ signal inducing the release of anti-inflammatory cytokines by phagocytes. An efficient and fast clearance of apoptotic cells is required to prevent secondary necrosis and leakage of intracellular danger signals into the surrounding tissue. Failure or prolongation of the clearance process leads to the release of intracellular antigens into the periphery provoking inflammation and development of systemic inflammatory autoimmune disease like systemic lupus erythematosus. Here we review the current findings concerning apoptosis-inducing pathways, important players of apoptotic cell recognition and clearance as well as the role of membrane remodeling in the engulfment of apoptotic cells by phagocytes.

  4. Surface science studies of model fuel cell electrocatalysts

    NASA Astrophysics Data System (ADS)

    Marković, N. M.; Ross, P. N.

    2002-04-01

    The purpose of this review is to discuss progress in the understanding of electrocatalytic reactions through the study of model systems with surface spectroscopies. Pure metal single crystals and well-characterized bulk alloys have been used quite successfully as models for real (commercial) electrocatalysts. Given the sheer volume of all work in electrocatalysis that is on fuel cell reactions, we will focus on electrocatalysts for fuel cells. Since Pt is the model fuel cell electrocatalyst, we will focus entirely on studies of pure Pt and Pt bimetallic alloys. The electrode reactions discussed include hydrogen oxidation/evolution, oxygen reduction, and the electrooxidation of carbon monoxide, formic acid, and methanol. Surface spectroscopies emphasized are FTIR, STM/AFM and surface X-ray scattering (SXS). The discussion focuses on the relation between the energetics of adsorption of intermediates and the reaction pathway and kinetics, and how the energetics and kinetics relate to the extrinsic properties of the model system, e.g. surface structure and/or composition. Finally, we conclude by discussing the limitations that are reached by using pure metal single crystals and well-characterized bulk alloys as models for real catalysts, and suggest some directions for developing more realistic systems.

  5. Analysis of cell surface alterations in Legionella pneumophila cells treated with human apolipoprotein E.

    PubMed

    Palusinska-Szysz, Marta; Zdybicka-Barabas, Agnieszka; Cytryńska, Małgorzata; Wdowiak-Wróbel, Sylwia; Chmiel, Elżbieta; Gruszecki, Wiesław I

    2015-03-01

    Binding of human apolipoprotein E (apoE) to Legionella pneumophila lipopolysaccharide was analysed at the molecular level by Fourier-transform infrared spectroscopy, thereby providing biophysical evidence for apoE-L. pneumophila lipopolysaccharide interaction. Atomic force microscopy imaging of apoE-exposed L. pneumophila cells revealed alterations in the bacterial cell surface topography and nanomechanical properties in comparison with control bacteria. The changes induced by apoE binding to lipopolysaccharide on the surface of L. pneumophila cells may participate in: (1) impeding the penetration of host cells by the bacteria; (2) suppression of pathogen intracellular growth and eventually; and (3) inhibition of the development of infection.

  6. 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.

  7. Ligand engagement on material surfaces is discriminated by cell mechanosensoring.

    PubMed

    Battista, Edmondo; Causa, Filippo; Lettera, Vincenzo; Panzetta, Valeria; Guarnieri, Daniela; Fusco, Sabato; Gentile, Francesco; Netti, Paolo A

    2015-03-01

    Peptide or protein ligands can be used for molecular decoration to enhance the functionality of synthetic materials. However, some skepticism has arisen about the efficacy of such strategy in practical contexts since serum proteins largely adsorb. To address this issue, it is crucial to ascertain whether a chemically conjugated integrin-binding peptide is fully recognized by a cell even if partially covered by a physisorbed layer of serum protein; in more general terms, if competitive protein fragments physisorbed onto the surface are distinguishable from those chemically anchored to it. Here, we engraft an RGD peptide on poly-ε-caprolactone (PCL) surfaces and follow the dynamics of focal adhesion (FA) and cytoskeleton assembly at different times and culture conditions using a variety of analytical tools. Although the presence of serum protein covers the bioconjugated RGD significantly, after the first adhesion phase cells dig into the physisorbed layer and reach the submerged signal to establish a more stable adhesion structure (mature FAs). Although the spreading area index is not substantially affected by the presence of the RGD peptide, cells attached to chemically bound signals develop a stronger adhesive interaction with the materials and assemble a mechanically stable cytoskeleton. This demonstrates that cells are able to discriminate, via mechanosensoring, between adhesive motives belonging to physisorbed proteins and those firmly anchored on the material surface.

  8. Quantum-radiative cooling for solar cells with textured surface

    NASA Astrophysics Data System (ADS)

    Gilman, Boris; Ivanov, Igor

    2004-11-01

    Efficient technique of Quantum Radiative Cooling (QRC) of textured Solar Cells and Modules is described that is capable of Solar Module (SM) temperature reduction by 5-20C, resulting in 3-10% efficiency increase. Novel methods are based on the quantum assisted IR emission from the surface covered by either multi-layer coatings made of Si-nitride, SiO or Si oxy-nitride films or specifically designed insulating sun-transparent chamber (QRC zone) that contains Selective Emissive (SE) gas or gas mix. QRC zone is mounted on the top of Solar Module replacing existing lamination coatings. To enhance the efficiency of QRC some specific methods and fabrication procedures are proposed to form an electricly charged textured surface that provide a high Electric Field at the surface thus enhancing IR emissivity from the surface. Such procedure can be also used to form the field Induced Surface Barriers in the Si-based Solar Cells that can substitute the existing diffused Emitters resulting in significant reduction of the Cycle Time as well as prospective Fabrication Cost.

  9. Surface modified alginate microcapsules for 3D cell culture

    NASA Astrophysics Data System (ADS)

    Chen, Yi-Wen; Kuo, Chiung Wen; Chueh, Di-Yen; Chen, Peilin

    2016-06-01

    Culture as three dimensional cell aggregates or spheroids can offer an ideal platform for tissue engineering applications and for pharmaceutical screening. Such 3D culture models, however, may suffer from the problems such as immune response and ineffective and cumbersome culture. This paper describes a simple method for producing microcapsules with alginate cores and a thin shell of poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) to encapsulate mouse induced pluripotent stem (miPS) cells, generating a non-fouling surface as an effective immunoisolation barrier. We demonstrated the trapping of the alginate microcapsules in a microwell array for the continuous observation and culture of a large number of encapsulated miPS cells in parallel. miPS cells cultured in the microcapsules survived well and proliferated to form a single cell aggregate. Droplet formation of monodisperse microcapsules with controlled size combined with flow cytometry provided an efficient way to quantitatively analyze the growth of encapsulated cells in a high-throughput manner. The simple and cost-effective coating technique employed to produce the core-shell microcapsules could be used in the emerging field of cell therapy. The microwell array would provide a convenient, user friendly and high-throughput platform for long-term cell culture and monitoring.

  10. Structural characterization of the N-linked pentasaccharide decorating glycoproteins of the halophilic archaeon Haloferax volcanii.

    PubMed

    Kandiba, Lina; Lin, Chia-Wei; Aebi, Markus; Eichler, Jerry; Guerardel, Yann

    2016-07-01

    N-Glycosylation is a post-translational modification performed in all three domains of life. In the halophilic archaea Haloferax volcanii, glycoproteins such as the S-layer glycoprotein are modified by an N-linked pentasaccharide assembled by a series of Agl (archaeal glycosylation) proteins. In the present study, mass spectrometry (MS) and nuclear magnetic resonance spectroscopy were used to define the structure of this glycan attached to at least four of the seven putative S-layer glycoprotein N-glycosylation sites, namely Asn-13, Asn-83, Asn-274 and Asn-279. Such approaches detected a trisaccharide corresponding to glucuronic acid (GlcA)-β1,4-GlcA-β1,4-glucose-β1-Asn, a tetrasaccharide corresponding to methyl-O-4-GlcA-β-1,4-galacturonic acid-α1,4-GlcA-β1,4-glucose-β1-Asn, and a pentasaccharide corresponding to hexose-1,2-[methyl-O-4-]GlcA-β-1,4-galacturonic acid-α1,4-GlcA-β1,4-glucose-β1-Asn, with previous MS and radiolabeling experiments showing the hexose at the non-reducing end of the pentasaccharide to be mannose. The present analysis thus corrects the earlier assignment of the penultimate sugar as a methyl ester of a hexuronic acid, instead revealing this sugar to be a methylated GlcA. The assignments made here are in good agreement with what was already known of the Hfx. volcanii N-glycosylation pathway from previous genetic and biochemical efforts while providing new insight into the process.

  11. Fragmentation Characteristics of Deprotonated N-linked Glycopeptides: Influences of Amino Acid Composition and Sequence

    NASA Astrophysics Data System (ADS)

    Nishikaze, Takashi; Kawabata, Shin-ichirou; Tanaka, Koichi

    2014-06-01

    Glycopeptide structural analysis using tandem mass spectrometry is becoming a common approach for elucidating site-specific N-glycosylation. The analysis is generally performed in positive-ion mode. Therefore, fragmentation of protonated glycopeptides has been extensively investigated; however, few studies are available on deprotonated glycopeptides, despite the usefulness of negative-ion mode analysis in detecting glycopeptide signals. Here, large sets of glycopeptides derived from well-characterized glycoproteins were investigated to understand the fragmentation behavior of deprotonated N-linked glycopeptides under low-energy collision-induced dissociation (CID) conditions. The fragment ion species were found to be significantly variable depending on their amino acid sequence and could be classified into three types: (i) glycan fragment ions, (ii) glycan-lost fragment ions and their secondary cleavage products, and (iii) fragment ions with intact glycan moiety. The CID spectra of glycopeptides having a short peptide sequence were dominated by type (i) glycan fragments (e.g., 2,4AR, 2,4AR-1, D, and E ions). These fragments define detailed structural features of the glycan moiety such as branching. For glycopeptides with medium or long peptide sequences, the major fragments were type (ii) ions (e.g., [peptide + 0,2X0-H]- and [peptide-NH3-H]-). The appearance of type (iii) ions strongly depended on the peptide sequence, and especially on the presence of Asp, Asn, and Glu. When a glycosylated Asn is located on the C-terminus, an interesting fragment having an Asn residue with intact glycan moiety, [glycan + Asn-36]-, was abundantly formed. Observed fragments are reasonably explained by a combination of existing fragmentation rules suggested for N-glycans and peptides.

  12. 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.

  13. Multiplexed Targeted Mass Spectrometry-Based Assays for the Quantification of N-Linked Glycosite-Containing Peptides in Serum

    PubMed Central

    Thomas, Stefani N.; Harlan, Robert; Chen, Jing; Aiyetan, Paul; Liu, Yansheng; Sokoll, Lori J.; Aebersold, Ruedi; Chan, Daniel W.; Zhang, Hui

    2016-01-01

    Protein glycosylation is one of the most common protein modifications, and the quantitative analysis of glycoproteins has the potential to reveal biological functions and their association with disease. However, the high throughput accurate quantification of glycoproteins is technically challenging due to the scarcity of robust assays to detect and quantify glycoproteins. Here we describe the development of multiplexed targeted MS assays to quantify N-linked glycosite-containing peptides in serum using parallel reaction monitoring (PRM). Each assay was characterized by its performance metrics and criteria established by the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (NCI CPTAC) to facilitate the widespread adoption of the assays in studies designed to confidently detect changes in the relative abundance of these analytes. An in-house developed software program, MRMPlus, was used to compute assay performance parameters including specificity, precision, and repeatability. We show that 43 selected N-linked glycosite-containing peptides identified in prostate cancer tissue studies carried out in our group were detected in the sera of prostate cancer patients within the quantitative range of the developed PRM assays. A total of 41 of these formerly N-linked glycosite-containing peptides (corresponding to 37 proteins) were reproducibly quantified based on their relative peak area ratios in human serum during PRM assay development, with 4 proteins showing differential significance in serum from nonaggressive (NAG) vs aggressive (AG) prostate cancer patient serum (n = 50, NAG vs AG). The data demonstrate that the assays can be used for the high throughput and reproducible quantification of a panel of formerly N-linked glycosite-containing peptides. The developed assays can also be used for the quantification of formerly N-linked glycosite-containing peptides in human serum irrespective of disease state. PMID:26451657

  14. Stable isotope labeling of oligosaccharide cell surface antigens

    SciTech Connect

    Unkefer, C.J.; Silks, L.A. III; Martinez, R.A.

    1998-12-31

    The overall goal of this Laboratory Directed Research and Development (LDRD) project was to develop new methods for synthesis of {sup 13}C-labeled oligosaccharides that are required for nuclear magnetic resonance (NMR) studies of their solution conformation. Oligosaccharides are components of the cell`s outer surface and are involved in important processes such as cell-cell recognition and adhesion. Recently, Danishefsky and coworkers at Slone-Kettering Cancer Center developed a method for the solid-phase chemical synthesis of oligosaccharides. The specific goal of this LDRD project was to prepare uniform {sup 13}C-labeled aldohexose precursors required for the solid-phase synthesis of the Lewis blood-group antigenic determinants. We report the synthesis of {sup 13}C-labeled D-glucal, D-galactal and Fucosyl precursors. We have been collaborating with the Danishefsky group on the synthesis of the Lewis oligosaccharides and the NMR analysis of their solution conformation.

  15. Glucocorticoids and the cell surface of human glioma cells: relationship to cytostasis.

    PubMed

    Mackie, A E; Freshney, R I; Akturk, F; Hunt, G

    1988-12-01

    The glucocorticoid hormones methyl prednisolone and dexamethasone were shown to be cytostatic, but not cytotoxic, at high cell densities for early passage and continuous cell lines from human glioma at 0.25 microM and above, in the presence or absence of serum. In the absence of serum both steroids at 2.5 nM increased the saturation density close to the level reached in serum. Examination of the iodinated glycoproteins of the cell surface by gel electrophoresis did not reveal any consistent change. However, gel exclusion chromatography of protease digests of the cell surface and of material released into the medium showed an increase in incorporation of 3H-glucosamine in pronase digests after treatment with methyl prednisolone. Ion exchange chromatography showed that sulphated glycosaminoglycans, particularly heparan sulphate, increased and hyaluronic acid decreased in response to steroids, and there was increased retention of GAGs on the cell surface relative to the released fraction. It was concluded that glucocorticoid hormones modify the cell surface of human glioma cells and that this may contribute to enhanced cell intraction and lead to increased density limitation of cell proliferation.

  16. Glucocorticoids and the cell surface of human glioma cells: relationship to cytostasis.

    PubMed Central

    Mackie, A. E.; Freshney, R. I.; Akturk, F.; Hunt, G.

    1988-01-01

    The glucocorticoid hormones methyl prednisolone and dexamethasone were shown to be cytostatic, but not cytotoxic, at high cell densities for early passage and continuous cell lines from human glioma at 0.25 microM and above, in the presence or absence of serum. In the absence of serum both steroids at 2.5 nM increased the saturation density close to the level reached in serum. Examination of the iodinated glycoproteins of the cell surface by gel electrophoresis did not reveal any consistent change. However, gel exclusion chromatography of protease digests of the cell surface and of material released into the medium showed an increase in incorporation of 3H-glucosamine in pronase digests after treatment with methyl prednisolone. Ion exchange chromatography showed that sulphated glycosaminoglycans, particularly heparan sulphate, increased and hyaluronic acid decreased in response to steroids, and there was increased retention of GAGs on the cell surface relative to the released fraction. It was concluded that glucocorticoid hormones modify the cell surface of human glioma cells and that this may contribute to enhanced cell intraction and lead to increased density limitation of cell proliferation. PMID:3254724

  17. Adherence of Candida albicans and Candida parapsilosis to epithelial cells correlates with fungal cell surface carbohydrates.

    PubMed

    Lima-Neto, Reginaldo G; Beltrão, Eduardo I C; Oliveira, Patrícia C; Neves, Rejane P

    2011-01-01

    Many studies have described the adherence of Candida albicans to epithelial cells but little is known about Candida parapsilosis adhesion and its role in host cell surface recognition. This study was designed to evaluate the correlation between the adherence of 20 C. albicans and 12 C. parapsilosis strains to human buccal epithelial cells and the expression of fungal cell surface carbohydrates using lectin histochemistry. Adherence assays were carried out by incubating epithelial cells in yeast suspensions (10(7) cells ml(-1) ) and peroxidase conjugated lectins (Con A, WGA, UEA I and PNA at 25 μg ml(-1) ) were used for lectin histochemistry. The results showed that adherence was overall greater for C. albicans than for C. parapsilosis (P < 0.01) and that the individual strain differences correlated with a high content of cell surface α-l-fucose residues as indicated by the UEA I staining pattern. Based on the saccharide specificity of the lectins used, these results suggest that l-fucose residues on cell surface glycoconjugates may represent recognition molecules for interactions between the yeast strain studied and the host (r = 0.6985, P = 0.0045). In addition, our results indicated the presence of α-d-glucose/α-d-mannose, N-acetyl-D-glucosamine/N-acetylneuraminic acid and D-galactose/N-acetyl-D-galactosamine in fungal cell wall.

  18. Interfacing biomembrane mimetic polymer surfaces with living cells Surface modification for reliable bioartificial liver

    NASA Astrophysics Data System (ADS)

    Iwasaki, Yasuhiko; Takami, Utae; Sawada, Shin-ichi; Akiyoshi, Kazunari

    2008-11-01

    The surface design used for reducing nonspecific biofouling is one of the most important issues for the fabrication of medical devices. We present here a newly synthesized a carbohydrate-immobilized phosphorylcholine polymer for surface modification of medical devices to control the interface with living cells. A random copolymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate (BMA), and 2-lactobionamidoethyl methacrylate (LAMA) was synthesized by conventional radical polymerization. The monomer feeding ratio in the copolymer was adjusted to 24/75/1 (MPC/BMA/LAMA). The copolymer (PMBL1.0) could be coated by solvent evaporation from an ethanol solution. Cells of the human hepatocellular liver carcinoma cell line (HepG2) having asialoglycoprotein receptors (ASGPRs) were seeded on PMBL1.0 or poly(BMA) (PBMA)-coated PET plates. On PBMA, many adherent cells were observed and were well spread with monolayer adhesion. HepG2 adhesion was observed on PMBL1.0 because the cell has ASGPRs. Furthermore, some of the cells adhering to PMBL1.0 had a spheroid formation and similarly shaped spheroids were scattered on the surface. According to confocal laser microscopic observation after 96 h cultivation, it was found that albumin production preferentially occurred in the center of the spheroid. The albumin production of the cells that adhered to PBMA was sparse. The amount of albumin production per unit cell that adhered to PMBL1.0 was determined by ELISA and was significantly higher than that which adhered to PBMA. Long-term cultivation of HepG2 was also performed using hollow fiber mini-modules coated with PMBL1.0. The concentration of albumin produced from HepG2 increased continuously for one month. In the mini-module, the function of HepG2 was effectively preserved for that period. On the hollow fiber membrane, spheroid formation of HepG2 cells was also observed. In conclusion, PMBL1.0 can provide a suitable surface for the cultivation of

  19. Probing and mapping electrode surfaces in solid oxide fuel cells.

    PubMed

    Blinn, Kevin S; Li, Xiaxi; Liu, Mingfei; Bottomley, Lawrence A; Liu, Meilin

    2012-09-20

    Solid oxide fuel cells (SOFCs) are potentially the most efficient and cost-effective solution to utilization of a wide variety of fuels beyond hydrogen (1-7). The performance of SOFCs and the rates of many chemical and energy transformation processes in energy storage and conversion devices in general are limited primarily by charge and mass transfer along electrode surfaces and across interfaces. Unfortunately, the mechanistic understanding of these processes is still lacking, due largely to the difficulty of characterizing these processes under in situ conditions. This knowledge gap is a chief obstacle to SOFC commercialization. The development of tools for probing and mapping surface chemistries relevant to electrode reactions is vital to unraveling the mechanisms of surface processes and to achieving rational design of new electrode materials for more efficient energy storage and conversion(2). Among the relatively few in situ surface analysis methods, Raman spectroscopy can be performed even with high temperatures and harsh atmospheres, making it ideal for characterizing chemical processes relevant to SOFC anode performance and degradation(8-12). It can also be used alongside electrochemical measurements, potentially allowing direct correlation of electrochemistry to surface chemistry in an operating cell. Proper in situ Raman mapping measurements would be useful for pin-pointing important anode reaction mechanisms because of its sensitivity to the relevant species, including anode performance degradation through carbon deposition(8, 10, 13, 14) ("coking") and sulfur poisoning(11, 15) and the manner in which surface modifications stave off this degradation(16). The current work demonstrates significant progress towards this capability. In addition, the family of scanning probe microscopy (SPM) techniques provides a special approach to interrogate the electrode surface with nanoscale resolution. Besides the surface topography that is routinely collected by AFM

  20. Analysis of Cell Surface Proteome Changes via Label-free, Quantitative Mass Spectrometry*S⃞

    PubMed Central

    Schiess, Ralph; Mueller, Lukas N.; Schmidt, Alexander; Mueller, Markus; Wollscheid, Bernd; Aebersold, Ruedi

    2009-01-01

    We present a mass spectrometry-based strategy for the specific detection and quantification of cell surface proteome changes. The method is based on the label-free quantification of peptide patterns acquired by high mass accuracy mass spectrometry using new software tools and the cell surface capturing technology that selectively enriches glycopeptides exposed to the cell exterior. The method was applied to monitor dynamic protein changes in the cell surface glycoproteome of Drosophila melanogaster cells. The results led to the construction of a cell surface glycoprotein atlas consisting of 202 cell surface glycoproteins of D. melanogaster Kc167 cells and indicated relative quantitative changes of cell surface glycoproteins in four different cellular states. Furthermore we specifically investigated cell surface proteome changes upon prolonged insulin stimulation. The data revealed insulin-dependent cell surface glycoprotein dynamics, including insulin receptor internalization, and linked these changes to intracellular signaling networks. PMID:19036722

  1. Basic surface properties of Aedes albopictus cells: effect of Mayaro virus infection on electrostatic charge and surface tension.

    PubMed

    Mezêncio, J M; Costa e Silva Filho, F; Rebello, M A

    1997-01-01

    Aedes albopictus cells possess a negative cell surface charge of -12.7 mV with an isoelectrophoretic point (IEP) located between pH 3.0 and 4.0. Infection with Mayaro virus rendered the surface of A. albopictus cells less negative reaching a zeta-potential value of -9.7 mV after 100 h of infection. Concomitantly, the IEP of the infected cells were also altered from 3.0-4.0 to 4.0-5.0. Furthermore, the contact angle measurements clearly showed qualitative alterations in the cell surface of infected cells.

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

    SciTech Connect

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

    2009-01-15

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

  3. 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.

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

    PubMed Central

    Hardy, Britta

    2014-01-01

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

  5. Polar/apolar compounds induce leukemia cell differentiation by modulating cell-surface potential.

    PubMed Central

    Arcangeli, A; Carlà, M; Del Bene, M R; Becchetti, A; Wanke, E; Olivotto, M

    1993-01-01

    The mechanism of action of polar/apolar inducers of cell differentiation, such as dimethyl sulfoxide and hexamethylene-bisacetamide, is still obscure. In this paper evidence is provided that their effects on murine erythroleukemia cells are modulated by various extracellular cations as a precise function of the cation effects on membrane surface potential. The interfacial effects of the inducers were directly measured on the charged electrode, showing that both dimethyl sulfoxide and hexamethylene-bisacetamide, at the effective concentrations for cell differentiation and within the physiological range of charge density, adsorb at the charged surface and produce a potential shift. A linear correlation was found between this shift and the inducer effects on cell differentiation. Besides offering a different interpretation of the mechanism of action of the inducers, these findings indicate that surface potential has a signaling function. They may also be relevant to cancer treatments based on tumor-cell commitment to terminal differentiation. Images Fig. 1 PMID:8516337

  6. 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.

  7. Proteomic analysis of cell surface proteins from Clostridium difficile.

    PubMed

    Wright, Anne; Wait, Robin; Begum, Shajna; Crossett, Ben; Nagy, Judit; Brown, Katherine; Fairweather, Neil

    2005-06-01

    Clostridium difficile is a bacterium that causes disease of the large intestine, particularly after treatment with antibiotics. The bacterium produces two toxins (A and B) that are responsible for the pathology of the disease. In addition, a number of bacterial virulence factors associated with adhesion to the gut have previously been identified, including the cell wall protein Cwp66, the high-molecular weight surface layer protein (HMW-SLP) and the flagella. As the genome sequence predicts many other cell wall associated proteins, we have investigated the diversity of proteins in cell wall extracts, with the aim of identifying further virulence factors. We have used a number of methods to remove the proteins associated with the cell wall of C. difficile. Two of the resulting extracts, obtained using low pH glycine treatment and lysozyme digestion of the cell wall, have been analysed in detail by two-dimensional electrophoresis and mass spectrometry. One hundred and nineteen spots, comprising 49 different proteins, have been identified. The two surface layer proteins (SLPs) are the most abundant proteins, and we have also found components of the flagellum. Interestingly, we have also determined that a number of paralogs of the HMW-SLP are expressed, and these could represent targets for further investigation as virulence factors.

  8. Nematic twist cell: Strong chirality induced at the surfaces

    NASA Astrophysics Data System (ADS)

    Lin, Tzu-Chieh; Nemitz, Ian R.; Pendery, Joel S.; Schubert, Christopher P. J.; Lemieux, Robert P.; Rosenblatt, Charles

    2013-04-01

    A nematic twist cell having a thickness gradient was filled with a mixture containing a configurationally achiral liquid crystal (LC) and chiral dopant. A chiral-based linear electrooptic effect was observed on application of an ac electric field. This "electroclinic effect" varied monotonically with d, changing sign at d =d0 where the chiral dopant exactly compensated the imposed twist. The results indicate that a significant chiral electrooptic effect always exists near the surfaces of a twist cell containing molecules that can be conformationally deracemized. Additionally, this approach can be used to measure the helical twisting power (HTP) of a chiral dopant in a liquid crystal.

  9. Mysterious hexagonal pyramids on the surface of Pyrobaculum cells.

    PubMed

    Rensen, Elena; Krupovic, Mart; Prangishvili, David

    2015-11-01

    In attempts to induce putative temperate viruses, we UV-irradiated cells of the hyperthermophilic archaeon Pyrobaculum oguniense. Virus replication could not be detected; however, we observed the development of pyramidal structures with 6-fold symmetry on the cell surface. The hexagonal basis of the pyramids was continuous with the cellular cytoplasmic membrane and apparently grew via the gradual expansion of the 6 triangular lateral faces, ultimately protruding through the S-layer. When the base of these isosceles triangles reached approximately 200 nm in length, the pyramids opened like flower petals. The origin and function of these mysterious nanostructures remain unknown.

  10. Active screen plasma nitriding enhances cell attachment to polymer surfaces

    NASA Astrophysics Data System (ADS)

    Kaklamani, Georgia; Bowen, James; Mehrban, Nazia; Dong, Hanshan; Grover, Liam M.; Stamboulis, Artemis

    2013-05-01

    Active screen plasma nitriding (ASPN) is a well-established technique used for the surface modification of materials, the result of which is often a product with enhanced functional performance. Here we report the modification of the chemical and mechanical properties of ultra-high molecular weight poly(ethylene) (UHMWPE) using 80:20 (v/v) N2/H2 ASPN, followed by growth of 3T3 fibroblasts on the treated and untreated polymer surfaces. ASPN-treated UHMWPE showed extensive fibroblast attachment within 3 h of seeding, whereas fibroblasts did not successfully attach to untreated UHMWPE. Fibroblast-coated surfaces were maintained for up to 28 days, monitoring their metabolic activity and morphology throughout. The chemical properties of the ASPN-treated UHMWPE surface were studied using X-ray photoelectron spectroscopy, revealing the presence of Csbnd N, Cdbnd N, and Ctbnd N chemical bonds. The elastic modulus, surface topography, and adhesion properties of the ASPN-treated UHMWPE surface were studied over 28 days during sample storage under ambient conditions and during immersion in two commonly used cell culture media.

  11. Exogenous cathepsin G upregulates cell surface MHC class I molecules on immune and glioblastoma cells

    PubMed Central

    Giese, Madleen; Turiello, Nadine; Molenda, Nicole; Palesch, David; Meid, Annika; Schroeder, Roman; Basilico, Paola; Benarafa, Charaf; Halatsch, Marc-Eric; Zimecki, Michal; Westhoff, Mike-Andrew; Wirtz, Christian Rainer; Burster, Timo

    2016-01-01

    Major histocompatibility complex (MHC) class I molecules present antigenic peptides to cytotoxic T cells. During an adaptive immune response, MHC molecules are regulated by several mechanisms including lipopolysaccharide (LPS) and interferon gamma (IFN-g). However, it is unclear whether the serine protease cathepsin G (CatG), which is generally secreted by neutrophils at the site of inflammation, might regulate MHC I molecules. We identified CatG, and to a higher extend CatG and lactoferrin (LF), as an exogenous regulator of cell surface MHC I expression of immune cells and glioblastoma stem cells. In addition, levels of MHC I molecules are reduced on dendritic cells from CatG deficient mice compared to their wild type counterparts. Furthermore, cell surface CatG on immune cells, including T cells, B cells, and NK cells triggers MHC I on THP-1 monocytes suggesting a novel mechanism for CatG to facilitate intercellular communication between infiltrating cells and the respective target cell. Subsequently, our findings highlight the pivotal role of CatG as a checkpoint protease which might force target cells to display their intracellular MHC I:antigen repertoire. PMID:27806341

  12. Surface enhanced Raman spectroscopy in breast cancer cells

    PubMed Central

    González-Solís, JL; Luévano-Colmenero, GH; Vargas-Mancilla, J

    2013-01-01

    Background and aims: Raman spectroscopy is a vibrational technique which provides information about the chemical structure. Nevertheless, since many chemicals are present in a cell at very low concentration, the Raman signal observed from a single cell is extremely weak. In surface enhanced Raman scattering (SERS), Raman signals can be enhanced by many orders of magnitude when nanoparticles are incorporated into the cell. Materials (subjects) and methods: The tumor biopsies were obtained from 5 patients who were clinically diagnosed with breast cancer. Breast cancer cells isolated from the biopsy were washed, centrifuged and seeded out. Cultivation took place in DMEM at 37°C in a humidified of 5% CO2 in air with addition of colloidal silver nanoparticles of 40 nm into the cell by sonication. Immediately, the washed cells were analyzed in phosphate buffered saline (PBS) at pH 7. Raman analysis was carried out on the Jobin-Yvon LabRAM HR800 microscope system, with a NIR 830 nm laser excitation source. Results: The strongly enhanced Raman signals allow Raman measurements of a single cell in the 200–1800 cm−1 range in relatively short collection times (5 second) using 17 mW near-infrared excitation. Observed spectral features differed across the cell, but chemical constituents in the cell nucleus and cytoplasm, such as DNA, RNA, and amino acids tyrosine and phenylalanine can be identified. Conclusions: Particularly strong field enhancement can be observed when nanoparticles form colloidal clusters. The results suggest that SERS could be a new technique for the identification of breast cancer cell. PMID:24155548

  13. Fibrin glue inhibits migration of ocular surface epithelial cells.

    PubMed

    Yeung, A M; Faraj, L A; McIntosh, O D; Dhillon, V K; Dua, H S

    2016-10-01

    PurposeFibrin glue has been used successfully in numerous ophthalmic surgical procedures. Recently, fibrin glue has been used in limbal stem cell transplantation to reduce both operative time and to negate the need for sutures. The aim of this study was to determine the effects of fibrin glue on epithelial cell migration in vitro.MethodsCorneoscleral rims were split to retain the epithelial layer, Bowman's layer, and anterior stroma. Rims were cut into eight equal-sized pieces and were placed directly on culture plates or affixed with fibrin glue. Rims were maintained in culture for 25 days and epithelial cell growth was monitored. Cells were photographed to measure area or growth and immunofluorescence staining of explants for fibrin was performed.ResultsExplants that were glued demonstrated significantly delayed epithelial cell growth and migration as compared with explants without glue. By day 16, all fibrin glue had dissolved and coincided with onset of cell growth from glued explants. Cell growth commenced between days 3 and 4 for control explants without glue and around days 14-16 for explants with fibrin glue.ConclusionsFibrin glue delays epithelial cell migration by acting as a physical barrier and can potentially interfere with explant-derived limbal epithelial cell migration on to the corneal surface. We propose that glue should be used to attach the conjunctival frill of the limbal explant but care should be taken to ensure that the glue does not wrap around the explant if used to secure the explant as well. Strategic use of glue, to attach the recessed conjunctiva, can be advantageous in delaying conjunctival cell migration and reducing the need for sequential sector conjunctival epitheliectomy.

  14. Cell-surface prion protein interacts with glycosaminoglycans.

    PubMed

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

    2002-11-15

    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.

  15. Surface Chemistry Regulates Valvular Interstitial Cell Differentiation In Vitro

    PubMed Central

    Rush, Matthew N.; Coombs, Kent E.; Hedberg-Dirk, Elizabeth L.

    2015-01-01

    The primary driver for valvular calcification is the differentiation of valvular interstitial cells (VICs) into a diseased phenotype. However, the factors leading to the onset of osteoblastic-like VICs (obVICs) and resulting calcification are not fully understood. This study isolates the effect of substrate surface chemistry on in vitro VIC differentiation and calcified tissue formation. Using ω-functionalized alkanethiol self-assembled monolayers (SAMs) on gold [CH3 (hydrophobic), OH (hydrophilic), COOH (COO−, negative at physiological pH), and NH2 (NH3+, positive at physiological pH)], we have demonstrated that surface chemistry modulates VIC phenotype and calcified tissue deposition independent of osteoblastic-inducing media additives. Over seven days VICs exhibited surface-dependent differences in cell proliferation (COO− = NH3+> OH > CH3), morphology, and osteoblastic potential. Both NH3+and CH3-terminated SAMs promoted calcified tissue formation while COO−-terminated SAMs showed no calcification. VICs on NH3+-SAMs exhibited the most osteoblastic phenotypic markers through robust nodule formation, up-regulated osteocalcin and α-smooth muscle actin expression, and adoption of a round/rhomboid morphology indicative of osteoblastic differentiation. With the slowest proliferation, VICs on CH3-SAMs promoted calcified aggregate formation through cell detachment and increased cell death indicative of dystrophic calcification. Furthermore, induction of calcified tissue deposition on NH3+ and CH3-SAMs was distinctly different than that of media induced osteoblastic VICs. These results demonstrate that substrate surface chemistry alters VIC behavior and plays an important role in calcified tissue formation. In addition, we have identified two novel methods of calcified VIC induction in vitro. Further study of these environments may yield new models for in vitro testing of therapeutics for calcified valve stenosis, although additional studies need to be conducted

  16. Surface Trafficking of APP and BACE in Live Cells.

    PubMed

    Bauereiss, Anna; Welzel, Oliver; Jung, Jasmin; Grosse-Holz, Simon; Lelental, Natalia; Lewczuk, Piotr; Wenzel, Eva M; Kornhuber, Johannes; Groemer, Teja W

    2015-06-01

    Amyloid-β (Aβ)-peptide, the major constituent of the plaques that develop during Alzheimer's disease, is generated via the cleavage of Aβ precursor protein (APP) by β-site APP-cleaving enzyme (BACE). Using live-cell imaging of APP and BACE labeled with pH-sensitive proteins, we could detect the release events of APP and BACE and their distinct kinetics. We provide kinetic evidence for the cleavage of APP by α-secretase on the cellular surface after exocytosis. Furthermore, simultaneous dual-color evanescent field illumination revealed that the two proteins are trafficked to the surface in separate compartments. Perturbing the membrane lipid composition resulted in a reduced frequency of exocytosis and affected BACE more strongly than APP. We propose that surface fusion frequency is a key factor regulating the aggregation of APP and BACE in the same membrane compartment and that this process can be modulated via pharmacological intervention.

  17. The Mesenchymal Precursor Cell Marker Antibody STRO-1 Binds to Cell Surface Heat Shock Cognate 70.

    PubMed

    Fitter, Stephen; Gronthos, Stan; Ooi, Soo Siang; Zannettino, Andrew C W

    2016-12-27

    Since its discovery more than 25 years ago, the STRO-1 antibody has played a fundamental role in defining the hierarchical nature of mesenchymal precursor cells (MPC) and their progeny. STRO-1 antibody binding remains a hallmark of immature pluripotent MPC. Despite the significance of STRO-1 in the MPC field, the identity of the antigen has remained elusive. Using a combination of two-dimensional gel electrophoresis, coupled with Western blotting and Tandem mass spectroscopy, we have identified the STRO-1 antigen as heat shock cognate 70 (HSC70;HSPA8). STRO-1 binds to immune-precipitated HSC70 and siRNA-mediated knock down of HSPA8 reduced STRO-1 binding. STRO-1 surface binding does not correlate with HSC70 expression and sequestration of cholesterol reduces STRO-1 surface binding, suggesting that the plasma membrane lipid composition may be an important determinant in the presentation of HSC70 on the cell surface. HSC70 is present on the surface of STRO-1(+) but not STRO-1(-) cell lines as assessed by cell surface biotinylation and recombinant HSC70 blocks STRO-1 binding to the cell surface. The STRO-1 epitope on HSC70 was mapped to the ATPase domain using a series of deletion mutants in combination with peptide arrays. Deletion of the first four amino acids of the consensus epitope negated STRO-1 binding. Notably, in addition to HSC70, STRO-1 cross-reacts with heat shock protein 70 (HSP70), however all the clonogenic cell activity is restricted to the STRO-1(BRIGHT) /HSP70(-) fraction. These results provide important insight into the properties that define multipotent MPC and provide the impetus to explore the role of cell surface HSC70 in MPC biology. Stem Cells 2016.

  18. SPARC regulates collagen interaction with cardiac fibroblast cell surfaces.

    PubMed

    Harris, Brett S; Zhang, Yuhua; Card, Lauren; Rivera, Lee B; Brekken, Rolf A; Bradshaw, Amy D

    2011-09-01

    Cardiac tissue from mice that do not express secreted protein acidic and rich in cysteine (SPARC) have reduced amounts of insoluble collagen content at baseline and in response to pressure overload hypertrophy compared with wild-type (WT) mice. However, the cellular mechanism by which SPARC affects myocardial collagen is not clearly defined. Although expression of SPARC by cardiac myocytes has been detected in vitro, immunohistochemistry of hearts demonstrated SPARC staining primarily associated with interstitial fibroblastic cells. Primary cardiac fibroblasts isolated from SPARC-null and WT mice were assayed for collagen I synthesis by [(3)H]proline incorporation into procollagen and by immunoblot analysis of procollagen processing. Bacterial collagenase was used to discern intracellular from extracellular forms of collagen I. Increased amounts of collagen I were found associated with SPARC-null versus WT cells, and the proportion of total collagen I detected on SPARC-null fibroblasts without propeptides [collagen-α(1)(I)] was higher than in WT cells. In addition, the amount of total collagen sensitive to collagenase digestion (extracellular) was greater in SPARC-null cells than in WT cells, indicating an increase in cell surface-associated collagen in the absence of SPARC. Furthermore, higher levels of collagen type V, a fibrillar collagen implicated in collagen fibril initiation, were found in SPARC-null fibroblasts. The absence of SPARC did not result in significant differences in proliferation or in decreased production of procollagen I by cardiac fibroblasts. We conclude that SPARC regulates collagen in the heart by modulating procollagen processing and interactions with fibroblast cell surfaces. These results are consistent with decreased levels of interstitial collagen in the hearts of SPARC-null mice being due primarily to inefficient collagen deposition into the extracellular matrix rather than to differences in collagen production.

  19. 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.

  20. Structure of a bacterial cell surface decaheme electron conduit.

    PubMed

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

    2011-06-07

    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. 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 intercytochrome electron exchange along "nanowire" appendages. We present a 3.2-Å crystal structure of one of these decaheme cytochromes, MtrF, that allows the spatial organization of the 10 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 octaheme chain transects the length of the protein and is bisected by a planar 45-Å tetraheme 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.

  1. Cell surface alpha 2,6 sialylation affects adhesion of breast carcinoma cells.

    PubMed

    Lin, Shaoqiang; Kemmner, Wolfgang; Grigull, Sabine; Schlag, Peter M

    2002-05-15

    Tumor-associated alterations of cell surface glycosylation play a crucial role in the adhesion and metastasis of carcinoma cells. The aim of this study was to examine the effect of alpha 2,6-sialylation on the adhesion properties of breast carcinoma cells. To this end mammary carcinoma cells, MDA-MB-435, were sense-transfected with sialyltransferase ST6Gal-I cDNA or antisense-transfected with a part of the ST6Gal-I sequence. Sense transfectants showed an enhanced ST6Gal-I mRNA expression and enzyme activity and an increased binding of the lectin Sambucus nigra agglutinin (SNA), specific for alpha 2,6-linked sialic acid. Transfection with ST6Gal-I in the antisense direction resulted in less enzyme activity and SNA reactivity. A sense-transfected clone carrying increased amounts of alpha 2,6-linked sialic acid adhered preferentially to collagen IV and showed reduced cell-cell adhesion and enhanced invasion capacity. In contrast, antisense transfection led to less collagen IV adhesion but enhanced homotypic cell-cell adhesion. In another approach, inhibition of ST6Gal-I enzyme activity by application of soluble antisense-oligodeoxynucleotides was studied. Antisense treatment resulted in reduced ST6 mRNA expression and cell surface 2,6-sialylation and significantly decreased collagen IV adhesion. Our results suggest that cell surface alpha 2,6-sialylation contributes to cell-cell and cell-extracellular matrix adhesion of tumor cells. Inhibition of sialytransferase ST6Gal-I by antisense-oligodeoxynucleotides might be a way to reduce the metastatic capacity of carcinoma cells.

  2. 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.

  3. Effects of molecular weight and surface functionalization on surface composition and cell adhesion to Hyaluronan coated titanium.

    PubMed

    Morra, M; Cassinelli, C; Carpi, A; Giardino, R; Fini, M

    2006-09-01

    This paper describes the effect of surface functionalization on surface composition and cell adhesion to titanium samples by high and low molecular weight Hyaluronan (HA). HA was covalently linked to aminated Ti surfaces obtained by two different surface functionalization techniques, that is polyethyleneimine (PEI) adsorption and deposition from allylamine plasma. The two approaches yield very different surface densities of available amino groups, affecting this way the number and frequency of surface-HA bonds and the configurational freedom of the latter. Results of cell adhesion test are dependent on the surface functionalization approach adopted, low molecular weight HA coupled to PEI functionalized Ti does not yield the same degree of resistance to cell adhesion found on other samples. These results indicate that the details of the surface functionalization step are crucial for surface engineering of implant devices by biological molecules.

  4. Interaction of Human Tumor Viruses with Host Cell Surface Receptors and Cell Entry

    PubMed Central

    Schäfer, Georgia; Blumenthal, Melissa J.; Katz, Arieh A.

    2015-01-01

    Currently, seven viruses, namely Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpes virus (KSHV), high-risk human papillomaviruses (HPVs), Merkel cell polyomavirus (MCPyV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human T cell lymphotropic virus type 1 (HTLV-1), have been described to be consistently associated with different types of human cancer. These oncogenic viruses belong to distinct viral families, display diverse cell tropism and cause different malignancies. A key to their pathogenicity is attachment to the host cell and entry in order to replicate and complete their life cycle. Interaction with the host cell during viral entry is characterized by a sequence of events, involving viral envelope and/or capsid molecules as well as cellular entry factors that are critical in target cell recognition, thereby determining cell tropism. Most oncogenic viruses initially attach to cell surface heparan sulfate proteoglycans, followed by conformational change and transfer of the viral particle to secondary high-affinity cell- and virus-specific receptors. This review summarizes the current knowledge of the host cell surface factors and molecular mechanisms underlying oncogenic virus binding and uptake by their cognate host cell(s) with the aim to provide a concise overview of potential target molecules for prevention and/or treatment of oncogenic virus infection. PMID:26008702

  5. Formation of nanofilms on cell surfaces to improve the insertion efficiency of a nanoneedle into cells

    SciTech Connect

    Amemiya, Yosuke; Kawano, Keiko; Matsusaki, Michiya; Akashi, Mitsuru; Nakamura, Noriyuki; Nakamura, Chikashi

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer We examined the insertion efficiency of nanoneedles into fibroblast and neural cells. Black-Right-Pointing-Pointer Nanofilms formed on cell surfaces improved the insertion efficiency of nanoneedles. Black-Right-Pointing-Pointer Nanofilms improved the insertion efficiency even in Y27632-treated cells. -- Abstract: A nanoneedle, an atomic force microscope (AFM) tip etched to 200 nm in diameter and 10 {mu}m in length, can be inserted into cells with the aid of an AFM and has been used to introduce functional molecules into cells and to analyze intracellular information with minimal cell damage. However, some cell lines have shown low insertion efficiency of the nanoneedle. Improvement in the insertion efficiency of a nanoneedle into such cells is a significant issue for nanoneedle-based cell manipulation and analysis. Here, we have formed nanofilms composed of extracellular matrix molecules on cell surfaces and found that the formation of the nanofilms improved insertion efficiency of a nanoneedle into fibroblast and neural cells. The nanofilms were shown to improve insertion efficiency even in cells in which the formation of actin stress fibers was inhibited by the ROCK inhibitor Y27632, suggesting that the nanofilms with the mesh structure directly contributed to the improved insertion efficiency of a nanoneedle.

  6. Interaction of human tumor viruses with host cell surface receptors and cell entry.

    PubMed

    Schäfer, Georgia; Blumenthal, Melissa J; Katz, Arieh A

    2015-05-22

    Currently, seven viruses, namely Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpes virus (KSHV), high-risk human papillomaviruses (HPVs), Merkel cell polyomavirus (MCPyV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human T cell lymphotropic virus type 1 (HTLV-1), have been described to be consistently associated with different types of human cancer. These oncogenic viruses belong to distinct viral families, display diverse cell tropism and cause different malignancies. A key to their pathogenicity is attachment to the host cell and entry in order to replicate and complete their life cycle. Interaction with the host cell during viral entry is characterized by a sequence of events, involving viral envelope and/or capsid molecules as well as cellular entry factors that are critical in target cell recognition, thereby determining cell tropism. Most oncogenic viruses initially attach to cell surface heparan sulfate proteoglycans, followed by conformational change and transfer of the viral particle to secondary high-affinity cell- and virus-specific receptors. This review summarizes the current knowledge of the host cell surface factors and molecular mechanisms underlying oncogenic virus binding and uptake by their cognate host cell(s) with the aim to provide a concise overview of potential target molecules for prevention and/or treatment of oncogenic virus infection.

  7. A possible model for cell-cell recognition via surface macromolecules.

    PubMed

    Burger, M M; Turner, R S; Kuhns, W J; Weinbaum, G

    1975-07-17

    Alternative possibilities for the establishment of the proper cell distribution during embryogenesis are summarized at the beginning, followed by an assessment of the examples known so far where cell-cell recognition is known to be mediated via cell surface components. In the second part the species-specific recognition process which occurs during the sorting-out of dissociated sponge cells is analysed since it may serve as a possible model for cell-cell recognition in higher animals. Three possible mechanisms for the establishment of proper cell distribution are considered. These include, first, chemotaxis: secondly, guidance of cell or cell sheet movement by extracellular matrix or by surrounding cells and thirdly, random movement followed by recognition at the final point of destination. Recognition is necessary for both of the two latter processes, i.e. for cell guidance as well as for locking the cells into their final position after random movement. Two basically different recognition mechanisms should be distinguished from each other. On the one hand cells may recognize each other with the help of macromolecules situated in or just outside of the plasmamembrane which fit to each other like enzymes and substrates or antibodies and antigens. On the other hand, cells may exchange information by exchanging cytoplasmatic components via vesicles or gap junctions. The species-specific aggregation of dissociated sponge cells is considered to be a possible model for cell-cell recognition in higher animals. A proteoglycan-like intercellular macromolecule called aggregation factor seems to mediate recognition of a given species of cells in the reaggregation process of dissociated cells. The data available at the present time suggest that a monovalent surface macromolecule (baseplate) may mediate the recognition process probably by recognizing the carbohydrate side chains of the multivalent proteoglycan aggregation factor. A cell-free system was devised to mimic this

  8. Advances in the theory and application of BSF cells. [Back Surface Field solar cells

    NASA Technical Reports Server (NTRS)

    Mandelkorn, J.; Lamneck, J. H.

    1975-01-01

    A study to determine the influence of fabrication processes and bulk material properties on the behavior of back surface field (BSF) cells is reported. It is concluded that a photovoltage is generated at the p(+), p back junction of the cell. The concept of majority carrier collection is proposed as a possible mechanism for this generation. Advantages accruing to the advent of BSF cells are outlined.

  9. Targeting Prostate Cancer Stem-Like Cells Through Cell Surface-Expressed GRP78

    DTIC Science & Technology

    2013-10-01

    hypothesis that cell surface GRP78 drives cancer stem-like behavior by activating an Akt/GSK-3/ Snail -1 signaling axis in prostate cancer stem-like...investigate the hypothesis that cell surface GRP78 drives cancer stem-like behavior by activating an Akt/GSK-3/ Snail -1 signaling axis in prostate cancer stem...investigate these signaling pathways in year 2. Task 4: Investigate the relative expression of Snail -1, a GSK-3 target, in adherent prostate cancer cells

  10. Cell Surface Protein Detection to Assess Receptor Internalization

    PubMed Central

    Czarnecka, Magdalena; Kitlinska, Joanna

    2017-01-01

    The migration of membrane receptors upon exposure to different stimulants/inhibitors is of great importance. Among others, the internalization of membrane receptors affects their accessibility to ligands and cell responsiveness to environmental cues. Experimentally, receptor internalization can be used as a measure of their activation. In our studies, we employed this approach to explore cross-talk between a seven transmembrane domain receptor for neuropeptide Y (NPY), Y5R, and a tyrosine kinase receptor for brain-derived neurotrophic factor (BDNF), TrkB. To this end, we measured the internalization of Y5R upon stimulation with the TrkB ligand, BDNF. Upon treatment with BDNF, the cells were exposed to a membrane impermeable, biotinylation reagent that selectively labels surface proteins. Subsequently, the biotinylated membrane proteins were affinity-purified on columns with avidin resins and analyzed by Western blot. Differences in the fraction of receptors present on the cell surface of control and ligand-treated cells served as a measure of their internalization and response to particular stimuli.

  11. Characteristic Changes in Cell Surface Glycosylation Accompany Intestinal Epithelial Cell (IEC) Differentiation: High Mannose Structures Dominate the Cell Surface Glycome of Undifferentiated Enterocytes.

    PubMed

    Park, Dayoung; Brune, Kristin A; Mitra, Anupam; Marusina, Alina I; Maverakis, Emanual; Lebrilla, Carlito B

    2015-11-01

    Changes in cell surface glycosylation occur during the development and differentiation of cells and have been widely correlated with the progression of several diseases. Because of their structural diversity and sensitivity to intra- and extracellular conditions, glycans are an indispensable tool for analyzing cellular transformations. Glycans present on the surface of intestinal epithelial cells (IEC) mediate interactions with billions of native microorganisms, which continuously populate the mammalian gut. A distinct feature of IECs is that they differentiate as they migrate upwards from the crypt base to the villus tip. In this study, nano-LC/ESI QTOF MS profiling was used to characterize the changes in glycosylation that correspond to Caco-2 cell differentiation. As Caco-2 cells differentiate to form a brush border membrane, a decrease in high mannose type glycans and a concurrent increase in fucosylated and sialylated complex/hybrid type glycans were observed. At day 21, when cells appear to be completely differentiated, remodeling of the cell surface glycome ceases. Differential expression of glycans during IEC maturation appears to play a key functional role in regulating the membrane-associated hydrolases and contributes to the mucosal surface innate defense mechanisms. Developing methodologies to rapidly identify changes in IEC surface glycans may lead to a rapid screening approach for a variety of disease states affecting the GI tract.

  12. 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

  13. 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.

  14. A hybrid biocathode: surface display of O2-reducing enzymes for microbial fuel cell applications.

    PubMed

    Szczupak, Alon; Kol-Kalman, Dan; Alfonta, Lital

    2012-01-04

    Laccase and bilirubin oxidase were successfully displayed on the surface of yeast cells. Subsequently, these modified yeast cells were used in the cathode compartment of a microbial fuel cell. The performance of the fuel cells is compared.

  15. Ovarian carcinoma cells synthesize both chondroitin sulfate and heparan sulfate cell surface proteoglycans that mediate cell adhesion to interstitial matrix.

    PubMed

    Kokenyesi, R

    Metastatic ovarian carcinoma metastasizes by intra-peritoneal, non-hematogenous dissemination. The adhesion of the ovarian carcinoma cells to extracellular matrix components, such as types I and III collagen and cellular fibronectin, is essential for intra-peritoneal dissemination. The purpose of this study was to determine whether cell surface proteoglycans (a class of matrix receptors) are produced by ovarian carcinoma cells, and whether these proteoglycans have a role in the adhesion of ovarian carcinoma cells to types I and III collagen and fibronectin. Proteoglycans were metabolically labeled for biochemical studies. Both phosphatidylinositol-anchored and integral membrane-type cell surface proteoglycans were found to be present on the SK-OV-3 and NIH:OVCAR-3 cell lines. Three proteoglycan populations of differing hydrodynamic size were detected in both SK-OV-3 and NIH:OVCAR-3 cells. Digestions with heparitinase and chondroitinase ABC showed that cell surface proteoglycans of SK-OV-3 cells had higher proportion of chondroitin sulfate proteoglycans (75:25 of chondroitin sulfate:heparan sulfate ratio), while NIH:OVCAR-3 cells had higher proportion of heparan sulfate proteoglycans (10:90 of chondroitin sulfate:heparan sulfate ratio). RT-PCR indicated the synthesis of a unique assortment of syndecans, glypicans, and CD44 by the two cell lines. In adhesion assays performed on matrix-coated titer plates both cell lines adhered to types I and III collagen and cellular fibronectin, and cell adhesion was inhibited by preincubation of the matrix with heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, or chondroitin glycosaminoglycans. Treatment of the cells with heparitinase, chondroitinase ABC, or methylumbelliferyl xyloside also interfered with adhesion confirming the role of both heparan sulfate and chondroitin sulfate cell surface proteoglycans as matrix receptors on ovarian carcinoma cells.

  16. Role of Site-Specific N-Glycans Expressed on GluA2 in the Regulation of Cell Surface Expression of AMPA-Type Glutamate Receptors.

    PubMed

    Takeuchi, Yusuke; Morise, Jyoji; Morita, Ippei; Takematsu, Hiromu; Oka, Shogo

    2015-01-01

    The AMPA-type glutamate receptor (AMPAR), which is a tetrameric complex composed of four subunits (GluA1-4) with several combinations, mediates the majority of rapid excitatory synaptic transmissions in the nervous system. Cell surface expression levels of AMPAR modulate synaptic plasticity, which is considered one of the molecular bases for learning and memory formation. To date, a unique trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc), human natural killer-1 (HNK-1) carbohydrate, was found expressed specifically on N-linked glycans of GluA2 and regulated the cell surface expression of AMPAR and the spine maturation process. However, evidence that the HNK-1 epitope on N-glycans of GluA2 directly affects these phenomena is lacking. Moreover, it is thought that other N-glycans on GluA2 also have potential roles in the regulation of AMPAR functions. In the present study, using a series of mutants lacking potential N-glycosylation sites (N256, N370, N406, and N413) within GluA2, we demonstrated that the mutant lacking the N-glycan at N370 strongly suppressed the intracellular trafficking of GluA2 from the endoplasmic reticulum (ER) in HEK293 cells. Cell surface expression of GluA1, which is a major subunit of AMPAR in neurons, was also suppressed by co-expression of the GluA2 N370S mutant. The N370S mutant and wild-type GluA2 were co-immunoprecipitated with GluA1, suggesting that N370S was properly associated with GluA1. Moreover, we found that N413 was the main potential site of the HNK-1 epitope that promoted the interaction of GluA2 with N-cadherin, resulting in enhanced cell surface expression of GluA2. The HNK-1 epitope on N-glycan at the N413 of GluA2 was also involved in the cell surface expression of GluA1. Thus, our data suggested that site-specific N-glycans on GluA2 regulate the intracellular trafficking and cell surface expression of AMPAR.

  17. Surface modification for interaction study with bacteria and preosteoblast cells

    NASA Astrophysics Data System (ADS)

    Song, Qing

    Surface modification plays a pivotal role in bioengineering. Polymer coatings can provide biocompatibility and biofunctionalities to biomaterials through surface modification. In this dissertation, initiated chemical vapor deposition (iCVD) was utilized to coat two-dimensional (2D) and three-dimensional (3D) substrates with differently charged polyelectrolytes in order to generate antimicrobial and osteocompatible biomaterials. ICVD is a modified CVD technique that enables surface modification in an all-dry condition without substrate damage and solvent contamination. The free-radical polymerization allows the vinyl polymers to conformally coat on various micro- and nano-structured substrates and maintains the delicate structure of the functional groups. The vapor deposition of polycations provided antimicrobial activity to planar and porous substrates through destroying the negatively charged bacterial membrane and brought about high contact-killing efficiency (99.99%) against Gram-positive Bacillus subtilis and Gram-negative Escherichia coli. Additionally, the polyampholytes synthesized by iCVD exhibited excellent antifouling performance against the adhesion of Gram-positive Listeria innocua and Gram-negative E. coli in phosphate buffered saline (PBS). Their antifouling activities were attributed to the electrostatic interaction and hydration layers that served as physical and energetic barriers to prevent bacterial adhesion. The contact-killing and antifouling polymers synthesized by iCVD can be applied to surface modification of food processing equipment and medical devices with the aim of reducing foodborne diseases and medical infections. Moreover, the charged polyelectrolyte modified 2D polystyrene surfaces displayed good osteocompatibility and enhanced osteogenesis of preosteoblast cells than the un-modified polystyrene surface. In order to promote osteoinduction of hydroxyapatite (HA) scaffolds, bioinspired polymer-controlled mineralization was conducted

  18. Fast solid-phase extraction of N-linked glycopeptides by amine-functionalized mesoporous silica nanoparticles.

    PubMed

    Miao, Weili; Zhang, Cheng; Cai, Yan; Zhang, Ying; Lu, Haojie

    2016-04-21

    Selective enrichment is a crucial step before the mass spectrometric analysis of glycoproteins. A new approach using 3-aminopropyltriethoxysilane (APTES)-functionalized mesoporous silica materials (SBA-15) was reported to enrich the glycoproteins. Selective extraction of glycopeptides was achieved through coupling the oxidized glycan chains on the glycopeptides with the amine groups on SBA-15 through a reductive amination reaction, then the captured glycopeptides were detached from the SBA-15 for the following MS analysis using the enzyme PNGase F. Because the mesoporous material has a confinement effect, the efficiency of enrichment and enzymatic deglycosylation was improved dramatically. The coupling time was shortened from 4 hours to 1 hour, and the deglycosylation time was greatly shortened from 6 hours to 3 hours. This approach was successfully applied to profile the N-glycoproteome of human colorectal cancer serum. 84 N-linked glycosylation sites from 56 N-linked glycoproteins were identified from as little as 5 μL serum.

  19. Functional mapping of cell surface proteins with localized stimulation of single cells

    NASA Astrophysics Data System (ADS)

    Sun, Bingyun; Chiu, Daniel T.

    2003-11-01

    This paper describes the development of using individual micro and nano meter-sized vesicles as delivery vessels to functionally map the distribution of cell surface proteins at the level of single cells. The formation of different sizes of vesicles from tens of nanometers to a few micrometers in diameter that contain the desired molecules is addressed. An optical trap is used to manipulate the loaded vesicle to specific cell morphology of interest, and a pulsed UV laser is used to photo-release the stimuli onto the cell membrane. Carbachol activated cellular calcium flux, upon binding to muscarinic acetylcholine receptors, is studied by this method, and the potential of using this method for the functional mapping of localized proteins on the cell surface membrane is discussed.

  20. Surface-labelling studies on skeletal-muscle cells in vitro. Heterogeneity of iodinated cell-surface proteins.

    PubMed Central

    Cates, G A; Holland, P C

    1980-01-01

    1. Two distinct classes of protein were detected at the surface of chick-embryo skeletal-muscle cells after iodination of the cells in monolayer culture. 2. The two classes of iodinated proteins differed in their ability to co-purify with a vesicular plasma-membrane fraction prepared from surface-labelled cells. 3. One class consisted of predominantly high-molecular-weight glycoproteins that co-purified with the plasma-membrane fraction, but showed no significant qualitative or quantitative alterations in labelling with 125I and lactoperoxidase during myogenesis. 4. A second class of predominantly lower-molecular-weight proteins showed reproducible quantitative alterations in 125I-labelling during myogenesis but failed to co-purify with the plasma-membrane fraction. 5. This second class of proteins may represent matrix proteins involved in intercellular adhesion or adhesion of cells to the substratum. They are unlikely to be directly required for the process of plasma-membrane fusion during myogenesis, since they do not copurify with a vesicular plasma-membrane fraction known to be capable of Ca2+-dependent fusion in vitro. PMID:7370009

  1. Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions.

    PubMed

    Purushothaman, Anurag; Bandari, Shyam Kumar; Liu, Jian; Mobley, James A; Brown, Elizabeth E; Sanderson, Ralph D

    2016-01-22

    Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression.

  2. Selection of Antibodies Interfering with Cell Surface Receptor Signaling Using Embryonic Stem Cell Differentiation.

    PubMed

    Melidoni, Anna N; Dyson, Michael R; McCafferty, John

    2016-01-01

    Antibodies able to bind and modify the function of cell surface signaling components in vivo are increasingly being used as therapeutic drugs. The identification of such "functional" antibodies from within large antibody pools is, therefore, the subject of intense research. Here we describe a novel cell-based expression and reporting system for the identification of functional antibodies from antigen-binding populations preselected with phage display. The system involves inducible expression of the antibody gene population from the Rosa-26 locus of embryonic stem (ES) cells, followed by secretion of the antibodies during ES cell differentiation. Target antigens are cell-surface signaling components (receptors or ligands) with a known effect on the direction of cell differentiation (FGFR1 mediating ES cell exit from self renewal in this particular protocol). Therefore, inhibition or activation of these components by functional antibodies in a few elite clones causes a shift in the differentiation outcomes of these clones, leading to their phenotypic selection. Functional antibody genes are then recovered from positive clones and used to produce the purified antibodies, which can be tested for their ability to affect cell fates exogenously. Identified functional antibody genes can be further introduced in different stem cell types. Inducible expression of functional antibodies has a temporally controlled protein-knockdown capability, which can be used to study the unknown role of the signaling pathway in different developmental contexts. Moreover, it provides a means for control of stem cell differentiation with potential in vivo applications.

  3. Tetraploid cells from cytokinesis failure induce aneuploidy and spontaneous transformation of mouse ovarian surface epithelial cells.

    PubMed

    Lv, Lei; Zhang, Tianwei; Yi, Qiyi; Huang, Yun; Wang, Zheng; Hou, Heli; Zhang, Huan; Zheng, Wei; Hao, Qiaomei; Guo, Zongyou; Cooke, Howard J; Shi, Qinghua

    2012-08-01

    Most ovarian cancers originate from the ovarian surface epithelium and are characterized by aneuploid karyotypes. Aneuploidy, a consequence of chromosome instability, is an early event during the development of ovarian cancers. However, how aneuploid cells are evolved from normal diploid cells in ovarian cancers remains unknown. In the present study, cytogenetic analyses of a mouse syngeneic ovarian cancer model revealed that diploid mouse ovarian surface epithelial cells (MOSECs) experienced an intermediate tetraploid cell stage, before evolving to aneuploid (mainly near-tetraploid) cells. Using long-term live-cell imaging followed by fluorescence in situ hybridization (FISH), we demonstrated that tetraploid cells originally arose from cytokinesis failure of bipolar mitosis in diploid cells, and gave rise to aneuploid cells through chromosome mis-segregation during both bipolar and multipolar mitoses. Injection of the late passage aneuploid MOSECs resulted in tumor formation in C57BL/6 mice. Therefore, we reveal a pathway for the evolution of diploid to aneuploid MOSECs and elucidate a mechanism for the development of near-tetraploid ovarian cancer cells.

  4. Endothelial cell labeling with indium-111-oxine as a marker of cell attachment to bioprosthetic surfaces

    SciTech Connect

    Sharefkin, J.B.; Lather, C.; Smith, M.; Rich, N.M.

    1983-03-01

    Canine vascular endothelium labeled with indium-111-oxine was used as a marker of cell attachment to vascular prosthetic surfaces with complex textures. Primarily cultured and freshly harvested endothelial cells both took up the label rapidly. An average of 72% of a 32 micro Ci labeling dose was taken up by 1.5 X 10(6) cells in 10 min in serum-free medium. Over 95% of freshly labeled cells were viable by trypan blue tests and only 5% of the label was released after 1 h incubations at 37 degrees C. Labeled and unlabeled cells had similar rates of attachment to plastic dishes. Scanning electron microscopic studies showed that labeled cells retained their ability to spread on tissue culture dishes even at low (1%) serum levels. Labeled endothelial cells seeded onto Dacron or expanded polytetrafluoroethylene vascular prostheses by methods used in current surgical models could be identified by autoradiography of microscopic sections of the prostheses, and the efficiency of cell attachment to the prosthesis could be measured by gamma counting. Indium-111 labeling affords a simple and rapid way to measure initial cell attachment to, and distribution on, vascular prosthetic materials. The method could also allow measurement of early cell loss from a flow surface in vivo by using external gamma imaging.

  5. Estrogen inhibits cell cycle progression and retinoblastoma phosphorylation in rhesus ovarian surface epithelial cell culture

    SciTech Connect

    Wright, Jay W.; Stouffer, Richard L.; Rodland, Karin D.

    2003-10-31

    Estrogen promotes the growth of some ovarian cancer cells at nanomolar concentrations, but has been shown to inhibit growth of normal ovarian surface epithelial (OSE) cells at micromolar concentrations (1μg/ml). OSE cells express the estrogen receptor (ER)-α, and are the source of 90% of various cancers. The potential sensitivity of OSE cells to estrogen stresses the importance of understanding the estrogen-dependent mechanisms at play in OSE proliferation and transformation, as well as in anticancer treatment. We investigated the effects of estradiol on cell proliferation in vitro, and demonstrate an intracellular locus of action of estradiol in cultured rhesus ovarian surface epithelial (RhOSE) cells. We show that ovarian and breast cells are growth-inhibited by micromolar concentration of estradiol and that this inhibition correlates with estrogen receptor expression. We further show that normal rhesus OSE cells do not activate ERK or Akt in response to estradiol nor does estradiol block the ability of serum to stimulate ERK or induce cyclin D expression. Contrarily, estradiol inhibits serum-dependent retinoblastoma protein (Rb) phosphorylation and blocks DNA synthesis. This inhibition does not formally arrest cells and is reversible within hours of estrogen withdrawal. Our data are consistent with growth inhibition by activation of Rb and indicate that sensitivity to hormone therapy in anticancer treatment can be modulated by cell cycle regulators downstream of the estrogen receptor.

  6. On-resin Convergent Synthesis of a Glycopeptide from HIV gp120 Containing a High Mannose N-linked Oligosaccharide

    PubMed Central

    Chen, Rui; Tolbert, Thomas J.

    2011-01-01

    Summary/Abstract This method describes a rapid and efficient approach for solid phase synthesis of N-linked glycopeptides which utilizes on-resin glycosylamine coupling to produce N-linked glycosylation sites (1). In this method, the full-length non-glycosylated peptide is first synthesized on solid phase using standard Fmoc chemistry. The glycosylation site is then introduced through an orthogonally protected 2-phenylisopropyl (PhiPr) Aspartic acid (Asp) residue. After selective deprotection of the Asp residue, a high mannose oligosaccharide glycosylamine is coupled on-resin to the free Asp side chain to form a N-glycosidic bond. Subsequent protecting group removal and peptide cleavage from the resin yield the desired glycopeptide. This strategy provides effective glycosylation reactions on the solid phase, simplifies glycopeptide purification relative to solution phase glycopeptide synthesis strategies, and enables recovery of potentially valuable, un-reacted oligosaccharides. This approach has been applied to the solid phase synthesis of the N-linked high mannose glycosylated form of peptide T (ASTTTNYT), a fragment of the HIV-1 envelope glycoprotein gp120 (2–3). PMID:21674342

  7. 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.

  8. A lectin-based cell microarray approach to analyze the mammalian granulosa cell surface glycosylation profile.

    PubMed

    Accogli, Gianluca; Desantis, Salvatore; Martino, Nicola Antonio; Dell'Aquila, Maria Elena; Gemeiner, Peter; Katrlík, Jaroslav

    2016-10-01

    The high complexity of glycome, the repertoire of glycans expressed in a cell or in an organism, is difficult to analyze and the use of new technologies has accelerated the progress of glycomics analysis. In the last decade, the microarray approaches, and in particular glycan and lectin microarrays, have provided new insights into evaluation of cell glycosylation status. Here we present a cell microarray method based on cell printing on microarray slides for the analysis of the glycosylation pattern of the cell glycocalyx. In order to demonstrate the reliability of the developed method, the glycome profiles of equine native uncultured mural granulosa cells (uGCs) and in vitro cultured mural granulosa cells (cGCs) were determined and compared. The method consists in the isolation of GCs, cell printing into arrays on microarray slide, incubation with a panel of biotinylated lectins, reaction with fluorescent streptavidin and signal intensity detection by a microarray scanner. Cell microarray technology revealed that glycocalyx of both uGCs and cGCs contains N-glycans, sialic acid terminating glycans, N-acetylglucosamine and O-glycans. The comparison of uGCs and cGCs glycan signals indicated an increase in the expression of sialic acids, N-acetylglucosamine, and N-glycans in cGCs. Glycan profiles determined by cell microarray agreed with those revealed by lectin histochemistry. The described cell microarray method represents a simple and sensitive procedure to analyze cell surface glycome in mammalian cells.

  9. Mapping cellular hierarchy by single cell analysis of the cell surface repertoire

    PubMed Central

    Guo, Guoji; Luc, Sidinh; Marco, Eugenio; Lin, Ta-Wei; Peng, Cong; Kerenyi, Marc A.; Beyaz, Semir; Kim, Woojin; Xu, Jian; Das, Partha Pratim; Neff, Tobias; Zou, Keyong; Yuan, Guo-Cheng; Orkin, Stuart H.

    2013-01-01

    SUMMARY Stem cell differentiation pathways are most often studied at the population level, whereas critical decisions are executed at the level of single cells. We have established a highly multiplexed, quantitative PCR assay to profile in an unbiased manner a panel of all commonly used cell surface markers (280 genes) from individual cells. With this method we analyzed over 1500 single cells throughout the mouse hematopoietic system, and illustrate its utility for revealing important biological insights. The comprehensive single cell dataset permits mapping of the mouse hematopoietic stem cell (HSC) differentiation hierarchy by computational lineage progression analysis. Further profiling of 180 intracellular regulators enabled construction of a genetic network to assign the earliest differentiation event during hematopoietic lineage specification. Analysis of acute myeloid leukemia elicited by MLL-AF9 uncovered a distinct cellular hierarchy containing two independent self-renewing lineages with different clonal activities. The strategy has broad applicability in other cellular systems. PMID:24035353

  10. MAGIC: an automated N-linked glycoprotein identification tool using a Y1-ion pattern matching algorithm and in silico MS² approach.

    PubMed

    Lynn, Ke-Shiuan; Chen, Chen-Chun; Lih, T Mamie; Cheng, Cheng-Wei; Su, Wan-Chih; Chang, Chun-Hao; Cheng, Chia-Ying; Hsu, Wen-Lian; Chen, Yu-Ju; Sung, Ting-Yi

    2015-02-17

    Glycosylation is a highly complex modification influencing the functions and activities of proteins. Interpretation of intact glycopeptide spectra is crucial but challenging. In this paper, we present a mass spectrometry-based automated glycopeptide identification platform (MAGIC) to identify peptide sequences and glycan compositions directly from intact N-linked glycopeptide collision-induced-dissociation spectra. The identification of the Y1 (peptideY0 + GlcNAc) ion is critical for the correct analysis of unknown glycoproteins, especially without prior knowledge of the proteins and glycans present in the sample. To ensure accurate Y1-ion assignment, we propose a novel algorithm called Trident that detects a triplet pattern corresponding to [Y0, Y1, Y2] or [Y0-NH3, Y0, Y1] from the fragmentation of the common trimannosyl core of N-linked glycopeptides. To facilitate the subsequent peptide sequence identification by common database search engines, MAGIC generates in silico spectra by overwriting the original precursor with the naked peptide m/z and removing all of the glycan-related ions. Finally, MAGIC computes the glycan compositions and ranks them. For the model glycoprotein horseradish peroxidase (HRP) and a 5-glycoprotein mixture, a 2- to 31-fold increase in the relative intensities of the peptide fragments was achieved, which led to the identification of 7 tryptic glycopeptides from HRP and 16 glycopeptides from the mixture via Mascot. In the HeLa cell proteome data set, MAGIC processed over a thousand MS(2) spectra in 3 min on a PC and reported 36 glycopeptides from 26 glycoproteins. Finally, a remarkable false discovery rate of 0 was achieved on the N-glycosylation-free Escherichia coli data set. MAGIC is available at http://ms.iis.sinica.edu.tw/COmics/Software_MAGIC.html .

  11. Genetic ablation of N-linked glycosylation reveals two key folding pathways for R345W fibulin-3, a secreted protein associated with retinal degeneration

    PubMed Central

    Hulleman, John D.; Kelly, Jeffery W.

    2015-01-01

    An R345W mutation in the N-glycoprotein, fibulin-3 (F3), results in inefficient F3 folding/secretion and higher intracellular F3 levels. Inheritance of this mutation causes the retinal dystrophy malattia leventinese. N-Linked glycosylation is a common cotranslational protein modification that can regulate protein folding efficiency and energetics. Therefore, we explored how N-glycosylation alters the protein homeostasis or proteostasis of wild-type (WT) and R345W F3 in ARPE-19 cells. Enzymatic and lectin binding assays confirmed that WT and R345W F3 are both primarily N-glycosylated at Asn249. Tunicamycin treatment selectively reduced R345W F3 secretion by 87% (vs. WT F3). Genetic elimination of F3 N-glycosylation (via an N249Q mutation) caused R345W F3 to aggregate intracellularly and adopt an altered secreted conformation. The endoplasmic reticulum (ER) chaperones GRP78 (glucose-regulated protein 78) and GRP94 (glucose-regulated protein 94), and the ER lectins calnexin and calreticulin were identified as F3 binding partners by immunoprecipitation. Significantly more N249Q and N249Q/R345W F3 interacted with GRP94, while substantially less N249Q and N249Q/R345W interacted with the ER lectins than their N-glycosylated counterparts. Inhibition of GRP94 ATPase activity reduced only N249Q/R345W F3 secretion (by 62%), demonstrating this variant’s unique reliance on GRP94 for secretion. These observations suggest that R345W F3, but not WT F3, requires N-glycosylation to acquire a stable, native-like structure.—Hulleman, J. D., Kelly, J. W. Genetic ablation of N-linked glycosylation reveals two key folding pathways for R345W fibulin-3, a secreted protein associated with retinal degeneration. PMID:25389134

  12. Cancer cell uptake behavior of Au nanoring and its localized surface plasmon resonance induced cell inactivation

    NASA Astrophysics Data System (ADS)

    Chu, Che-Kuan; Tu, Yi-Chou; Chang, Yu-Wei; Chu, Chih-Ken; Chen, Shih-Yang; Chi, Ting-Ta; Kiang, Yean-Woei; Yang, Chih-Chung

    2015-02-01

    Au nanorings (NRIs), which have the localized surface plasmon resonance (LSPR) wavelength around 1058 nm, either with or without linked antibodies, are applied to SAS oral cancer cells for cell inactivation through the LSPR-induced photothermal effect when they are illuminated by a laser of 1065 nm in wavelength. Different incubation times of cells with Au NRIs are considered for observing the variations of cell uptake efficiency of Au NRI and the threshold laser intensity for cell inactivation. In each case of incubation time, the cell sample is washed for evaluating the total Au NRI number per cell adsorbed and internalized by the cells based on inductively coupled plasma mass spectrometry measurement. Also, the Au NRIs remaining on cell membrane are etched with KI/I2 solution to evaluate the internalized Au NRI number per cell. The threshold laser intensities for cell inactivation before washout, after washout, and after KI/I2 etching are calibrated from the circular area sizes of inactivated cells around the illuminated laser spot center with various laser power levels. By using Au NRIs with antibodies, the internalized Au NRI number per cell increases monotonically with incubation time up to 24 h. However, the number of Au NRI remaining on cell membrane reaches a maximum at 12 h in incubation time. The cell uptake behavior of an Au NRI without antibodies is similar to that with antibodies except that the uptake NRI number is significantly smaller and the incubation time for the maximum NRI number remaining on cell membrane is delayed to 20 h. By comparing the threshold laser intensities before and after KI/I2 etching, it is found that the Au NRIs remaining on cell membrane cause more effective cancer cell inactivation, when compared with the internalized Au NRIs.

  13. Surface and allied studies in silicon solar cells

    NASA Technical Reports Server (NTRS)

    Lindholm, F. A.

    1984-01-01

    Measuring small-signal admittance versus frequency and forward bias voltage together with a new transient measurement apparently provides the most reliable and flexible method available for determining back surface recombination velocity and low-injection lifetime of the quasineutral base region of silicon solar cells. The new transient measurement reported here is called short-circuit-current decay (SCCD). In this method, forward voltage equal to about the open-circuit or the maximum power voltage establishes excess holes and electrons in the junction transition region and in the quasineutral regions. The sudden application of a short circuit causes an exiting of the excess holes and electrons in the transition region within about ten picoseconds. From observing the slope and intercept of the subsequent current decay, the base lifetime and surface recombination velocity can be determined. The admittance measurement previously mentioned then enters to increase accuracy particularly for devices for which the diffusion length exceeds the base thickness.

  14. Surface modification of carbon fuels for direct carbon fuel cells

    NASA Astrophysics Data System (ADS)

    Li, Xiang; Zhu, Zhonghua; Chen, Jiuling; De Marco, Roland; Dicks, Andrew; Bradley, John; Lu, Gaoqing

    The direct carbon fuel cell (DCFC) is a promising power-generation device that has much higher efficiency (80%) and less emissions than conventional coal-fired power plants. Two commercial carbons (activated carbon and carbon black) pre-treated with HNO 3, HCl or air plasma are tested in a DCFC. The correlation between the surface properties and electrochemical performance of the carbon fuels is explored. The HNO 3-treated carbon fuels have the highest electrochemical reactivity in the DCFC due to the largest degree of surface oxygen functional groups. The overall effect on changing the electrochemical reactivity of carbon fuels is in the order HNO 3 > air plasma ≈ HCl. Product gas analysis indicates that complete oxidation of carbon to CO 2 can be achieved at 600-700 °C.

  15. Islet Cell Surface Antibodies from Insulin-dependent Diabetics Bind Specifically to Pancreatic B Cells

    PubMed Central

    Van De Winkel, M.; Smets, G.; Gepts, W.; Pipeleers, D.

    1982-01-01

    Viable rat islet cells were used to detect islet cell surface antibodies (ICSA) in the sera of diabetic and control patients. ICSA were present in almost all recent-onset insulin-dependent diabetics younger than 30 yr (15/16); their incidence in other diabetics (6/22) was also higher than in normal controls (1/18) or in patients with autoimmune thyroiditis (1/12). The varying specificity of the ICSA for the different islet cell types led to the recognition of class I sera, whose ICSA bind exclusively to B cells, class II sera, binding only to A and pancreatic polypeptide (PP) cells and class III sera, reacting with the three islet cell types but not with D cells. Most recent-onset insulin-dependent diabetics younger than 30 contained class I-ICSA, which is consistent with an autoimmune basis of their disease and with an involvement of surface antibodies in the B cell destruction. The presence of class II ICSA in three older diabetics and in one normal control raises the question whether autoimmune reactions against A and PP cells exist and are associated with a distinct entity in islet disease. It is concluded that the autoimmune form of diabetes mellitus represents a heterogeneous group, in which ICSA-positive patients can be distinguished on the basis of their ICSA-binding to one or more islet cell types. Three techniques can be used for the further identification of circulating ICSA, namely binding experiments with purified A or B cells, electron microscopical analysis of ICSA-binding islet cells purified by fluorescence-activated cell sorting, and the immunocytochemical characterization of ICSA-positive cells. Images PMID:6123526

  16. Role of N-glycosylation in cell surface expression and protection against proteolysis of the intestinal anion exchanger SLC26A3.

    PubMed

    Hayashi, Hisayoshi; Yamashita, Yukari

    2012-03-01

    SLC26A3 is a Cl(-)/HCO(3)(-) exchanger that plays a major role in Cl(-) absorption from the intestine. Its mutation causes congenital chloride-losing diarrhea. It has been shown that SLC26A3 are glycosylated, with the attached carbohydrate being extracellular and perhaps modulating function. However, the role of glycosylation has yet to be clearly determined. We used the approaches of biochemical modification and site-directed mutagenesis to prevent glycosylation. Deglycosylation experiments with glycosidases indicated that the mature glycosylated form of SLC26A3 exists at the plasma membrane, and a putative large second extracellular loop contains all of the N-linked carbohydrates. Deglycosylation of SLC26A3 causes depression of transport activity compared with wild-type, although robust intracellular pH changes were still observed, suggesting that N-glycosylation is not absolutely necessary for transport activity. To localize glycosylation sites, we mutated the five consensus sites by replacing asparagine (N) with glutamine. Immnoblotting suggests that SLC26A3 is glycosylated at N153, N161, and N165. Deglycosylation of SLC26A3 causes a defect in cell surface processing with decreased cell surface expression. We also assessed whether SLC26A3 is protected from tryptic digestion. While the mature glycosylated SLC26A3 showed little breakdown after treatment with trypsin, deglycosylated SLC26A3 exhibited increased susceptibility to trypsin, suggesting that the oligosaccharides protect SLC26A3 from tryptic digestion. In conclusion, our data indicate that N-glycosylation of SLC26A3 is important for cell surface expression and for protection from proteolytic degradation that may contribute to the understanding of pathogenesis of congenital disorders of glycosylation.

  17. SPE (tm) regenerative hydrogen/oxygen fuel cells for extraterrestrial surface and microgravity applications

    NASA Technical Reports Server (NTRS)

    Mcelroy, J. F.

    1990-01-01

    Viewgraphs on SPE regenerative hydrogen/oxygen fuel cells for extraterrestrial surface and microgravity applications are presented. Topics covered include: hydrogen-oxygen regenerative fuel cell energy storage system; electrochemical cell reactions; SPE cell voltage stability; passive water removal SPE fuel cell; fuel cell performance; SPE water electrolyzers; hydrophobic oxygen phase separator; hydrophilic/electrochemical hydrogen phase separator; and unitized regenerative fuel cell.

  18. 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.

  19. Cell receptor and surface ligand density effects on dynamic states of adhering circulating tumor cells.

    PubMed

    Zheng, Xiangjun; Cheung, Luthur Siu-Lun; Schroeder, Joyce A; Jiang, Linan; Zohar, Yitshak

    2011-10-21

    Dynamic states of cancer cells moving under shear flow in an antibody-functionalized microchannel are investigated experimentally and theoretically. The cell motion is analyzed with the aid of a simplified physical model featuring a receptor-coated rigid sphere moving above a solid surface with immobilized ligands. The motion of the sphere is described by the Langevin equation accounting for the hydrodynamic loadings, gravitational force, receptor-ligand bindings, and thermal fluctuations; the receptor-ligand bonds are modeled as linear springs. Depending on the applied shear flow rate, three dynamic states of cell motion have been identified: (i) free motion, (ii) rolling adhesion, and (iii) firm adhesion. Of particular interest is the fraction of captured circulating tumor cells, defined as the capture ratio, via specific receptor-ligand bonds. The cell capture ratio decreases with increasing shear flow rate with a characteristic rate. Based on both experimental and theoretical results, the characteristic flow rate increases monotonically with increasing either cell-receptor or surface-ligand density within certain ranges. Utilizing it as a scaling parameter, flow-rate dependent capture ratios for various cell-surface combinations collapse onto a single curve described by an exponential formula.

  20. Cell surface sialylation affects binding of enterovirus 71 to rhabdomyosarcoma and neuroblastoma cells

    PubMed Central

    2012-01-01

    Background Enterovirus 71 (EV71) is a major causative agent of hand-foot-and-mouth disease (HFMD), and infection of EV71 to central nerve system (CNS) may result in a high mortality in children less than 2 years old. Although there are two highly glycosylated membrane proteins, SCARB2 and PSGL-1, which have been identified as the cellular and functional receptors of EV71, the role of glycosylation in EV71 infection is still unclear. Results We demonstrated that the attachment of EV71 to RD and SK-N-SH cells was diminished after the removal of cell surface sialic acids by neuraminidase. Sialic acid specific lectins, Maackia amurensis (MAA) and Sambucus Nigra (SNA), could compete with EV71 and restrained the binding of EV71 significantly. Preincubation of RD cells with fetuin also reduced the binding of EV71. In addition, we found that SCARB2 was a sialylated glycoprotein and interaction between SCARB2 and EV71 was retarded after desialylation. Conclusions In this study, we demonstrated that cell surface sialic acids assist in the attachment of EV71 to host cells. Cell surface sialylation should be a key regulator that facilitates the binding and infection of EV71 to RD and SK-N-SH cells. PMID:22853823

  1. Fibronectin biosynthesis and cell-surface expression by cardiac and non-cardiac endothelial cells.

    PubMed Central

    Johnson, C. M.; Helgeson, S. C.

    1993-01-01

    We examined the biosynthesis and surface expression of fibronectin, an adhesive glycoprotein, in several types of cultured porcine endothelial cells: pulmonary artery, thoracic aorta, coronary artery, aortic valve, and mitral valve. We used immunocytochemical staining to compare the levels of fibronectin present in these same tissues in vivo. Using endogenous radiolabeling, we found that all cell types except aortic valve endothelial cells synthesized and released into the culture media substantial quantities of fibronectin. Using radioiodination of intact cells, we found that, whereas both thoracic aorta and pulmonary artery cells had measurable fibronectin on the surface, aortic valve, mitral valve, and coronary artery cells had little cell-surface fibronectin present. Immunocytochemical staining showed that all endothelial regions except aortic valve had substantial quantities of immunoreactive fibronectin in vivo. These data suggest that the aortic valve endothelium may be distinct from other endothelia. Such differences could be important for the pathogenesis of valvular disease. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8494044

  2. Identification of a cell-surface antigen selectively expressed on the natural killer cell

    PubMed Central

    1977-01-01

    We have studied the cell-surface phenotype of natural killer (NK) cells of NZB and B6 mice which react to an MuLV+ lymphoid tumor. (a) NK cells do not express Thy1, Ly2, or Ig surface markers. (b) NK cells express an antigen recognized by C3H anti-CE antiserum ('anti-Ly1.2 antiserum'). Inasmuch as NK activity of spleen cells from B6 and B6/Ly1.1 congenic strains were both equally sensitive to C3H anti-CE antiserum, the NK antigen is distinct from Ly1.2. This point was confirmed by the observation that alphaNK activity was removed by absorption of C3H anti-CE antiserum with spleen cells from either B6 or B6/Ly1.1 congenic strains. Absorption of C3H alphaCE serum with BALB/c thymocytes and spleen cells (which are Ly1.2+NK-) removed anti-Ly1.2 activity and left anti-NK activity intact. This absorption step could be circumvented by inserting the BALB/c genotype into the recipient immunized to CE cells (i.e., (C3H X BALB/c)F1 alphaCE spleen cells). This antiserum, provisionally termed 'anti-NK', defines a new subclass of lymphocytes which may play a central role in the immunosurveillance against tumors. PMID:187714

  3. Differential carbohydrate binding and cell surface glycosylation of human cancer cell lines.

    PubMed

    Arndt, Nadia X; Tiralongo, Joe; Madge, Paul D; von Itzstein, Mark; Day, Christopher J

    2011-09-01

    Currently there is only a modest level knowledge of the glycosylation status of immortalised cell lines that are commonly used in cancer biology as well as their binding affinities to different glycan structures. Through use of glycan and lectin microarray technology, this study has endeavoured to define the different bindings of cell surface carbohydrate structures to glycan-binding lectins. The screening of breast cancer MDA-MB435 cells, cervical cancer HeLa cells and colon cancer Caco-2, HCT116 and HCT116-FM6 cells was conducted to determine their differential bindings to a variety of glycan and lectin structures printed on the array slides. An inverse relationship between the number of glycan structures recognised and the variety of cell surface glycosylation was observed. Of the cell lines tested, it was found that four bound to sialylated structures in initial screening. Secondary screening in the presence of a neuraminidase inhibitor (4-deoxy-4-guanidino-Neu5Ac2en) significantly reduced sialic acid binding. The array technology has proven to be useful in determining the glycosylation signatures of various cell-lines as well as their glycan binding preferences. The findings of this study provide the groundwork for further investigation into the numerous glycan-lectin interactions that are exhibited by immortalised cell lines.

  4. Surface and allied studies in silicon solar cells

    NASA Technical Reports Server (NTRS)

    Lindholm, F. A.

    1984-01-01

    Significant improvements were made in the short-circuit current-decay method of measuring the recombination lifetime tau and the back surface recombination velocity S of the quasineutral base of silicon solar cells. The improvements include a circuit implementation that increases the speed of switching from the forward-voltage to the short-circuit conditions. They also include a supplementation of this method by some newly developed techniques employing small-signal admittance as a function of frequency omega. This supplementation is highly effective for determining tau for cases in which the diffusion length L greatly exceeds the base thickness W. Representative results on different solar cells are reported. Some advances made in the understanding of passivation provided by the polysilicon/silicon heterojunction are outlined. Recent measurements demonstrate that S 10,000 cm/s derive from this method of passivation.

  5. 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.

  6. Cell/surface interactions and adhesion on bioactive glass 45S5.

    PubMed

    Levy, S; Van Dalen, M; Agonafer, S; Soboyejo, W O

    2007-01-01

    This paper examines the effects of surface texture (smooth versus rough) on cell/surface interactions on the bioactive glass, 45S5. The cell surface interactions associated with cell spreading are studied using cell culture experiments. Subsequent energy dispersive x-ray spectroscopy is also used to reveal the distributions of calcium, phosphorous, sodium and oxygen on the surfaces of the bioactive glasses. The implications of the results are then discussed for the applications of textured bioactive glasses in medicine.

  7. Measurement of diffusion length and surface recombination velocity in Interdigitated Back Contact (IBC) and Front Surface Field (FSF) solar cells

    NASA Astrophysics Data System (ADS)

    Verlinden, Pierre; Van de Wiele, Fernand

    1985-03-01

    A method is proposed for measuring the diffusion length and surface recombination velocity of Interdigitated Back Contact (IBC) solar cells by means of a simple linear regression on experimental quantum efficiency values versus the inverse of the absorption coefficient. This method is extended to the case of Front Surface Field (FSF) solar cells. Under certain conditions, the real or the effective surface recombination velocity may be measured.

  8. Cell-surface translational dynamics of nicotinic acetylcholine receptors

    PubMed Central

    Barrantes, Francisco J.

    2014-01-01

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

  9. Control of cell behavior on PTFE surface using ion beam irradiation

    NASA Astrophysics Data System (ADS)

    Kitamura, Akane; Kobayashi, Tomohiro; Meguro, Takashi; Suzuki, Akihiro; Terai, Takayuki

    2009-05-01

    A polytetrafluoroethylene (PTFE) surface is smooth and biologically inert, so that cells cannot attach to it. Ion beam irradiation of the PTFE surface forms micropores and a melted layer, and the surface is finally covered with a large number of small protrusions. Recently, we found that cells could adhere to this irradiated PTFE surface and spread over the surface. Because of their peculiar attachment behavior, these surfaces can be used as biological tools. However, the factors regulating cell adhesion are still unclear, although some new functional groups formed by irradiation seem to contribute to this adhesion. To control cell behavior on PTFE surfaces, we must determine the effects of the outermost irradiated surface on cell adhesion. In this study, we removed the thin melted surface layer by postirradiation annealing and investigated cell behavior on the surface. On the surface irradiated with 3 × 1016 ions/cm2, cells spread only on the remaining parts of the melted layer. From these results, it is clear that the melted layer had a capacity for cell attachment. When the surface covered with protrusions was irradiated with a fluence of 1 × 1017 ions/cm2, the distribution of cells changed after the annealing process from 'sheet shaped' into multicellular aggregates with diameters of around 50 μm. These results indicate that we can control cell behavior on PTFE surfaces covered with protrusions using irradiation and subsequent annealing. Multicellular spheroids can be fabricated for tissue engineering using this surface.

  10. 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.

  11. Targeting Cell Surface Proteins in Molecular Photoacoustic Imaging to Detect Ovarian Cancer Early

    DTIC Science & Technology

    2012-07-01

    10-1-0422 TITLE: Targeting Cell Surface Proteins in Molecular Photoacoustic Imaging to Detect Ovarian Cancer Early PRINCIPAL...molecular imaging 7 cdrescher@fhcrc.org Targeting Cell Surface Proteins in Molecular Photoacoustic Imaging to Detect Ovarian Cancer Early Page 3...Targeting Cell Surface Proteins in Molecular Photoacoustic Imaging to Detect Ovarian Cancer Early Charles W Drescher, MD, Principle Investigator

  12. Proteomic Analyses Reveal Common Promiscuous Patterns of Cell Surface Proteins on Human Embryonic Stem Cells and Sperms

    PubMed Central

    Gu, Bin; Zhang, Jiarong; Wu, Ying; Zhang, Xinzong; Tan, Zhou; Lin, Yuanji; Huang, Xiao; Chen, Liangbiao; Yao, Kangshou; Zhang, Ming

    2011-01-01

    Background It has long been proposed that early embryos and reproductive organs exhibit similar gene expression profiles. However, whether this similarity is propagated to the protein level remains largely unknown. We have previously characterised the promiscuous expression pattern of cell surface proteins on mouse embryonic stem (mES) cells. As cell surface proteins also play critical functions in human embryonic stem (hES) cells and germ cells, it is important to reveal whether a promiscuous pattern of cell surface proteins also exists for these cells. Methods and Principal Findings Surface proteins of hES cells and human mature sperms (hSperms) were purified by biotin labelling and subjected to proteomic analyses. More than 1000 transmembrane or secreted cell surface proteins were identified on the two cell types, respectively. Proteins from both cell types covered a large variety of functional categories including signal transduction, adhesion and transporting. Moreover, both cell types promiscuously expressed a wide variety of tissue specific surface proteins, and some surface proteins were heterogeneously expressed. Conclusions/Significance Our findings indicate that the promiscuous expression of functional and tissue specific cell surface proteins may be a common pattern in embryonic stem cells and germ cells. The conservation of gene expression patterns between early embryonic cells and reproductive cells is propagated to the protein level. These results have deep implications for the cell surface signature characterisation of pluripotent stem cells and germ cells and may lead the way to a new area of study, i.e., the functional significance of promiscuous gene expression in pluripotent and germ cells. PMID:21559292

  13. Responses of endothelial cells, smooth muscle cells, and platelets dependent on the surface topography of polytetrafluoroethylene.

    PubMed

    Lamichhane, Sujan; Anderson, Jordan A; Remund, Tyler; Sun, Hongli; Larson, Mark K; Kelly, Patrick; Mani, Gopinath

    2016-09-01

    In this study, the effect of different structures (flat, expanded, and electrospun) of polytetrafluoroethylene (PTFE) on the interactions of endothelial cells (ECs), smooth muscle cells (SMCs), and platelets was investigated. In addition, the mechanisms that govern the interactions between ECs, SMCs, and platelets with different structures of PTFE were discussed. The surface characterizations showed that the different structures of PTFE have the same surface chemistry, similar surface wettability and zeta potential, but uniquely different surface topography. The viability, proliferation, morphology, and phenotype of ECs and SMCs interacted with different structures of PTFE were investigated. Expanded PTFE (ePTFE) provided a relatively better surface for the growth of ECs. In case of SMC interactions, although all the different structures of PTFE inhibited SMC growth, a maximum inhibitory effect was observed for ePTFE. In case of platelet interactions, the electrospun PTFE provided a better surface for preventing the adhesion and activation of platelets. Thus, this study demonstrated that the responses of ECs, SMCs, and platelets strongly dependent on the surface topography of the PTFE. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2291-2304, 2016.

  14. Neisseria lactamica selectively induces mitogenic proliferation of the naive B cell pool via cell surface Ig.

    PubMed

    Vaughan, Andrew T; Brackenbury, Louise S; Massari, Paola; Davenport, Victoria; Gorringe, Andrew; Heyderman, Robert S; Williams, Neil A

    2010-09-15

    Neisseria lactamica is a commensal bacteria that colonizes the human upper respiratory tract mucosa during early childhood. In contrast to the closely related opportunistic pathogen Neisseria meningitidis, there is an absence of adaptive cell-mediated immunity to N. lactamica during the peak age of carriage. Instead, outer membrane vesicles derived from N. lactamica mediate a B cell-dependent proliferative response in mucosal mononuclear cells that is associated with the production of polyclonal IgM. We demonstrate in this study that this is a mitogenic human B cell response that occurs independently of T cell help and any other accessory cell population. The ability to drive B cell proliferation is a highly conserved property and is present in N. lactamica strains derived from diverse clonal complexes. CFSE staining of purified human tonsillar B cells demonstrated that naive IgD(+) and CD27(-) B cells are selectively induced to proliferate by outer membrane vesicles, including the innate CD5(+) subset. Neither purified lipooligosaccharide nor PorB from N. lactamica is likely to be responsible for this activity. Prior treatment of B cells with pronase to remove cell-surface Ig or treatment with BCR-specific Abs abrogated the proliferative response to N. lactamica outer membrane vesicles, suggesting that this mitogenic response is dependent upon the BCR.

  15. Engineered antifouling microtopographies: surface pattern effects on cell distribution.

    PubMed

    Decker, Joseph T; Sheats, Julian T; Brennan, Anthony B

    2014-12-23

    Microtopography has been observed to lead to altered attachment behavior for marine fouling organisms; however, quantification of this phenomenon is lacking in the scientific literature. Here, we present quantitative measurement of the disruption of normal attachment behavior of the fouling algae Ulva linza by antifouling microtopographies. The distribution of the diatom Navicula incerta was shown to be unaffected by the presence of topography. The radial distribution function was calculated for both individual zoospores and cells as well as aggregates of zoospores from attachment data for a variety topographic configurations and at a number of different attachment densities. Additionally, the screening distance and maximum values were mapped according to the location of zoospore aggregates within a single unit cell. We found that engineered topographies decreased the distance between spore aggregates compared to that for a smooth control surface; however, the distributions for individual spores were unchanged. We also found that the local attachment site geometry affected the screening distance for aggregates of zoospores, with certain geometries decreasing screening distance and others having no measurable effect. The distribution mapping techniques developed and explored in this article have yielded important insight into the design parameters for antifouling microtopographies that can be implemented in the next generation of antifouling surfaces.

  16. Neutrophil cell surface receptors and their intracellular signal transduction pathways☆

    PubMed Central

    Futosi, Krisztina; Fodor, Szabina; Mócsai, Attila

    2013-01-01

    Neutrophils play a critical role in the host defense against bacterial and fungal infections, but their inappropriate activation also contributes to tissue damage during autoimmune and inflammatory diseases. Neutrophils express a large number of cell surface receptors for the recognition of pathogen invasion and the inflammatory environment. Those include G-protein-coupled chemokine and chemoattractant receptors, Fc-receptors, adhesion receptors such as selectins/selectin ligands and integrins, various cytokine receptors, as well as innate immune receptors such as Toll-like receptors and C-type lectins. The various cell surface receptors trigger very diverse signal transduction pathways including activation of heterotrimeric and monomeric G-proteins, receptor-induced and store-operated Ca2 + signals, protein and lipid kinases, adapter proteins and cytoskeletal rearrangement. Here we provide an overview of the receptors involved in neutrophil activation and the intracellular signal transduction processes they trigger. This knowledge is crucial for understanding how neutrophils participate in antimicrobial host defense and inflammatory tissue damage and may also point to possible future targets of the pharmacological therapy of neutrophil-mediated autoimmune or inflammatory diseases. PMID:23994464

  17. Cell surface glycoproteins of CHO cells. I. Internalization and rapid recycling

    SciTech Connect

    Raub, T.J.; Denny, J.B.; Roberts, R.M.

    1986-01-01

    The major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 4/sup 0/C with the membrane-impermeant reagent, trinitrobenzenesulfonate (TNBS). Immunoprecipitation and subsequent two-dimensional, sodiumdodecyl sulfate, polyacrylamide gel electrophoresis (SDS-PAGE) of proteins from derivatized cells that had been labelled previously with (/sup 3/H)D-glucosamine or (/sup 3/H)L-leucine showed that TNBS reacted with most of the high molecular weight (HMW) acidic glycoproteins that became labelled with iodine by the lactoperoxidase technique and that bind the lectin, wheat germ agglutinin (WGA). After warming the cells to allow endocytosis to proceed, molecule haptenized with trinitrophenol (TNP) groups were followed radio-chemically by means of (/sup 125/I)anti-DNP antibodies. Within 15 min at 37/sup 0/C, a steady-state between surface and cytoplasmic label was reached, with about 65% of the hapten located internally. Recycling of internalized TNP groups back to the cell surface also occurred rapidly (t/sub 1/2/ approx. 5 min). Our results are consistent with the view that the majority of plasma membrane glycoproteins are continuously being internalized and recycled at a high rate.

  18. Heat-transfer-method-based cell culture quality assay through cell detection by surface imprinted polymers.

    PubMed

    Eersels, Kasper; van Grinsven, Bart; Khorshid, Mehran; Somers, Veerle; Püttmann, Christiane; Stein, Christoph; Barth, Stefan; Diliën, Hanne; Bos, Gerard M J; Germeraad, Wilfred T V; Cleij, Thomas J; Thoelen, Ronald; De Ceuninck, Ward; Wagner, Patrick

    2015-02-17

    Previous work has indicated that surface imprinted polymers (SIPs) allow for highly specific cell detection through macromolecular cell imprints. The combination of SIPs with a heat-transfer-based read-out technique has led to the development of a selective, label-free, low-cost, and user-friendly cell detection assay. In this study, the breast cancer cell line ZR-75-1 is used to assess the potential of the platform for monitoring the quality of a cell culture in time. For this purpose, we show that the proposed methodology is able to discriminate between the original cell line (adherent growth, ZR-75-1a) and a descendant cell line (suspension growth, ZR-75-1s). Moreover, ZR-75-1a cells were cultured for a prolonged period of time and analyzed using the heat-transfer method (HTM) at regular time intervals. The results of these experiments demonstrate that the thermal resistance (Rth) signal decays after a certain number of cell culture passages. This can likely be attributed to a compromised quality of the cell culture due to cross-contamination with the ZR-75-1s cell line, a finding that was confirmed by classical STR DNA profiling. The cells do not express the same functional groups on their membrane, resulting in a weaker bond between cell and imprint, enabling cell removal by mechanical friction, provided by flushing the measuring chamber with buffer solution. These findings were further confirmed by HTM and illustrate that the biomimetic sensor platform can be used as an assay for monitoring the quality of cell cultures in time.

  19. Cell surface receptor targeted biomimetic apatite nanocrystals for cancer therapy.

    PubMed

    Iafisco, Michele; Delgado-Lopez, Josè Manuel; Varoni, Elena Maria; Tampieri, Anna; Rimondini, Lia; Gomez-Morales, Jaime; Prat, Maria

    2013-11-25

    Nanosized drug carriers functionalized with moieties specifically targeting tumor cells are promising tools in cancer therapy, due to their ability to circulate in the bloodstream for longer periods and their selectivity for tumor cells, enabling the sparing of healthy tissues. Because of its biocompatibility, high bioresorbability, and responsiveness to pH changes, synthetic biomimetic nanocrystalline apatites are used as nanocarriers to produce multifunctional nanoparticles, by coupling them with the chemotherapeutic drug doxorubicin (DOXO) and the DO-24 monoclonal antibody (mAb) directed against the Met/Hepatocyte Growth Factor receptor (Met/HGFR), which is over-expressed on different types of carcinomas and thus represents a useful tumor target. The chemical-physical features of the nanoparticles are fully investigated and their interaction with cells expressing (GTL-16 gastric carcinoma line) or not expressing (NIH-3T3 fibroblasts) the Met/HGFR is analyzed. Functionalized nanoparticles specifically bind to and are internalized in cells expressing the receptor (GTL-16) but not in the ones that do not express it (NIH-3T3). Moreover they discharge DOXO in the targeted GTL-16 cells that reach the nucleus and display cytotoxicity as assessed in an MTT assay. Two different types of ternary nanoparticles are prepared, differing for the sequence of the functionalization steps (adsorption of DOXO first and then mAb or vice versa), and it is found that the ones in which mAb is adsorbed first are more efficient under all the examined aspects (binding, internalization, cytotoxicity), possibly because of a better mAb orientation on the nanoparticle surface. These multifunctional nanoparticles could thus be useful instruments for targeted local or systemic drug delivery, allowing a reduction in the therapeutic dose of the drug and thus adverse side effects. Moreover, this work opens new perspectives in the use of nanocrystalline apatites as a new platform for theranostic

  20. 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

    2014-12-09

    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.

  1. Engineering Cell Instructive Materials To Control Cell Fate and Functions through Material Cues and Surface Patterning.

    PubMed

    Ventre, Maurizio; Netti, Paolo A

    2016-06-22

    Mastering the interaction between cells and extracellular environment is a fundamental prerequisite in order to engineer functional biomaterial interfaces able to instruct cells with specific commands. Such advanced biomaterials might find relevant application in prosthesis design, tissue engineering, diagnostics and stem cell biology. Because of the highly complex, dynamic, and multifaceted context, a thorough understanding of the cell-material crosstalk has not been achieved yet; however, a variety of material features including biological cues, topography, and mechanical properties have been proved to impact the strength and the nature of the cell-material interaction, eventually affecting cell fate and functions. Although the nature of these three signals may appear very different, they are equated by their participation in the same material-cytoskeleton crosstalk pathway as they regulate cell adhesion events. In this work we present recent and relevant findings on the material-induced cell responses, with a particular emphasis on how the presentation of biochemical/biophysical signals modulates cell behavior. Finally, we summarize and discuss the literature data to draw out unifying elements concerning cell recognition of and reaction to signals displayed by material surfaces.

  2. Hyaluronan synthesis induces microvillus-like cell surface protrusions.

    PubMed

    Kultti, Anne; Rilla, Kirsi; Tiihonen, Riikka; Spicer, Andrew P; Tammi, Raija H; Tammi, Markku I

    2006-06-09

    Hyaluronan synthases (HASs) are plasma membrane enzymes that simultaneously elongate, bind, and extrude the growing hyaluronan chain directly into extracellular space. In cells transfected with green fluorescent protein (GFP)-tagged Has3, the dorsal surface was decorated by up to 150 slender, 3-20-microm-long microvillus-type plasma membrane protrusions, which also contained filamentous actin, the hyaluronan receptor CD44, and lipid raft microdomains. Enzymatic activity of HAS was required for the growth of the microvilli, which were not present in cells transfected with other GFP proteins or inactive GFP-Has3 mutants or in cells incubated with exogenous soluble hyaluronan. The microvilli induced by HAS3 were gradually withered by introduction of an inhibitor of hyaluronan synthesis and rapidly retracted by hyaluronidase digestion, whereas they were not affected by competition with hyaluronan oligosaccharides and disruption of the CD44 gene, suggesting independence of hyaluronan receptors. The data bring out the novel concept that the glycocalyx created by dense arrays of hyaluronan chains, tethered to HAS during biosynthesis, can induce and maintain prominent microvilli.

  3. Decreased tumorigenicity correlates with expression of altered cell surface carbohydrates in Lec9 CHO cells.

    PubMed Central

    Ripka, J; Shin, S; Stanley, P

    1986-01-01

    To investigate a role for surface carbohydrates in cellular malignancy, 15 different glycosylation-defective CHO cell mutants were examined for their tumorigenic and metastatic capacities after subcutaneous injection into nude mice. Most of the glycosylation mutants displayed similar or slightly decreased tumorigenicity compared with parental CHO cells. Neither parental CHO cells nor any of the mutants were observed to metastasize. However, independent isolates of one mutant type, Lec9, showed a dramatic reduction in tumor formation. The altered carbohydrates expressed at the surface of Lec9 cells appeared to be responsible for their loss of tumorigenicity, because revertants for lectin resistance were able to form tumors, and a double mutant (Lec9.Lec1) that expressed a Lec1 glycosylation phenotype also formed tumors. Finally, Lec9 cells were able to form tumors in gamma-irradiated nude mice, suggesting that recognition by an irradiation-sensitive host cell(s) was responsible for their reduced tumorigenicity in untreated nude mice. PMID:3785164

  4. Separation and reformation of cell surface dopamine receptor oligomers visualized in cells.

    PubMed

    O'Dowd, Brian F; Ji, Xiaodong; Alijaniaram, Mohammad; Nguyen, Tuan; George, Susan R

    2011-05-11

    We previously showed that dopamine receptors existed as homo- and heterooligomers, in cells and in brain tissue. We developed a method designed to study the formation and regulation of G protein coupled receptor (GPCR) oligomers in cells, using a GPCR into which a nuclear localization sequence (NLS) had been inserted. Unlike wildtype GPCRs, in the presence of agonist/antagonist ligands the GPCR-NLS is retained at the cell surface, and following ligand removal, the GPCR-NLS translocated from the cell surface. The D(1) dopamine receptor expressed with either D(2)-NLS or D(1-)NLS receptors translocated to the nucleus, indicating hetero- or homo-oligomerization with the NLS-containing receptor. Using these tools, we now demonstrate that D(1)-D(2) dopamine heterooligomers can be disrupted and the component receptors separated by dopamine and selective agonists that occupied one or both binding pockets. Subsequent agonist removal allowed the reformation of the heterooligomer. D(1) receptor homooligomers could also be disrupted by agonist, but at higher concentrations than that required for the disruption of the D(1)-D(2) heteromer. Dopamine D(1) or D(2) receptor antagonists had no effect on the integrity of the homo- or heterooligomer. We have also determined that the D(1)-D(2) heterooligomer contains D(1) homooligomers. These studies indicate that the populations of dopamine receptor oligomers at the cell surface are subject to conformational changes following agonist occupancy and are likely dynamically regulated following agonist activation.

  5. High cell-surface density of HER2 deforms cell membranes

    PubMed Central

    Chung, Inhee; Reichelt, Mike; Shao, Lily; Akita, Robert W.; Koeppen, Hartmut; Rangell, Linda; Schaefer, Gabriele; Mellman, Ira; Sliwkowski, Mark X.

    2016-01-01

    Breast cancers (BC) with HER2 overexpression (referred to as HER2 positive) progress more aggressively than those with normal expression. Targeted therapies against HER2 can successfully delay the progression of HER2-positive BC, but details of how this overexpression drives the disease are not fully understood. Using single-molecule biophysical approaches, we discovered a new effect of HER2 overexpression on disease-relevant cell biological changes in these BC. We found HER2 overexpression causes deformation of the cell membranes, and this in turn disrupts epithelial features by perturbing cell–substrate and cell–cell contacts. This membrane deformation does not require receptor signalling activities, but results from the high levels of HER2 on the cell surface. Our finding suggests that early-stage morphological alterations of HER2-positive BC cells during cancer progression can occur in a physical and signalling-independent manner. PMID:27599456

  6. HOS cell adhesion on Ti6Al4V surfaces texturized by laser engraving

    NASA Astrophysics Data System (ADS)

    Sandoval Amador, A.; Carreño Garcia, H.; Escobar Rivero, P.; Peña Ballesteros, D. Y.; Estupiñán Duran, H. A.

    2016-02-01

    The cell adhesion of the implant is determinate by the chemical composition, topography, wettability, surface energy and biocompatibility of the biomaterial. In this work the interaction between human osteosarcoma HOS cells and textured Ti6Al4V surfaces were evaluated. Ti6Al4V surfaces were textured using a CO2 laser in order to obtain circular spots on the surfaces. Test surfaces were uncoated (C1) used as a control surface, and surfaces with points obtained by laser engraving, with 1mm spacing (C2) and 0.5mm (C3). The HOS cells were cultured in RPMI-1640 medium with 10% fetal bovine serum and 1% antibiotics. No cells toxicity after one month incubation time occurred. The increased cell adhesion and cell spreading was observed after 1, 3 and 5 days without significant differences between the sample surfaces (C2 and C3) and control (uncoated) at the end of the experiment.

  7. Back surface cell structures for reducing recombination in CZ silicon solar cells

    NASA Astrophysics Data System (ADS)

    King, R. R.; Mitchell, K. W.; Gee, J. M.

    1994-12-01

    Mass-produced terrestrial CZ silicon solar cells are currently entering the domain in which bulk diffusion length is comparable to the cell thickness, so that recombination at the back surface can have a significant effect on device performance. Three manufacturable processes that address the problem of back recombination are examined here: boron diffusion from a deposited doped SiO2, layer; Al-alloyed layers using screen-printed paste; and use of a collecting n* layer on the back interdigitated with the positive electrode. 104 sq cm cells fabricated at Siemens Solar Industries using these back surface structures are characterized by current-voltage, spectral response, photoconductivity decay, and SIMS measurements.

  8. Nutrient-deprived cancer cells preferentially use sialic acid to maintain cell surface glycosylation.

    PubMed

    Badr, Haitham A; AlSadek, Dina M M; Mathew, Mohit P; Li, Chen-Zhong; Djansugurova, Leyla B; Yarema, Kevin J; Ahmed, Hafiz

    2015-11-01

    Cancer is characterized by abnormal energy metabolism shaped by nutrient deprivation that malignant cells experience during various stages of tumor development. This study investigated the response of nutrient-deprived cancer cells and their non-malignant counterparts to sialic acid supplementation and found that cells utilize negligible amounts of this sugar for energy. Instead cells use sialic acid to maintain cell surface glycosylation through complementary mechanisms. First, levels of key metabolites (e.g., UDP-GlcNAc and CMP-Neu5Ac) required for glycan biosynthesis are maintained or enhanced upon Neu5Ac supplementation. In concert, sialyltransferase expression increased at both the mRNA and protein levels, which facilitated increased sialylation in biochemical assays that measure sialyltransferase activity as well as at the whole cell level. In the course of these experiments, several important differences emerged that differentiated the cancer cells from their normal counterparts including resistant to sialic acid-mediated energy depletion, consistently more robust sialic acid-mediated glycan display, and distinctive cell surface vs. internal vesicle display of newly-produced sialoglycans. Finally, the impact of sialic acid supplementation on specific markers implicated in cancer progression was demonstrated by measuring levels of expression and sialylation of EGFR1 and MUC1 as well as the corresponding function of sialic acid-supplemented cells in migration assays. These findings both provide fundamental insight into the biological basis of sialic acid supplementation of nutrient-deprived cancer cells and open the door to the development of diagnostic and prognostic tools.

  9. Nutrient-deprived cancer cells preferentially use sialic acid to maintain cell surface glycosylation

    PubMed Central

    Badr, Haitham A.; AlSadek, Dina M.M.; Mathew, Mohit P.; Li, Chen-Zhong; Djansugurova, Leyla B.; Yarema, Kevin J.; Ahmed, Hafiz

    2015-01-01

    Cancer is characterized by abnormal energy metabolism shaped by nutrient deprivation that malignant cells experience during various stages of tumor development. This study investigated the response of nutrient-deprived cancer cells and their non-malignant counterparts to sialic acid supplementation and found that cells utilize negligible amounts of this sugar for energy. Instead cells use sialic acid to maintain cell surface glycosylation through complementary mechanisms. First, levels of key metabolites (e.g., UDP-GlcNAc and CMP-Neu5Ac) required for glycan biosynthesis are maintained or enhanced upon Neu5Ac supplementation. In concert, sialyltransferase expression increased at both the mRNA and protein levels, which facilitated increased sialylation in biochemical assays that measure sialyltransferase activity as well as at the whole cell level. In the course of these experiments, several important differences emerged that differentiated the cancer cells from their normal counterparts including resistant to sialic acid-mediated energy depletion, consistently more robust sialic acid-mediated glycan display, and distinctive cell surface vs. internal vesicle display of newly-produced sialoglycans. Finally, the impact of sialic acid supplementation on specific markers implicated in cancer progression was demonstrated by measuring levels of expression and sialylation of EGFR1 and MUC1 as well as the corresponding function of sialic acid-supplemented cells in migration assays. These findings both provide fundamental insight into the biological basis of sialic acid supplementation of nutrient-deprived cancer cells and open the door to the development of diagnostic and prognostic tools. PMID:26295436

  10. Cell Surface Glycan Alterations in Epithelial Mesenchymal Transition Process of Huh7 Hepatocellular Carcinoma Cell

    PubMed Central

    Kang, Xiaonan; Sun, Chun; Jiang, Kai; Huang, Li; Lu, Yu; Sui, Jingzhe; Qin, Xue; Liu, Yinkun

    2013-01-01

    Background and Objective Due to recurrence and metastasis, the mortality of Hepatocellular carcinoma (HCC) is high. It is well known that the epithelial mesenchymal transition (EMT) and glycan of cell surface glycoproteins play pivotal roles in tumor metastasis. The goal of this study was to identify HCC metastasis related differential glycan pattern and their enzymatic basis using a HGF induced EMT model. Methodology HGF was used to induce HCC EMT model. Lectin microarray was used to detect the expression of cell surface glycan and the difference was validated by lectin blot and fluorescence cell lectin-immunochemistry. The mRNA expression levels of glycotransferases were determined by qRT-PCR. Results After HGF treatment, the Huh7 cell lost epithelial characteristics and obtained mesenchymal markers. These changes demonstrated that HGF could induce a typical cell model of EMT. Lectin microarray analysis identified a decreased affinity in seven lectins ACL, BPL, JAC, MPL, PHA-E, SNA, and SBA to the glycan of cell surface glycoproteins. This implied that glycan containing T/Tn-antigen, NA2 and bisecting GlcNAc, Siaα2-6Gal/GalNAc, terminal α or βGalNAc structures were reduced. The binding ability of thirteen lectins, AAL, LCA, LTL, ConA, NML, NPL, DBA, HAL, PTL II, WFL, ECL, GSL II and PHA-L to glycan were elevated, and a definite indication that glycan containing terminal αFuc and ± Sia-Le, core fucose, α-man, gal-β(α) GalNAc, β1,6 GlcNAc branching and tetraantennary complex oligosaccharides structures were increased. These results were further validated by lectin blot and fluorescence cell lectin-immunochemistry. Furthermore, the mRNA expression level of Mgat3 decreased while that of Mgat5, FucT8 and β3GalT5 increased. Therefore, cell surface glycan alterations in the EMT process may coincide with the expression of glycosyltransferase. Conclusions The findings of this study systematically clarify the alterations of cell surface glycan in cancer EMT, and

  11. Silyl protection in the solid-phase synthesis of N-linked glycopeptides. Preparation of glycosylated fluorogenic substrates for subtilisins.

    PubMed

    Christiansen-Brams, I; Jansson, A M; Meldal, M; Breddam, K; Bock, K

    1994-11-01

    The trimethylsilyl (TMS) group was used for protection of the hydroxy groups of three disaccharide 1-amino-alditols and of the glycosylamines of glucose, maltotriose and maltoheptose. The per-O-trimethylsilylated derivatives were coupled with N alpha-Fmoc-Asp(Cl)-OPfp 7 to give six glycosylated building blocks for the solid-phase synthesis of N-linked glycopeptides. Building block 8 was used in the synthesis of five internally quenched fluorescent substrates which were studied by enzymatic hydrolysis with savinase, a subtilisin-type enzyme.

  12. Encouraging Progress in the ω-Aspartylation of Complex Oligosaccharides as a General Route to β-N-Linked Glycopolypeptides

    PubMed Central

    Wang, Ping; Li, Xuechen; Zhu, Jianglong; Chen, Jin; Yuan, Yu; Wu, Xiangyang; Danishefsky, Samuel J.

    2011-01-01

    Described herein is a method for the joining of complex peptides to complex oligosaccharides via an N-linkage. The ω-aspartylation is conducted with coupling fully deprotected glycosylamine with a peptide containing a unique thioacid at the ω-aspartate carboxyl. In the presence of HOBT, under conditions which, in principle, allow for oxidation, complex components are combined in encouraging yields to produce structurally and stereochemically defined N-linked glycopolypeptides wherein the carbohydrate domain can be quite complex. Various mechanisms for oxidative coupling are proposed. PMID:21207981

  13. Expression of functionally relevant cell surface markers in dibutyltin-exposed human natural killer cells.

    PubMed

    Odman-Ghazi, Sabah O; Hatcher, Frank; Whalen, Margaret M

    2003-07-25

    Butyltin (BT) compounds are known for their worldwide contamination. Dibutyltin (DBT) is used as a stabilizer in plastic products, and as a deworming agent in poultry. Poultry products have been shown to contain measurable levels of DBT. Drinking water has also been reported to contain BTs due to leaching from PVC pipes. We, and others, have found measurable levels of DBT in human blood. BTs appear to increase the risk of cancer and other viral infections in exposed individuals. In previous studies we have shown that the tumor killing function of natural killer (NK) lymphocytes was greatly diminished after as little as a 1 h exposure to DBT and the inhibition continued even after removal of the compound. We also showed that there was a significant decrease in NK cell lysis of K562 target cells after an exposure to 1.5 microM DBT for 24 h. This 24 h exposure also decreased the ability of NK cells to bind to tumor cells. Loss of binding function was not seen when NK cells were exposed to 5-10 microM DBT for 1 h. However, NK cells exposed to 5 microM DBT for 1 h and then incubated in DBT-free media for 24, 48, or 96 h, showed a significant loss of tumor-binding function within 24 h. The effects of DBT exposure on seven cell surface molecules that are involved in NK-cell interactions with target cells were investigated. The results indicated that the exposure of NK cells to 1.5 microM DBT for 24 h decreased the expression of CD2, CD11a, CD16, CD11c. There was no decrease in expression of any of the markers studied when NK cells were exposed to 5 microM DBT for 1 h, consistent with the fact that a 1-h exposure had no effect on the ability of NK cells to bind tumor cells. However, when NK cells were exposed to 5 microM DBT for 1 h followed by 24, 48 or 96 h incubations in DBT-free media there was decreased expression of several of the cells surface molecules with the most dramatic decreases being in CD16 and CD56.

  14. Studies on the function of cell surface glycoproteins. II. Possible role of surface glycoproteins in the control of cytoskeletal organization and surface morphology

    PubMed Central

    1979-01-01

    Immunoglobulin from goat antiserum directed against purified surface membranes from transformed BHK21/C13 cells (anti-M) has been shown to cause both control and transformed hamster cells to round and detach from the substrate (see accompanying paper). This paper documents the effects of the antiserum on the cytoskeletal organization and cell surface morphology of control BHK21/C13 cells examined by scanning and transmission electron microscopy. As a result of antiserum-induced rounding, the normally smooth cell surface becomes covered with filopodia and blebs, and the organization of all three components of the filamentous cytoskeleton is altered. In terms of cell surface morphology and cytoskeletal organization, the cells resemble rounded, postmitotic or trypsinized BHK cells rather than cells treated with either anticytoskeletal drugs or lectins. Immunocytochemical and radioimmune assay experiments support the suggestion that the rounding reaction induced by anti-M serum results from the specific interaction of antibodies with molecules on the cell surface. It is suggested that anti-M serum induces alterations in cytoskeletal organization via a transmembrane signal and that cytoskeletal reorganization is a fundamental part of the rounding and detachment process. PMID:457749

  15. Role of the Bp35 cell surface polypeptide in human B-cell activation.

    PubMed Central

    Clark, E A; Shu, G; Ledbetter, J A

    1985-01-01

    A 35-kDa polypeptide, Bp35, expressed on the surface of all B cells, plays a role in B-cell activation. Monoclonal antibodies to Bp35 stimulate human tonsillar B cells to proliferate. The activation induced by anti-Bp35 is similar to anti-Ig-mediated in several ways: the activation does not require T cells but is augmented by T-cell-derived allogeneic factors; monovalent Fab fragments to Bp35 do not trigger proliferation but instead block activation by whole antibody, indicating that cross-linking is required; and induction by anti-Bp35, like the induction by anti-Ig, is inhibited by monoclonal anti-IgM via an Fc domain-dependent mechanism. However, several features of anti-Bp35-mediated proliferation are clearly different from activation by anti-Ig: anti-Bp35 monoclonal antibodies do not require attachment to beads to function, the proliferation induced by anti-Bp35 and anti-Ig is additive, and Fab fragments of anti-Bp35 augment proliferation induced by anti-Ig. Models for the possible function of the Bp35 polypeptide as either a "bridge" or a "second signal" with surface Ig in B-cell activation are discussed. PMID:3872456

  16. Surface Plasmon Resonance Effect in Inverted Perovskite Solar Cells.

    PubMed

    Cui, Jin; Chen, Cheng; Han, Junbo; Cao, Kun; Zhang, Wenjun; Shen, Yan; Wang, Mingkui

    2016-03-01

    This work reports on incorporation of spectrally tuned gold/silica (Au/SiO2) core/shell nanospheres and nanorods into the inverted perovskite solar cells (PVSC). The band gap of hybrid lead halide iodide (CH3NH3PbI3) can be gradually increased by replacing iodide with increasing amounts of bromide, which can not only offer an appreciate solar radiation window for the surface plasmon resonance effect utilization, but also potentially result in a large open circuit voltage. The introduction of localized surface plasmons in CH3NH3PbI2.85Br0.15-based photovoltaic system, which occur in response to electromagnetic radiation, has shown dramatic enhancement of exciton dissociation. The synchronized improvement in photovoltage and photocurrent leads to an inverted CH3NH3PbI2.85Br0.15 planar PVSC device with power conversion efficiency of 13.7%. The spectral response characterization, time resolved photoluminescence, and transient photovoltage decay measurements highlight the efficient and simple method for perovskite devices.

  17. Surface Plasmon Resonance Effect in Inverted Perovskite Solar Cells

    PubMed Central

    Cui, Jin; Chen, Cheng; Han, Junbo; Cao, Kun; Zhang, Wenjun; Shen, Yan

    2016-01-01

    This work reports on incorporation of spectrally tuned gold/silica (Au/SiO2) core/shell nanospheres and nanorods into the inverted perovskite solar cells (PVSC). The band gap of hybrid lead halide iodide (CH3NH3PbI3) can be gradually increased by replacing iodide with increasing amounts of bromide, which can not only offer an appreciate solar radiation window for the surface plasmon resonance effect utilization, but also potentially result in a large open circuit voltage. The introduction of localized surface plasmons in CH3NH3PbI2.85Br0.15‐based photovoltaic system, which occur in response to electromagnetic radiation, has shown dramatic enhancement of exciton dissociation. The synchronized improvement in photovoltage and photocurrent leads to an inverted CH3NH3PbI2.85Br0.15 planar PVSC device with power conversion efficiency of 13.7%. The spectral response characterization, time resolved photoluminescence, and transient photovoltage decay measurements highlight the efficient and simple method for perovskite devices. PMID:28174678

  18. Archaeal surface layer proteins contain beta propeller, PKD, and beta helix domains and are related to metazoan cell surface proteins.

    PubMed

    Jing, Hua; Takagi, Junichi; Liu, Jin-huan; Lindgren, Sara; Zhang, Rong-guang; Joachimiak, A; Wang, Jia-huai; Springer, Timothy A

    2002-10-01

    The surface layer of archaeobacteria protects cells from extreme environments and, in Methanosarcina, may regulate cell adhesion. We identify three domain types that account for the complete architecture of numerous Methanosarcina surface layer proteins (SLPs). We solve the crystal structure for two of these domains, which correspond to the two N-terminal domains of an M. mazei SLP. One domain displays a unique, highly symmetrical, seven-bladed beta propeller fold, and the other belongs to the polycystic kidney disease (PKD) superfamily fold. The third domain is predicted to adopt a beta helix fold. These domains have homologs in metazoan cell surface proteins, suggesting remarkable relationships between domains in archaeal SLPs and metazoan cell surface proteins.

  19. Mechanical Trap Surface-Enhanced Raman Spectroscopy for Three-Dimensional Surface Molecular Imaging of Single Live Cells.

    PubMed

    Jin, Qianru; Li, Ming; Polat, Beril; Paidi, Santosh K; Dai, Aimee; Zhang, Amy; Pagaduan, Jayson V; Barman, Ishan; Gracias, David H

    2017-03-27

    Reported is a new shell-based spectroscopic platform, named mechanical trap surface-enhanced Raman spectroscopy (MTSERS), for simultaneous capture, profiling, and 3D microscopic mapping of the intrinsic molecular signatures on the membrane of single live cells. By leveraging the functionalization of the inner surfaces of the MTs with plasmonic gold nanostars, and conformal contact of the cell membrane, MTSERS permits excellent signal enhancement, reliably detects molecular signatures, and allows non-perturbative, multiplex 3D surface imaging of analytes, such as lipids and proteins on the surface of single cells. The demonstrated ability underscores the potential of MTSERS to perform 3D spectroscopic microimaging and to furnish biologically interpretable, quantitative, and dynamic molecular maps in live cell populations.

  20. Modulation of human multipotent and pluripotent stem cells using surface nanotopographies and surface-immobilised bioactive signals: A review.

    PubMed

    Wang, Peng-Yuan; Thissen, Helmut; Kingshott, Peter

    2016-11-01

    The ability to control the interactions of stem cells with synthetic surfaces is proving to be effective and essential for the quality of passaged stem cells and ultimately the success of regenerative medicine. The stem cell niche is crucial for stem cell self-renewal and differentiation. Thus, mimicking the stem cell niche, and here in particular the extracellular matrix (ECM), in vitro is an important goal for the expansion of stem cells and their applications. Here, surface nanotopographies and surface-immobilised biosignals have been identified as major factors that control stem cell responses. The development of tailored surfaces having an optimum nanotopography and displaying suitable biosignals is proposed to be essential for future stem cell culture, cell therapy and regenerative medicine applications. While early research in the field has been restricted by the limited availability of micro- and nanofabrication techniques, new approaches involving the use of advanced fabrication and surface immobilisation methods are starting to emerge. In addition, new cell types such as induced pluripotent stem cells (iPSCs) have become available in the last decade, but have not been fully understood. This review summarises significant advances in the area and focuses on the approaches that are aimed at controlling the behavior of human stem cells including maintenance of their self-renewal ability and improvement of their lineage commitment using nanotopographies and biosignals. More specifically, we discuss developments in biointerface science that are an important driving force for new biomedical materials and advances in bioengineering aiming at improving stem cell culture protocols and 3D scaffolds for clinical applications. Cellular responses revolve around the interplay between the surface properties of the cell culture substrate and the biomolecular composition of the cell culture medium. Determination of the precise role played by each factor, as well as the

  1. Primordial germ cell differentiation of nuclear transfer embryonic stem cells using surface modified electroconductive scaffolds.

    PubMed

    Eslami-Arshaghi, Tarlan; Vakilian, Saeid; Seyedjafari, Ehsan; Ardeshirylajimi, Abdolreza; Soleimani, Masoud; Salehi, Mohammad

    2016-12-30

    A combination of nanotopographical cues and surface modification of collagen and fibronectin is a potential platform in primordial germ cells (PGCs) differentiation. In the present study, the synergistic effect of nanotopography and surface modification on differentiation of nuclear transfer embryonic stem cells (nt-ESCs) toward PGC lineage was investigated. In order to achieve this goal, poly-anyline (PANi) was mix within poly-L-lactic acid (PLLA). Afterward, the random composite mats were fabricated using PLLA and PANi mix solution. The nanofiber topography notably upregulated the expressions of prdm14, mvh and c-kit compared with tissue culture polystyrene (TCP). Moreover, the combination of nanofiber topography and surface modification resulted in more enhancement of PGCs differentiation compared with non-modified nanofibrous scaffold. Additionally, gene expression results showed that mvh and c-kit were expressed at higher intensity in cells exposed to collagen and fibronectin rather than collagen or fibronectin solitary. These results demonstrated the importance of combined effect of collagen and fibronectin in order to develop a functional extracellular matrix (ECM) mimic in directing stem cell fate and the potential of such biofunctional scaffolds for treatment of infertility.

  2. Cell surface-mediated cellular interactions: effects of B104 neuroblastoma surface determinants on C6 glioma cellular properties.

    PubMed

    Ciment, G; de Vellis, J

    1982-01-01

    To study the influence of cell surface-associated molecules on intercellular communication, C6 glioma cells were cultured both on plastic and on substrata of paraformaldehyde-fixed B104 neuroblastoma cells. By then comparing the phenotypic expression of these "cocultured" C6 cells with cells cultured on tissue culture plastic, the influence of the cellular substratum was determined. The beta-adrenergic-responsive cyclic AMP-generating system of C6 cells was compared on these various substrata. We found that fixed beds of dibutyryl cyclic AMP (dbcAMP)-treated B104 cells uncoupled beta-receptors from adenylate cyclase, whereas fixed beds of similarly treated C6 cells did not. However, other cellular properties were not affected by growth atop fixed dbcAMP-treated B104 cell beds including the rate of C6 cellular proliferation and their rate of protein synthesis. The cell surface-associated determinant on B104 cells capable of uncoupling the beta-responsive cyclase system of C6 cells is probably a protein, as judged by its susceptibility to protease treatment. Other properties of C6 cells were also affected by the various substrata including basal and hydrocortisone-induced levels of glycerol phosphate dehydrogenase (GPDH; an oligodendroglial marker) and the rate of RNA synthesis in these cells.

  3. Affinity adsorption of cells to surfaces and strategies for cell detachment.

    PubMed

    Hubble, John

    2007-01-01

    The use of bio-specific interactions for the separation and recovery of bio-molecules is now widely established and in many cases the technique has successfully crossed the divide between bench and process scale operation. Although the major specificity advantage of affinity-based separations also applies to systems intended for cell fractionation, developments in this area have been slower. Many of the problems encountered result from attempts to take techniques developed for molecular systems and, with only minor modification to the conditions used, apply them for the separation of cells. This approach tends to ignore or at least trivialise the problems, which arise from the heterogeneous nature of a cell suspension and the multivalent nature of the cell/surface interaction. To develop viable separation processes on a larger scale, effective contacting strategies are required in separators that also allow detachment or recovery protocols that overcome the enhanced binding strength generated by multivalent interactions. The effects of interaction valency on interaction strength needs to be assessed and approaches developed to allow effective detachment and recovery of adsorbed cells without compromising cell viability. This article considers the influence of operating conditions on cell attachment and the extent to which multivalent interactions determine the strength of cell binding and subsequent detachment.

  4. Human Diversity in a Cell Surface Receptor that Inhibits Autophagy.

    PubMed

    Chaudhary, Anu; Leite, Mara; Kulasekara, Bridget R; Altura, Melissa A; Ogahara, Cassandra; Weiss, Eli; Fu, Wenqing; Blanc, Marie-Pierre; O'Keeffe, Michael; Terhorst, Cox; Akey, Joshua M; Miller, Samuel I

    2016-07-25

    Mutations in genes encoding autophagy proteins have been associated with human autoimmune diseases, suggesting that diversity in autophagy responses could be associated with disease susceptibility or severity. A cellular genome-wide association study (GWAS) screen was performed to explore normal human diversity in responses to rapamycin, a microbial product that induces autophagy. Cells from several human populations demonstrated variability in expression of a cell surface receptor, CD244 (SlamF4, 2B4), that correlated with changes in rapamycin-induced autophagy. High expression of CD244 and receptor activation with its endogenous ligand CD48 inhibited starvation- and rapamycin-induced autophagy by promoting association of CD244 with the autophagy complex proteins Vps34 and Beclin-1. The association of CD244 with this complex reduced Vps34 lipid kinase activity. Lack of CD244 is associated with auto-antibody production in mice, and lower expression of human CD244 has previously been implicated in severity of human rheumatoid arthritis and systemic lupus erythematosus, indicating that increased autophagy as a result of low levels of CD244 may alter disease outcomes.

  5. Inorganic Surface Engineering to Enhance Perovskite Solar Cell Efficiency.

    PubMed

    Aeineh, Naemeh; Barea, Eva M; Behjat, Abbas; Sharifi, Nafiseh; Mora-Sero, Ivan

    2017-03-29

    The photoconversion efficiency of perovskite solar cells (PSCs) has been enhanced by the deposition of inorganic nanoparticles (NPs) at the interface between the compact TiO2 electron selective contact and the mesoporous TiO2 film. The NPs used have been core/shell Au@SiO2, where a thin SiO2 coating protects the Au core from the direct chemical interaction with CH3NH3PbI3 halide perovskite used as light harvesting material. Samples prepared with the Au@SiO2 NPs exhibits higher external quantum efficiency in all the complete wavelength range at which perovskite presents light absorption and not just at the wavelengths at which Au@SiO2 NPs presents their absorption peak. This fact rules out a direct plasmonic process as the responsible on cell performance enhancement. A detail characterization by photoluminescence, impedance spectroscopy and open circuit voltage decay unveil a modification of the interfacial properties with an augmentation of the interfacial electrostatic potential that increases both photovoltage and photocurrent. This work highlights the dramatic role of interfaces in PSC performance. The use of reduced quantities of highly stable inorganic compounds to modify the PSC interface instead of the extensively used organic compounds opens the door to a new surface engineering based on inorganic compounds.

  6. 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

  7. A simplified model for dynamics of cell rolling and cell-surface adhesion

    SciTech Connect

    Cimrák, Ivan

    2015-03-10

    We propose a three dimensional model for the adhesion and rolling of biological cells on surfaces. We study cells moving in shear flow above a wall to which they can adhere via specific receptor-ligand bonds based on receptors from selectin as well as integrin family. The computational fluid dynamics are governed by the lattice-Boltzmann method. The movement and the deformation of the cells is described by the immersed boundary method. Both methods are fully coupled by implementing a two-way fluid-structure interaction. The adhesion mechanism is modelled by adhesive bonds including stochastic rules for their creation and rupture. We explore a simplified model with dissociation rate independent of the length of the bonds. We demonstrate that this model is able to resemble the mesoscopic properties, such as velocity of rolling cells.

  8. Flow cytometry detection of planktonic cells with polycyclic aromatic hydrocarbons sorbed to cell surfaces.

    PubMed

    Cerezo, Maria I; Linden, Matthew; Agustí, Susana

    2017-02-17

    Polycyclic aromatic hydrocarbons are very important components of oil pollution. These pollutants tend to sorb to cell surfaces, exerting toxic effects on organisms. Our study developed a flow cytometric method for the detection of PAHs sorbed to phytoplankton by exploiting their spectral characteristics. We discriminated between cells with PAHs from cells free of PAHs. Clear discrimination was observed with flow cytometer provided with 375 or 405nm lasers in addition to the standard 488nm laser necessary to identify phytoplankton. Using this method, we measured the relationship between the percentages of phytoplankton organisms with PAHs, with the decrease in the growth rate. Moreover, the development of this method could be extended to facilitate the study of PAHs impact on cell cultures from a large variety of organisms.

  9. Cellular glycosylation affects Herceptin binding and sensitivity of breast cancer cells to doxorubicin and growth factors

    PubMed Central

    Peiris, Diluka; Spector, Alexander F.; Lomax-Browne, Hannah; Azimi, Tayebeh; Ramesh, Bala; Loizidou, Marilena; Welch, Hazel; Dwek, Miriam V.

    2017-01-01

    Alterations in protein glycosylation are a key feature of oncogenesis and have been shown to affect cancer cell behaviour perturbing cell adhesion, favouring cell migration and metastasis. This study investigated the effect of N-linked glycosylation on the binding of Herceptin to HER2 protein in breast cancer and on the sensitivity of cancer cells to the chemotherapeutic agent doxorubicin (DXR) and growth factors (EGF and IGF-1). The interaction between Herceptin and recombinant HER2 protein and cancer cell surfaces (on-rate/off-rate) was assessed using a quartz crystal microbalance biosensor revealing an increase in the accessibility of HER2 to Herceptin following deglycosylation of cell membrane proteins (deglycosylated cells Bmax: 6.83 Hz; glycosylated cells Bmax: 7.35 Hz). The sensitivity of cells to DXR and to growth factors was evaluated using an MTT assay. Maintenance of SKBR-3 cells in tunicamycin (an inhibitor of N-linked glycosylation) resulted in an increase in sensitivity to DXR (0.1 μM DXR P < 0.001) and a decrease in sensitivity to IGF-1 alone and to IGF-1 supplemented with EGF (P < 0.001). This report illustrates the importance of N-linked glycosylation in modulating the response of cancer cells to chemotherapeutic and biological treatments and highlights the potential of glycosylation inhibitors as future combination treatments for breast cancer. PMID:28223691

  10. Scanning electron microscopy of kidney cells in culture: surface features of polarized epithelia.

    PubMed

    McAteer, J A; Dougherty, G S; Gardner, K D; Evan, A P

    1986-01-01

    We have used scanning electron microscopy (SEM) to examine the surface morphology of the renal epithelial cell lines MDCK and LLC-PKl to determine the influence of alternative culture substrate conditions on cell polarity. We observed that regardless of physical culture conditions, cells established and maintained polarity, expressed by the characteristics of apical and basal surfaces. Culture conditions did, however, influence the orientation of cell polarity in vitro. MDCK cells were grown within collagen gel, in which individual cells exhibited clonal growth to form fluid-filled epithelial cysts. The cells of MDCK-cysts were polarized with apical surface facing the lumen and basal surface against the surrounding collagen gel. This configuration made it possible to gain direct visual access, by SEM, to the basal surface by removing the supportive collagen lattice. The apical surface of MDCK-cysts was lined by short microvilli. Each cell possessed a solitary cilium. In comparison, the basal surface had few appendages, although cell boundaries were marked by interdigitating short processes. LLC-PKl cells in monolayer culture bore solitary cilia and long microvilli at their apical surface. The basal surface of cells involved in dome formation was observed to possess only a sparse population of short, blunt processes. When LLC-PKl cells were raised in stationary suspension culture or in monolayer atop non-culture grade plastic, they formed cysts with the cell apex facing the surrounding medium. These cells showed variable apical morphology. The cells of large, highly expanded cysts were often attenuated and had a relatively smooth apical surface. The basal surface of cells of fractured LLC-PKl cysts commonly was also smooth, without prominent appendages.

  11. Nerve cells culture from lumbar spinal cord on surfaces modified by plasma pyrrole polymerization.

    PubMed

    Zuñiga-Aguilar, E; Olayo, R; Ramírez-Fernández, O; Morales, J; Godínez, R

    2014-01-01

    Currently, there are several techniques for modified cell culture surfaces under research to improve cell growth and adhesion. Recently, different methods have been used for surface coating, using biomolecules that enhance cell attachment and growth of nerve cells from spinal cord, such as the use of Poly-DL-Ornithine/Laminin. Plasma-polymerized pyrrole (PPy)-treated surfaces have showed improvement on surfaces biocompatibility with the cells in culture since they do not interfere with any of the biological cell functions. In the present work, we present a novel mouse nerve cell culture technique, using PPy-treated cell culture surfaces. A comparative study of cell survival using Poly-DL-Ornithine/Laminin-treated surfaces was performed. Our results of cell survival when compared with data already reported by other investigators, show that cells cultured on the PPy-modified surface increased survival up to 21 days when compared with Poly-DL-Ornithine/Laminin-coated culture, where 8 days cell survival was obtained. There were electrical and morphological differences in the nerve cells grown in the different surfaces. By comparing the peak ion currents of Poly-DL-Ornithine/Laminin-seeded cells for 8 days with cells grown for 21 days on PPy, an increase of 516% in the Na(+) current and 127% in K(+) currents in cells seeded on PPy were observed. Immunofluorescence techniques showed the presence of cell synapses and culture viability after 21 days. Our results then showed that PPy-modified surfaces are an alternative culture method that increases nerve cells survival from lumbar spinal cord cell culture by preserving its electrical and morphological features.

  12. Strategic addition of an N-linked glycan to a monoclonal antibody improves its HIV-1-neutralizing activity.

    PubMed

    Song, Ruijiang; Oren, Deena A; Franco, David; Seaman, Michael S; Ho, David D

    2013-11-01

    Ibalizumab is a humanized monoclonal antibody that binds human CD4--a key receptor for HIV--and blocks HIV-1 infection. However, HIV-1 strains with mutations resulting in loss of an N-linked glycan from the V5 loop of the envelope glycoprotein gp120 are resistant to ibalizumab. Previous structural analysis suggests that this glycan fills a void between the gp120 V5 loop and the ibalizumab light chain, perhaps causing steric hindrance that disrupts viral entry. If this void contributes to HIV-1 resistance to ibalizumab, we reasoned that 'refilling' it by engineering an N-linked glycan into the ibalizumab light chain at a position spatially proximal to gp120 V5 may restore susceptibility to ibalizumab. Indeed, one such ibalizumab variant neutralized 100% of 118 diverse HIV-1 strains tested in vitro, including 10 strains resistant to parental ibalizumab. These findings demonstrate that the strategic placement of a glycan in the variable region of a monoclonal antibody can substantially enhance its activity.

  13. Strategic addition of an N-linked glycan to a monoclonal antibody improves its HIV-1-neutralizing activity

    PubMed Central

    Song, Ruijiang; Oren, Deena A.; Franco, David; Seaman, Michael S.; Ho, David D.

    2013-01-01

    Ibalizumab is a humanized monoclonal antibody that binds human CD4—a key receptor for HIV—and blocks HIV-1 infection. However, HIV-1 strains with mutations resulting in loss of an N-linked glycan from the V5 loop of the envelope protein gp120 are resistant to ibalizumab. Previous structural analysis suggests that this glycan fills a void between the gp120 V5 loop and the ibalizumab L chain, perhaps causing steric hindrance that disrupts viral entry. If this void contributes to HIV-1 resistance to ibalizumab, we reasoned that ‘refilling’ it by engineering an N-linked glycan into the ibalizumab L chain at a position spatially proximal to gp120 V5 may restore susceptibility to ibalizumab. Indeed, one such ibalizumab variant neutralized 100% of 118 tested diverse HIV-1 strains in vitro, including ten strains resistant to parental ibalizumab. These findings demonstrate that the strategic placement of a glycan in the variable region of a monoclonal antibody can substantially enhance its activity. PMID:24097413

  14. Perturbing the folding energy landscape of the bacterial immunity protein Im7 by site-specific N-linked glycosylation

    PubMed Central

    Chen, Mark M.; Bartlett, Alice I.; Nerenberg, Paul S.; Friel, Claire T.; Hackenberger, Christian P. R.; Stultz, Collin M.; Radford, Sheena E.; Imperiali, Barbara

    2010-01-01

    N-linked glycosylation modulates protein folding and stability through a variety of mechanisms. As such there is considerable interest in the development of general rules to predict the structural consequences of site-specific glycosylation and to understand how these effects can be exploited in the design and development of modified proteins with advantageous properties. In this study, expressed protein ligation is used to create site-specifically glycosylated variants of the bacterial immunity protein Im7 modified with the chitobiose disaccharide (GlcNAc-GlcNAc). Glycans were introduced at seven solvent exposed sites within the Im7 sequence and the kinetic and thermodynamic consequences of N-linked glycosylation analyzed. The values for glycan incorporation were found to range from +5.2 to -3.8 kJ·mol-1. In several cases, glycosylation influences folding by modulating the local conformational preferences of the glycosylated sequence. These locally mediated effects are most prominent in the center of α-helices where glycosylation negatively effects folding and in compact turn motifs between segments of ordered secondary structure where glycosylation promotes folding and enhances the overall stability of the native protein. The studies also provide insight into why glycosylation is commonly identified at the transition between different types of secondary structure and when glycosylation may be used to elaborate protein structure to protect disordered sequences from proteolysis or immune system recognition. PMID:21148421

  15. Tyrosine derivatization and preparative purification of the sialyl and asialy-N-linked oligosaccharides from porcine fibrinogen.

    PubMed

    Da Silva, M L; Tamura, T; McBroom, T; Rice, K G

    1994-07-01

    The N-linked oligosaccharides from porcine fibrinogen were purified following their release from glycopeptides using N-glycosidase F. In separate experiments, both sialyl and asialyl oligosaccharides were prepared from 5 g of fibrinogen. The reducing oligosaccharides were reacted with ammonium bicarbonate to form/oligosaccharide-glycosylamines and then derivatized with tert-butoxycarbonyl-L-tyrosine N-hydroxysuccinimidyl ester. Tyrosinamide--oligosaccharides were purified first by gel filtration chromatography and then by reverse-phase HPLC and the products were characterized by proton NMR and fast atom bombardment-MS. Porcine fibrinogen was found to have predominantly a single asialyl biantennary oligosaccharide containing a fucose linked alpha 1-6 to GlcNAc 1. The oligosaccharide possesses two sialylation patterns with a major form (70%) having a single N-acetyl neuraminic acid (NeuAc) residue linked alpha 2-6 to galactose on only one antenna and a minor form (30%) possessing two NeuAc residues linked alpha 2-6 to both terminal galactose residues. In addition to developing an isolation procedure and establishing the structures of porcine fibrinogen oligosaccharides, this study improves on the tyrosine derivatization technique as a general approach to isolate structurally diverse N-linked oligosaccharides from glycoproteins.

  16. Natural bactericidal surfaces: mechanical rupture of Pseudomonas aeruginosa cells by cicada wings.

    PubMed

    Ivanova, Elena P; Hasan, Jafar; Webb, Hayden K; Truong, Vi Khanh; Watson, Gregory S; Watson, Jolanta A; Baulin, Vladimir A; Pogodin, Sergey; Wang, James Y; Tobin, Mark J; Löbbe, Christian; Crawford, Russell J

    2012-08-20

    Natural superhydrophobic surfaces are often thought to have antibiofouling potential due to their self-cleaning properties. However, when incubated on cicada wings, Pseudomonas aeruginosa cells are not repelled; instead they are penetrated by the nanopillar arrays present on the wing surface, resulting in bacterial cell death. Cicada wings are effective antibacterial, as opposed to antibiofouling, surfaces.

  17. Human epithelial cells exposed to functionalized multiwalled carbon nanotubes: interactions and cell surface modifications.

    PubMed

    Fanizza, C; Casciardi, S; Incoronato, F; Cavallo, D; Ursini, C L; Ciervo, A; Maiello, R; Fresegna, A M; Marcelloni, A M; Lega, D; Alvino, A; Baiguera, S

    2015-09-01

    With the expansion of the production and applications of multiwalled carbon nanotubes (MWCNTs) in several industrial and science branches, the potential adverse effects on human health have attracted attention. Numerous studies have been conducted to evaluate how chemical functionalization may affect MWCNT effects; however, controversial data have been reported, showing either increased or reduced toxicity. In particular, the impact of carboxylation on MWCNT cytotoxicity is far from being completely understood. The aim of this work was the evaluation of the modifications induced by carboxylated-MWCNTs (MWCNTs-COOH) on cell surface and the study of cell-MWCNT-COOH interactions by means of field emission scanning electron microscope (FESEM). Human pulmonary epithelial cells (A549) were incubated with MWCNTs-COOH for different exposure times and concentrations (10 μg/mL for 1, 2, 4 h; 5, 10, 20 μg/mL for 24 h). At short incubation time, MWCNTs-COOH were easily observed associated with plasma membrane and in contact with microvilli. After 24 h exposure, FESEM analysis revealed that MWCNTs-COOH induced evident changes in the cellular surface in comparison to control cells: treated cells showed blebs, holes and a depletion of the microvilli density in association with structure modifications, such as widening and/or lengthening. In particular, an increase of cells showing holes and microvilli structure alterations was observed at 20 μg/mL concentration. FESEM analysis showed nanotube agglomerates, of different sizes, entering into the cell with two different mechanisms: inward bending of the membrane followed by nanotube sinking, and nanotube internalization directly through holes. The observed morphological microvilli modifications, induced by MWCNTs-COOH, could affect epithelial functions, such as the control of surfactant production and secretion, leading to pathological conditions, such as alveolar proteinosis. More detailed studies will be, however, necessary to

  18. Surface topography during neural stem cell differentiation regulates cell migration and cell morphology.

    PubMed

    Czeisler, Catherine; Short, Aaron; Nelson, Tyler; Gygli, Patrick; Ortiz, Cristina; Catacutan, Fay Patsy; Stocker, Ben; Cronin, James; Lannutti, John; Winter, Jessica; Otero, José Javier

    2016-12-01

    We sought to determine the contribution of scaffold topography to the migration and morphology of neural stem cells by mimicking anatomical features of scaffolds found in vivo. We mimicked two types of central nervous system scaffolds encountered by neural stem cells during development in vitro by constructing different diameter electrospun polycaprolactone (PCL) fiber mats, a substrate that we have shown to be topographically similar to brain scaffolds. We compared the effects of large fibers (made to mimic blood vessel topography) with those of small-diameter fibers (made to mimic radial glial process topography) on the migration and differentiation of neural stem cells. Neural stem cells showed differential migratory and morphological reactions with laminin in different topographical contexts. We demonstrate, for the first time, that neural stem cell biological responses to laminin are dependent on topographical context. Large-fiber topography without laminin prevented cell migration, which was partially reversed by treatment with rock inhibitor. Cell morphology complexity assayed by fractal dimension was inhibited in nocodazole- and cytochalasin-D-treated neural precursor cells in large-fiber topography, but was not changed in small-fiber topography with these inhibitors. These data indicate that cell morphology has different requirements on cytoskeletal proteins dependent on the topographical environment encountered by the cell. We propose that the physical structure of distinct scaffolds induces unique signaling cascades that regulate migration and morphology in embryonic neural precursor cells. J. Comp. Neurol. 524:3485-3502, 2016. © 2016 Wiley Periodicals, Inc.

  19. N-Linked Protein Glycosylation is Required for Full Competence in Campylobacter jejuni 81-176

    DTIC Science & Technology

    2004-10-01

    Agrobacterium tumefaciens into plant cells. J. Bacteriol. 179:78– 89. 5. Benz, I., and M. A. Schmidt. 2001. Glycosylation with heptose residues mediated by...glycosylation sites present in the H. pylori and Agrobacterium tumefaciens homologs, respectively. The A. tu- mefaciens homolog of VirB10 has been previously...complementation analysis of the Agrobacterium tumefaciens virB operon: virB2 through virB11 are essen- tial virulence genes. J. Bacteriol. 176:3646

  20. Cell surface alteration in Epstein-Barr virus-transformed cells from patients with extreme insulin resistance

    SciTech Connect

    Gorden, D.L.; Robert, A.; Moncada, V.Y.; Taylor, S.I.; Muehlhauser, J.C.; Carpentier, J.L. )

    1990-08-01

    An abnormality was detected in the morphology of the cell surface of Epstein-Barr virus-transformed lymphocytes of patients with genetic forms of insulin resistance. In cells from two patients with leprechaunism and two patients with type A extreme insulin resistance, scanning electron microscopy demonstrated a decrease in the percentage of the cell surface occupied by microvilli in cells from the patients with leprechaunism and type A insulin resistance compared with control cells. When cells from a healthy control subject and one of the patients with leprechaunism (Lep/Ark-1) were incubated with {sup 125}I-labeled insulin, there was a decrease in the percentage of {sup 125}I-insulin associated with microvilli on the cell surface. Thus, the decreased localization of insulin receptors with the microvillous region of the cell surface was in proportion to the decrease in microvilli.

  1. Effect of surface potential on epithelial cell adhesion, proliferation and morphology.

    PubMed

    Chang, Hsun-Yun; Kao, Wei-Lun; You, Yun-Wen; Chu, Yi-Hsuan; Chu, Kuo-Jui; Chen, Peng-Jen; Wu, Chen-Yi; Lee, Yu-Hsuan; Shyue, Jing-Jong

    2016-05-01

    Cell adhesion is the basis of individual cell survival, division and motility. Hence, understanding the effects that the surface properties have on cell adhesion, proliferation and morphology are crucial. In particular, surface charge/potential has been identified as an important factor that affects cell behavior. However, how cells respond to incremental changes in surface potential remains unclear. By using binary self-assembled monolayer (SAM) modified Au surfaces that are similar in mechanical/chemical properties and provide a series of surface potentials, the effect of surface potential on the behavior of cells can be studied. In this work, the effect of surface potential on epithelial cells, including human embryonic kidney (HEK293T) and human hepatocellular carcinoma (HepG2), were examined. The results showed that the adhesion density of epithelial cells increased with increasing surface potential, which is similar to but varied more significantly compared with fibroblasts. The proliferation rate is found to be independent of surface potential in both cell types. Furthermore, epithelial cells show no morphological change with respect to surface potential, whereas the morphology of the fibroblasts clearly changed with the surface potential. These differences between the cell types were rationalized by considering the difference in extracellular matrix composition. Laminin-dominant epithelial cells showed higher adhesion density and less morphological change than did fibronectin-dominant fibroblasts because the more significant adsorption of positively charged laminin on the surface enhanced the adhesion of epithelial cells. In contrast, due to the dominance of negatively charged fibronectin that adsorbed weakly on the surface, fibroblasts had to change their morphology to fit the inhomogeneous fibronectin-adsorbed area.

  2. On the relation between surface roughness of metallic substrates and adhesion of human primary bone cells.

    PubMed

    Anselme, K; Bigerelle, M

    2014-01-01

    Surface characteristics of materials, whether their topography, chemistry, or surface energy, play an essential part in osteoblast adhesion on biomaterials. Thus, the quality of cell adhesion will influence the cell's capacity to proliferate and differentiate in contact with a biomaterial. We have developed for more than ten years numerous studies on the influence of topography and chemistry of metallic substrates on the response of primary human bone cells. The originality of our approach is that contrary to most of other authors, we quantified the adhesion of primary human bone cells on metallic substrates with perfectly characterized surface topography after some hours but also over 21 days. Moreover, we have developed original statistical approaches for characterizing the relation between surface roughness and cell-adhesion parameters. In this article, we will illustrate different studies we did these last ten years concerning the development of a new adhesion parameter, the adhesion power; the correlation between short-term adhesion, long-term adhesion, and proliferation; the influence of roughness organization on cell adhesion and the development of the order parameter; our modeling approach of cell adhesion on surface topography; the relative influence of surface chemistry and topography on cell adhesion and contact angle; the relation between surface features dimensions and cell adhesion. Further, some considerations will be given on the methods for scanning surface topography for cell-adhesion studies. Finally, perspectives will be given to elucidate these intracellular mechanotransduction mechanisms induced by the deformation of cells on model sinusoidal peaks-or-valleys surfaces.

  3. Coupling Binding to Catalysis – Using Yeast Cell Surface Display to Select Enzymatic Activities

    PubMed Central

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

    2015-01-01

    Summary 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

  4. Influence of carbon source on cell surface topology of Thermomonospora curvata.

    PubMed Central

    Hostalka, F; Moultrie, A; Stutzenberger, F

    1992-01-01

    The appearance of cell surface protuberances in Thermomonospora curvata correlated with cell-bound exoenzymes which could be removed by brief sonication. Mycelia grown on cellulose or xylan had numerous protuberances and retained 20 to 25% of endoglucanase and endoxylanase at cell surfaces, while those grown on pectin or starch had few protuberances and negligible bound pectinase or amylase. Images PMID:1400256

  5. 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

  6. Evidence for cell surface control of macronuclear DNA synthesis in Stentor.

    PubMed

    de Terra, N

    1975-11-27

    In cell grafts, Stentor macronuclei associated with separate regions of cell surface can be made asynchronous with regard to morphology and DNA synthesis even though they demonstrably share a common endoplasm. These results suggest a mechanism for nuclear differentiation within a single cytoplasmic compartment, based on cell surface differences.

  7. 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.

  8. 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.

  9. Surface complexation of neptunium (V) onto whole cells and cell componets of Shewanella alga

    SciTech Connect

    Reed, Donald Timothy; Deo, Randhir P; Rittmann, Bruce E; Songkasiri, Warinthorn

    2008-01-01

    We systematically quantified surface complexation of neptunium(V) onto whole cells of Shewanella alga strain BrY and onto cell wall and extracellular polymeric substances (EPS) of S. alga. We first performed acid and base titrations and used the mathematical model FITEQL with constant-capacitance surface-complexation to determine the concentrations and deprotonation constants of specific surface functional groups. Deprotonation constants most likely corresponded to a carboxyl site associated with amino acids (pK{sub a} {approx} 2.4), a carboxyl group not associated with amino acids (pK{sub a} {approx} 5), a phosphoryl site (pK{sub a} {approx} 7.2), and an amine site (pK{sub a} > 10). We then carried out batch sorption experiments with Np(V) and each of the S. alga components at different pHs. Results show that solution pH influenced the speciation of Np(V) and each of the surface functional groups. We used the speciation sub-model of the biogeochemical model CCBATCH to compute the stability constants for Np(V) complexation to each surface functional group. The stability constants were similar for each functional group on S. alga bacterial whole cells, cell walls, and EPS, and they explain the complicated sorption patterns when they are combined with the aqueous-phase speciation of Np(V). For pH < 8, NpO{sub 2}{sup +} was the dominant form of Np(V), and its log K values for the low-pK{sub a} carboxyl, other carboxyl, and phosphoryl groups were 1.75, 1.75, and 2.5 to 3.1, respectively. For pH greater than 8, the key surface ligand was amine >XNH3+, which complexed with NpO{sub 2}(CO{sub 3}){sub 3}{sup 5-}. The log K for NpO{sub 2}(CO{sub 3}){sub 3}{sup 5-} complexed onto the amine groups was 3.1 to 3.6. All of the log K values are similar to those of Np(V) complexes with aqueous carboxyl and N-containing carboxyl ligands. These results point towards the important role of surface complexation in defining key actinide-microbiological interactions in the subsurface.

  10. Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing

    PubMed Central

    Szober, Christoph M.; Hauck, Stefanie M.; Euler, Kerstin N.; Fröhlich, Kristina J. H.; Alge-Priglinger, Claudia; Ueffing, Marius; Deeg, Cornelia A.

    2012-01-01

    The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses’ vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS), and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP) and retinal pigment epithelium-specific protein 65kDa (RPE65). Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies. PMID:23203049

  11. New insights into the nanometer-scaled cell-surface interspace by cell-sensor measurements

    SciTech Connect

    Lehmann, Mirko . E-mail: mirko.lehmann@micronas.com; Baumann, Werner

    2005-05-01

    The culture of adherent cells on solid surfaces is an established in vitro method, and the adhesion process of a cell is considered as an important trigger for many cellular processes (e.g., polarity and tumor genesis). However, not all of the eliciting biochemical or biophysical reactions are yet understood. Interestingly, there are not much experimental data about the impact that the interspace between an adherent cell and the (solid) substrate has on the cell's behavior. This interspace is mainly built by the basolateral side of epithelial cells and the substrate. This paper gives some new results of non-invasive and non-optical measurements in the interspace. The measurements were made with silicon cell-sensor hybrids. Measurements of acidification, adhesion, and respiration are analyzed in view of the situation in the interspace. The results show that, in general, the release of an ion or molecule on the basolateral side can have much more influence on the biophysical situation than a release of an ion or molecule on the apical side. In particular, the apical acidification (i.e., amount of extruded protons) of, e.g., epithelial tumor cells is several orders of magnitude higher than the basolateral acidification. These experimental results are a simple consequence of the fact that the basolateral volume of the interspace is several orders of magnitudes smaller than the apical volume. These results have the following consequences for the cell adhesion:a)static situation: if a cell is already adhered to a solid substrate, the basolateral and apical release and uptake of molecules have to be considered in a very differentiated way; b)dynamic situation: if the cell is adhering to the substrate, the then built basolateral side changes in a much stronger way than the apical side. This effect is here discussed as a possible eliciting and general mechanism for essential intracellular changes.

  12. Nano-mechanical exploration of the surface and sub-surface of hydrated cells of Staphylococcus epidermidis.

    PubMed

    Méndez-Vilas, Antonio; Gallardo-Moreno, Amparo M; González-Martín, M Luisa

    2006-01-01

    The surface of hydrated cells of Staphylococcus epidermidis has been probed using an atomic force microscope. While local force measurements over the surface of bacteria reveal a heterogeneous chemical surface, with heterogeneous mechanical properties, different kinds of force curves appear with high frequency, and are thought to provide information on features contributing strongly to the overall mechanical and surface behaviour of the cell. Force curves often present two different mechanical regimes, being the first one (outer) of about 48 nm thick, and presenting a local relative elasticity of about 0.08 N/m, which is about a third of the relative elasticity of the inner part of the cell wall, harder, with a relative elasticity of about 0.24 N/m, in water. Both regimes appears as straight lines in the force versus distance curves (the 'corresponding' stress-strain curves in contact mechanics), but hysteresis is observed between the approach and the retraction line in the inner regime, indicating a degree of viscoelasticity. No viscoelasticity is observed in the outer regime, however, which presents quite linear and juxtaposed approach-retraction lines. These kinds of force curves do not present measurable pull-off forces nor snap-in forces, which indicates an almost null interaction between tip and bacterial surface, which could be in agreement with the measured very high hydrophobicity of this strain. Another kind of force curve has been observed recurrently, showing peaks in the retraction curves. Adhesive pull-off forces were measured giving an average of about 2 nN. Interestingly, however, these force curves appear only when quite irregular and wavy retraction curves are present, from the very beginning of its trace (maximum indentation). This leads us to think that these pull-off forces measured by our AFM do not give information on surface forces-unbinding events at the surface of the bacteria, but could be related to events at the sub-surface of the cell

  13. Functionalized Magnetic Nanoparticles for the Detection and Quantitative Analysis of Cell Surface Antigen

    PubMed Central

    Shahbazi-Gahrouei, Daryoush; Abdolahi, Mohammad; Zarkesh-Esfahani, Sayyed Hamid; Laurent, Sophie; Sermeus, Corine; Gruettner, Cordula

    2013-01-01

    Cell surface antigens as biomarkers offer tremendous potential for early diagnosis, prognosis, and therapeutic response in a variety of diseases such as cancers. In this research, a simple, rapid, accurate, inexpensive, and easily available in vitro assay based on magnetic nanoparticles and magnetic cell separation principle was applied to identify and quantitatively analyze the cell surface antigen expression in the case of prostate cancer cells. Comparing the capability of the assay with flow cytometry as a gold standard method showed similar results. The results showed that the antigen-specific magnetic cell separation with antibody-coated magnetic nanoparticles has high potential for quantitative cell surface antigen detection and analysis. PMID:23484112

  14. Cell-surface proteoglycan in sea urchin primary mesenchyme cell migration

    SciTech Connect

    Lane, M.C.

    1989-01-01

    Early in the development of the sea urchin embryo, the primary mesenchyme cells (PMC) migrate along the basal lamina of the blastocoel. Migration is inhibited in L. pictus embryos cultured in sulfate-free seawater and in S. purpuratus embryos exposed to exogenous {beta}-D-xylosides. An in vitro assay was developed to test the migratory capacity of normal PMC on normal and treated blastocoelic matrix. Sulfate deprivation and exposure to exogenous xyloside render PMC nonmotile on either matrix. Materials removed from the surface of normal PMC by treatment with 1 M urea restored migratory ability to defective cells, whereas a similar preparation isolated from the surface of epithelial cells at the same stage did not. Migration also resumed when cells were removed from the xyloside or returned to normal seawater. The urea extract was partially purified and characterized by radiolabeling, gel electrophoresis, fluorography, ion exchange chromatography, and western blotting. The PMC synthesize a large chondroitin sulfate/dermatan sulfate proteoglycan that is present in an active fraction isolated by chromatography. Chondroitinase ABC digestion of live cells blocked migration reversibly, further supporting the identification of the chondroitin sulfate/dermatan sulfate proteoglycan as the active component in the urea extract. Much of the incorporated sulfate was distributed along the filopodia in {sup 35}SO{sub 4}-labelled PMC by autoradiography. The morphology of normal and treated S. purpuratus PMC was examined by scanning electron microscopy, and differences in spreading, particularly of the extensive filopodia present on the cells, was observed. A model for the role of the chondroitin sulfate/dermatan sulfate proteoglycan in cell detachment during migration is proposed.

  15. Mating pheromone-induced alteration of cell surface proteins in the heterobasidiomycetous yeast Tremella mesenterica.

    PubMed

    Miyakawa, T; Kadota, T; Okubo, Y; Hatano, T; Tsuchiya, E; Fukui, S

    1984-06-01

    Mating pheromone-induced alteration of the cell surface proteins of haploid cells, presumed to play crucial roles in the specific cell-cell interactions during sexual conjugation of Tremella mesenterica , was investigated. Exposed surface proteins were revealed by lactoperoxidase-catalyzed iodination in combination with polyacrylamide gel electrophoresis and autoradiography. From comparison of the molecular species of 125I-labeled surface proteins of the vegetative and the gamete (mating pheromone-treated) cells of the two compatible mating types (ab and AB), it was suggested that a striking change in cell surface structure occurs during the differentiation; although labeled protein species of the vegetative cells of the two mating types were indistinguishable, several new species, both mating type specific and nonspecific, appeared in the gamete cells. Turnover of the labeled proteins of the vegetative cells was negligible, whereas that of the gamete cells was rapid with release of low-molecular-weight labeled proteins in the medium. A role for the labeled surface proteins of the gamete cells in the cell-cell interactions during sexual conjugation was suggested by the following: the surface changes were induced by mating pheromone; the labeled proteins were preferentially localized on the surface of the mating tube; the labeled species appeared sequentially during the differentiation; and mating type-specific species were present in both mating types.

  16. Cell surface display of functional human MHC class II proteins: yeast display versus insect cell display

    PubMed Central

    Wen, Fei; Sethi, Dhruv K.; Wucherpfennig, Kai W.; Zhao, Huimin

    2011-01-01

    Reliable and robust systems for engineering functional major histocompatibility complex class II (MHCII) proteins have proved elusive. Availability of such systems would enable the engineering of peptide-MHCII (pMHCII) complexes for therapeutic and diagnostic applications. In this paper, we have developed a system based on insect cell surface display that allows functional expression of heterodimeric DR2 molecules with or without a covalently bound human myelin basic protein (MBP) peptide, which is amenable to directed evolution of DR2–MBP variants with improved T cell receptor (TCR)-binding affinity. This study represents the first example of functional display of human pMHCII complexes on insect cell surface. In the process of developing this pMHCII engineering system, we have also explored the potential of using yeast surface display for the same application. Our data suggest that yeast display is a useful system for analysis and engineering of peptide binding of MHCII proteins, but not suitable for directed evolution of pMHC complexes that bind with low affinity to self-reactive TCRs. PMID:21752831

  17. An accessible protocol for solid-phase extraction of N-linked glycopeptides through reductive amination by amine-functionalized magnetic nanoparticles.

    PubMed

    Zhang, Ying; Kuang, Min; Zhang, Lijuan; Yang, Pengyuan; Lu, Haojie

    2013-06-04

    In light of the significance of glycosylation for wealthy biological events, it is important to prefractionate glycoproteins/glycopeptides from complex biological samples. Herein, we reported a novel protocol of solid-phase extraction of glycopeptides through a reductive amination reaction by employing the easily accessible 3-aminopropyltriethoxysilane (APTES)-functionalized magnetic nanoparticles. The amino groups from APTES, which were assembled onto the surface of the nanoparticles through a one-step silanization reaction, could conjugate with the aldehydes from oxidized glycopeptides and, therefore, completed the extraction. To the best of our knowledge, this is the first example of applying the reductive amination reaction into the isolation of glycopeptides. Due to the elimination of the desalting step, the detection limit of glycopeptides was improved by 2 orders of magnitude, compared to the traditional hydrazide chemistry-based solid phase extraction, while the extraction time was shortened to 4 h, suggesting the high sensitivity, specificity, and efficiency for the extraction of N-linked glycopeptides by this method. In the meantime, high selectivity toward glycoproteins was also observed in the separation of Ribonuclease B from the mixtures contaminated with bovine serum albumin. What's more, this technique required significantly less sample volume, as demonstrated in the successful mapping of glycosylation of human colorectal cancer serum with the sample volume as little as 5 μL. Because of all these attractive features, we believe that the innovative protocol proposed here will shed new light on the research of glycosylation profiling.

  18. Evidence that cell surface heparan sulfate is involved in the high affinity thrombin binding to cultured porcine aortic endothelial cells.

    PubMed Central

    Shimada, K; Ozawa, T

    1985-01-01

    It has been postulated that thrombin binds to endothelial cells through, at least in part, cell surface glycosaminoglycans such as heparan sulfate, which could serve as antithrombin cofactor on the endothelium. In the present study, we have directly evaluated the binding of 125I-labeled bovine thrombin to cultured porcine aortic endothelial cells. The thrombin binding to the cell surface was rapid, reversible, and displaced by enzymatically inactive diisopropylphosphoryl-thrombin. The concentration of thrombin at half-maximal binding was approximately 20 nM. Both specific and nonspecific binding of 125I-thrombin to the endothelial cell surface was partially inhibited in the presence of protamine sulfate, after the removal of cell surface heparan sulfate by the treatment of cells with crude Flavobacterium heparinum enzyme or purified heparitinase. The binding as a function of the concentration of thrombin revealed that the maximal amount of specific binding was reduced by approximately 50% with little alteration in binding affinity by these enzymatic treatments. The reversibility and active-site independence as well as the rate of the binding did not change after heparitinase treatment. Whereas removal of chondroitin sulfates by chondroitin ABC lyase treatment of cells did not affect the binding, identical enzymatic treatments of [35S]sulfate-labeled cells showed that either heparan sulfate or chondroitin sulfate was selectively and completely removed from the cell surface by heparitinase or chondroitin ABC lyase treatment, respectively. Furthermore, proteolysis of cell surface proteins by the purified glycosaminoglycan lyases was excluded by the identical enzymatic treatments of [3H]leucine-labeled or cell surface radioiodinated cells. Our results provide the first direct evidence that heparan sulfate on the cell surface is involved in the high-affinity, active site-independent thrombin binding by endothelial cells, and also suggest the presence of thrombin

  19. Surface engineering tumor cells with adjuvant-loaded particles for use as cancer vaccines.

    PubMed

    Ahmed, Kawther K; Geary, Sean M; Salem, Aliasger K

    2017-02-28

    Cell surface engineering is an expanding field and whilst extensive research has been performed decorating cell surfaces with biomolecules, the engineering of cell surfaces with particles has been a largely unexploited area. This study reports on the assembly of cell-particle hybrids where irradiated tumor cells were surface engineered with adjuvant-loaded, biodegradable, biocompatible, polymeric particles, with the aim of generating a construct capable of functioning as a therapeutic cancer vaccine. Successfully assembled cell-particle hybrids presented here comprised either melanoma cells or prostate cancer cells stably adorned with Toll-like receptor-9 ligand-loaded particles using streptavidin-biotin cross-linking. Both cell-particle assemblies were tested in vivo for their potential as therapeutic cancer vaccines yielding promising therapeutic results for the prostate cancer model. The ramifications of results obtained for both tumor models are openly discussed.

  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. Nano-patterned SU-8 surface using nanosphere-lithography for enhanced neuronal cell growth.

    PubMed

    Kim, Eunhee; Yoo, Seung-Jun; Kim, Eunjung; Kwon, Tae-Hwan; Zhang, Li; Moon, Cheil; Choi, Hongsoo

    2016-04-29

    Mimicking the nanoscale surface texture of the extracellular matrix can affect the regulation of cellular behavior, including adhesion, differentiation, and neurite outgrowth. In this study, SU-8-based polymer surfaces with well-ordered nanowell arrays were fabricated using nanosphere lithography with polystyrene nanoparticles. We show that the SU-8 surface with nanowells resulted in similar neuronal development of rat pheochromocytoma (PC12) cells compared with an unpatterned poly-L-lysine (PLL)-coated SU-8 surface. Additionally, even after soaking the substrate in cell culture medium for two weeks, cells on the nanowell SU-8 surface showed long-term neurite outgrowth compared to cells on the PLL-coated SU-8 surface. The topographical surface modification of the nanowell array demonstrates potential as a replacement for cell adhesive material coatings such as PLL, for applications requiring long-term use of polymer-based implantable devices.

  2. Plasma modification of PMMA films: surface free energy and cell-attachment studies.

    PubMed

    Ozcan, Canturk; Hasirci, Nesrin

    2007-01-01

    The surface of a material is the most important part determining the acceptance by and compatibility with the environment. In many cases, although the bulk properties are excellent for a specific application, the surface may require to be modified and engineered in the desired direction. This is especially important for materials used in biological media, since the surface charge, hydophilicity and wettability are important for thrombosis formation, cell attachment or cell proliferation. In this study, poly(methyl methacrylate) films were prepared by solvent casting and their surfaces were modified by oxygen plasma treatment by applying powers of 20, 100 and 300 W. The effects of surface chemistry alterations on hydophilicity, work of adhesion, surface free energy and cell adhesion were examined. Cell attachment and proliferation are especially important for the materials used for tissue-engineering purposes. The results demonstrated that there is an optimum value for hydrophilicity and surface free energy which enhance cell attachment.

  3. Surface free energy predominates in cell adhesion to hydroxyapatite through wettability.

    PubMed

    Nakamura, Miho; Hori, Naoko; Ando, Hiroshi; Namba, Saki; Toyama, Takeshi; Nishimiya, Nobuyuki; Yamashita, Kimihiro

    2016-05-01

    The initial adhesion of cells to biomaterials is critical in the regulation of subsequent cell behaviors. The purpose of this study was to investigate a mechanism through which the surface wettability of biomaterials can be improved and determine the effects of biomaterial surface characteristics on cellular behaviors. We investigated the surface characteristics of various types of hydroxyapatite after sintering in different atmospheres and examined the effects of various surface characteristics on cell adhesion to study cell-biomaterial interactions. Sintering atmosphere affects the polarization capacity of hydroxyapatite by changing hydroxide ion content and grain size. Compared with hydroxyapatite sintered in air, hydroxyapatite sintered in saturated water vapor had a higher polarization capacity that increased surface free energy and improved wettability, which in turn accelerated cell adhesion. We determined the optimal conditions of hydroxyapatite polarization for the improvement of surface wettability and acceleration of cell adhesion.

  4. LapF and Its Regulation by Fis Affect the Cell Surface Hydrophobicity of Pseudomonas putida

    PubMed Central

    Lahesaare, Andrio; Ainelo, Hanna; Teppo, Annika; Kivisaar, Maia; Heipieper, Hermann J.; Teras, Riho

    2016-01-01

    The ability of bacteria to regulate cell surface hydrophobicity is important for the adaptation to different environmental conditions. The hydrophobicity of cell surface can be determined by several factors, including outer membrane and surface proteins. In this study, we report that an adhesin LapF influences cell surface hydrophobicity of Pseudomonas putida. Cells lacking LapF are less hydrophobic than wild-type cells in stationary growth phase. Moreover, the overexpression of the global regulator Fis decreases surface hydrophobicity by repressing the expression of lapF. Flow cytometry analysis revealed that bacteria producing LapF are more viable when confronted with methanol (a hydrophilic compound) but are more susceptible to 1-octanol (a hydrophobic compound). Thus, these results revealed that LapF is the hydrophobicity factor for the cell surface of P. putida. PMID:27812186

  5. Global Transcriptomic Analysis of Model Human Cell Lines Exposed to Surface-Modified Gold Nanoparticles: The Effect of Surface Chemistry

    PubMed Central

    Grzincic, E. M.; Yang, J. A.; Drnevich, J.; Falagan-Lotsch, P.; Murphy, C. J.

    2015-01-01

    Gold nanoparticles (Au NPs) are attractive for biomedical applications not only for their remarkable physical properties, but also for the ease of which their surface chemistry can be manipulated. Many applications involve functionalization of the Au NP surface in order to improve biocompatibility, attach targeting ligands or carry drugs. However, changes in cells exposed to Au NPs of different surface chemistries have been observed, and little is known about how Au NPs and their surface coatings may impact cellular gene expression. The gene expression of two model human cell lines, human dermal fibroblasts (HDF) and prostate cancer cells (PC3) was interrogated by microarray analysis of over 14,000 human genes. The cell lines were exposed to four differently functionalized Au NPs: citrate, poly(allylamine hydrochloride) (PAH), and lipid coatings combined with alkanethiols or PAH. Gene functional annotation categories and weighted gene correlation network analysis were used in order to connect gene expression changes to common cellular functions and to elucidate expression patterns between Au NP samples. Coated Au NPs affect genes implicated in proliferation, angiogenesis, and metabolism in HDF cells, and inflammation, angiogenesis, proliferation apoptosis regulation, survival and invasion in PC3 cells. Subtle changes in surface chemistry, such as the initial net charge, lability of the ligand, and underlying layers greatly influence the degree of expression change and the type of cellular pathway affected. PMID:25491924

  6. Quantification of macrophage cell surface molecules in rheumatoid arthritis.

    PubMed Central

    Hessian, P A; Highton, J; Palmer, D G

    1989-01-01

    The response of macrophages to stimulation by interferon-gamma (IFN-gamma) in vitro is characterized by an increase in the cell surface expression of MHC class II HLA-DR antigen (HLA-DR) and the high-affinity Fc-receptor for immunoglobulin G (FcRI) while the expression of the C3b-receptor (CR1) is reduced. Based on these observations, we have examined further the possibility that IFN-gamma may modulate the activation of mononuclear phagocytes (Mph) in patients with rheumatoid arthritis (RA). As reported by others, we found low levels of IFN-gamma in the synovial fluid of these patients (less than 0.3 IU/ml using radioimmunoassay). As an alternative means of establishing whether Mph are influenced by levels of IFN-gamma too low to measure directly, we have quantified the expression of membrane associated HLA-DR, FcRI and CR1 on cell populations isolated from synovial fluid and peripheral blood. The expression of these molecules by Mph is known to be influenced by IFN-gamma. We found that Mph isolated from the synovial fluid of patients with RA showed a significantly increased HLA-DR expression. Significantly less CR1 was associated with the synovial fluid Mph than with peripheral blood monocytes. However the expression of the FcRI by the synovial fluid Mph and peripheral blood monocyte populations was similar. The quantitative changes in HLA-DR and CR1 expression by synovial fluid Mph (but not those of FcRI) were consistent with those seen following IFN-gamma activation of monocytes in vitro. While these results indicate that IFN-gamma may have a role in activating the Mph present in synovial fluid, the apparent independent regulation of FcRI observed suggests other mediators may also be involved. PMID:2527651

  7. Nanoindentation characterisation of human colorectal cancer cells considering cell geometry, surface roughness and hyperelastic constitutive behaviour.

    PubMed

    Boccaccio, Antonio; Uva, Antonio E; Papi, Massimiliano; Fiorentino, Michele; De Spirito, Marco; Monno, Giuseppe

    2017-01-27

    Characterisation of the mechanical behaviour of cancer cells is an issue of crucial importance as specific cell mechanical properties have been measured and utilized as possible biomarkers of cancer progression. Atomic force microscopy certainly occupies a prominent place in the field of the mechanical characterisation devices. We developed a hybrid approach to characterise different cell lines (SW620 and SW480) of the human colon carcinoma submitted to nanoindentation measurements. An ad hoc algorithm was written that compares the force-indentation curves experimentally retrieved with those predicted by a finite element model that simulates the nanoindentation process and reproduces the cell geometry and the surface roughness. The algorithm perturbs iteratively the values of the cell mechanical properties implemented in the finite element model until the difference between the experimental and numerical force-indentation curves reaches the minimum value. The occurrence of this indicates that the implemented material properties are very close to the real ones. Different hyperelastic constitutive models, such as Arruda-Boyce, Mooney-Rivlin and Neo-Hookean were utilized to describe the structural behaviour of indented cells. The algorithm was capable of separating, for all the cell lines investigated, the mechanical properties of cell cortex and cytoskeleton. Material properties determined via the algorithm were different with respect to those obtained with the Hertzian contact theory. This demonstrates that factors such as: the cell geometry/anatomy and the hyperelastic constitutive behaviour, which are not contemplated in the Hertz's theory hypotheses, do affect the nanoindentation measurements. The proposed approach represents a powerful tool that, only on the basis of nanoindentation measurements, is capable of characterising material at the subcellular level.

  8. Nanoindentation characterisation of human colorectal cancer cells considering cell geometry, surface roughness and hyperelastic constitutive behaviour

    NASA Astrophysics Data System (ADS)

    Boccaccio, Antonio; Uva, Antonio E.; Papi, Massimiliano; Fiorentino, Michele; De Spirito, Marco; Monno, Giuseppe

    2017-01-01

    Characterisation of the mechanical behaviour of cancer cells is an issue of crucial importance as specific cell mechanical properties have been measured and utilized as possible biomarkers of cancer progression. Atomic force microscopy certainly occupies a prominent place in the field of the mechanical characterisation devices. We developed a hybrid approach to characterise different cell lines (SW620 and SW480) of the human colon carcinoma submitted to nanoindentation measurements. An ad hoc algorithm was written that compares the force-indentation curves experimentally retrieved with those predicted by a finite element model that simulates the nanoindentation process and reproduces the cell geometry and the surface roughness. The algorithm perturbs iteratively the values of the cell mechanical properties implemented in the finite element model until the difference between the experimental and numerical force-indentation curves reaches the minimum value. The occurrence of this indicates that the implemented material properties are very close to the real ones. Different hyperelastic constitutive models, such as Arruda-Boyce, Mooney-Rivlin and Neo-Hookean were utilized to describe the structural behaviour of indented cells. The algorithm was capable of separating, for all the cell lines investigated, the mechanical properties of cell cortex and cytoskeleton. Material properties determined via the algorithm were different with respect to those obtained with the Hertzian contact theory. This demonstrates that factors such as: the cell geometry/anatomy and the hyperelastic constitutive behaviour, which are not contemplated in the Hertz’s theory hypotheses, do affect the nanoindentation measurements. The proposed approach represents a powerful tool that, only on the basis of nanoindentation measurements, is capable of characterising material at the subcellular level.

  9. Polymer surface functionalities that control human embryoid body cell adhesion revealed by high throughput surface characterization of combinatorial material microarrays.

    PubMed

    Yang, Jing; Mei, Ying; Hook, Andrew L; Taylor, Michael; Urquhart, Andrew J; Bogatyrev, Said R; Langer, Robert; Anderson, Daniel G; Davies, Martyn C; Alexander, Morgan R

    2010-12-01

    High throughput materials discovery using combinatorial polymer microarrays to screen for new biomaterials with new and improved function is established as a powerful strategy. Here we combine this screening approach with high throughput surface characterization (HT-SC) to identify surface structure-function relationships. We explore how this combination can help to identify surface chemical moieties that control protein adsorption and subsequent cellular response. The adhesion of human embryoid body (hEB) cells to a large number (496) of different acrylate polymers synthesized in a microarray format is screened using a high throughput procedure. To determine the role of the polymer surface properties on hEB cell adhesion, detailed HT-SC of these acrylate polymers is carried out using time of flight secondary ion mass spectrometry (ToF SIMS), X-ray photoelectron spectroscopy (XPS), pico litre drop sessile water contact angle (WCA) measurement and atomic force microscopy (AFM). A structure-function relationship is identified between the ToF SIMS analysis of the surface chemistry after a fibronectin (Fn) pre-conditioning step and the cell adhesion to each spot using the multivariate analysis technique partial least squares (PLS) regression. Secondary ions indicative of the adsorbed Fn correlate with increased cell adhesion whereas glycol and other functionalities from the polymers are identified that reduce cell adhesion. Furthermore, a strong relationship between the ToF SIMS spectra of bare polymers and the cell adhesion to each spot is identified using PLS regression. This identifies a role for both the surface chemistry of the bare polymer and the pre-adsorbed Fn, as-represented in the ToF SIMS spectra, in controlling cellular adhesion. In contrast, no relationship is found between cell adhesion and wettability, surface roughness, elemental or functional surface composition. The correlation between ToF SIMS data of the surfaces and the cell adhesion demonstrates

  10. Isolation of cell surface proteins for mass spectrometry-based proteomics.

    PubMed

    Elschenbroich, Sarah; Kim, Yunee; Medin, Jeffrey A; Kislinger, Thomas

    2010-02-01

    Defining the cell surface proteome has profound importance for understanding cell differentiation and cell-cell interactions, as well as numerous pathogenic abnormalities. Owing to their hydrophobic nature, plasma membrane proteins that reside on the cell surface pose analytical challenges and, despite efforts to overcome difficulties, remain under-represented in proteomic studies. Limitations in the classically employed ultracentrifugation-based approaches have led to the invention of more elaborate techniques for the purification of cell surface proteins. Three of these methods--cell surface coating with cationic colloidal silica beads, biotinylation and chemical capture of surface glycoproteins--allow for marked enrichment of this subcellular proteome, with each approach offering unique advantages and characteristics for different experiments. In this article, we introduce the principles of each purification method and discuss applications from the recent literature.

  11. Heparanase Facilitates Cell Adhesion and Spreading by Clustering of Cell Surface Heparan Sulfate Proteoglycans

    PubMed Central

    Levy-Adam, Flonia; Feld, Sari; Suss-Toby, Edith; Vlodavsky, Israel; Ilan, Neta

    2008-01-01

    Heparanase is a heparan sulfate (HS) degrading endoglycosidase participating in extracellular matrix degradation and remodeling. Apart of its well characterized enzymatic activity, heparanase was noted to exert also enzymatic-independent functions. Non-enzymatic activities of heparanase include enhanced adhesion of tumor-derived cells and primary T-cells. Attempting to identify functional domains of heparanase that would serve as targets for drug development, we have identified heparin binding domains of heparanase. A corresponding peptide (residues Lys158-Asp171, termed KKDC) was demonstrated to physically associate with heparin and HS, and to inhibit heparanase enzymatic activity. We hypothesized that the pro-adhesive properties of heparanase are mediated by its interaction with cell surface HS proteoglycans, and utilized the KKDC peptide to examine this possibility. We provide evidence that the KKDC peptide interacts with cell membrane HS, resulting in clustering of syndecan-1 and syndecan-4. We applied classical analysis of cell morphology, fluorescent and time-lapse microscopy and demonstrated that the KKDC peptide efficiently stimulates the adhesion and spreading of various cell types, mediated by PKC, Src, and the small GTPase Rac1. These results support, and further substantiate the notion that heparanase function is not limited to its enzymatic activity. PMID:18545691

  12. Binding motifs of CBP2 a potential cell surface target for carcinoma cells.

    PubMed

    Sauk, J J; Coletta, R D; Norris, K; Hebert, C

    2000-05-01

    Previously we have shown (Hebert et al. [1999] J. Cell Biochem. 73:248-258) that among many cell lines the CBP2 gene product, Hsp47, eludes its retention receptor, erd2P, resulting in the appearance of Hsp47 on the cell surface associated with the tetraspanin protein CD9. Since Hsp47 possesses a highly restricted binding cleft, random peptide display libraries were used to characterize peptides binding to Hsp47 and then to target this protein on carcinoma cell lines in vitro. Comparison of the clones obtained from panning revealed little specific homology based on sequence alone. To determine whether carcinoma cells expressing Hsp47 could selectively take up the selected bacteriophages, traditional immunofluorescence and confocal microscopy were employed. These studies revealed that phage-displaying Hsp47 binding peptides bound to cell lines expressing Hsp47 and that the peptides were rapidly taken up to a location coincident with Hsp47 staining. These observations were confirmed by cytometric analyses. These data indicate that CBP2 product may provide a molecular target for chemotherapy and/or imaging of malignancies.

  13. Human red blood cell aging: correlative changes in surface charge and cell properties.

    PubMed

    Huang, Yao-Xiong; Wu, Zheng-Jie; Mehrishi, Jitendra; Huang, Bao-Tian; Chen, Xing-Yao; Zheng, Xin-Jing; Liu, Wen-Jing; Luo, Man

    2011-12-01

    Red blood cells (RBCs) during microcirculation, aging and storage, lose N-acetylneuraminic acid (NANA) and other biomaterials thereby altering cell structures, some properties and functions. Such cell damage very likely underlies the serious adverse effects of blood transfusion. However, a controversy has remained since 1961-1977 as to whether with aging, the RBCs, suffering loss of NANA, do have a decreased charge density. Any correlation between the changes in the cell properties with cell aging is also not clear. Therefore, to remove the ambiguity and uncertainty, we carried out multiparameteric studies on Percoll fractions of blood of 38 volunteers (lightest-young-Y-RBCs, densest-old-O-RBCs, two middle fractions).We found that there were striking differences between the properties of Y-RBCs and O-RBCs. The ζ-potential of Y-RBCs decreased gradually with aging. Studies in parallel on RBC fractions incubated with both positively charged quantum dots and Sambucus Nigra-fluorescein isothiocyanate (FITC) along with their ζ-potentials provide for the first time direct visual evidence about the lesser amount of charge density and NANA on O-RBCs, and a collinear decrease in their respective ζ-potentials. Close correlation was found between the surface charge on an aging RBC and its structure and functions, from the cell morphology, the membrane deformability to the intracellular Hb structure and oxidation ability. This quantitative approach not only clarifies the picture but also has implications in biology and medicine.

  14. Enhanced cell surface polymer grafting in concentrated and nonreactive aqueous polymer solutions.

    PubMed

    Rossi, Nicholas A A; Constantinescu, Iren; Brooks, Donald E; Scott, Mark D; Kizhakkedathu, Jayachandran N

    2010-03-17

    Macromolecular cell surface modification techniques have shown tremendous utility in various biomedical applications. However, a major drawback concerns inefficient cell surface modification caused by the poor association of hydrophilic macromolecules with cell surfaces. Here, a novel, highly efficient, and universal strategy in which nonreactive "additive" macromolecules are used to modulate the grafting efficiency of cell surface reactive, hydrophilic macromolecules is described. Unprecedented enhanced cell surface modifications by up to 10-fold were observed when various concentrations of a suitable "additive" polymer was present with a constant and low concentration of a "reactive" macromolecule. The importance of this increased efficiency and the possible mechanisms involved are discussed. The cell compatible technique is demonstrated in the case of four different cell types--red blood cells (RBC), leukocytes, platelets, and Jurkat cells. A practical application of grafting macromolecules to cell surfaces in concentrated polymer solutions is demonstrated by the enhanced camouflage of RBC surface antigens for the development of RhD null RBC. In principle, the technique can be adapted to various macromolecular systems and cell types, with significant potential for biomedical applications such as live cell based technologies.

  15. Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology

    PubMed Central

    2012-01-01

    Background Cell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg) from Pichia pastoris expression cells were optimized using response surface methodology (RSM) based on the central composite design (CCD). The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells. Results The proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW) and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR) and HBsAg concentration of 29.20 mg/L respectively. Conclusions The model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing. PMID:23039947

  16. Surface and allied studies in silicon solar cells

    NASA Technical Reports Server (NTRS)

    Lindholm, F. A.

    1983-01-01

    Two main results are presented. The first deals with a simple method that determines the minority-carrier lifetime and the effective surface recombination velocity of the quasi-neutral base of silicon solar cells. The method requires the observation of only a single transient, and is amenable to automation for in-process monitoring in manufacturing. This method, which is called short-circuit current decay, avoids distortion in the observed transient and consequent inacccuracies that arise from the presence of mobile holes and electrons stored in the p/n junction spacecharge region at the initial instant of the transient. The second main result consists in a formulation of the relevant boundary-value problems that resembles that used in linear two-port network theory. This formulation enables comparisons to be made among various contending methods for measuring material parameters of p/n junction devices, and enables the option of putting the description in the time domain of the transient studies in the form of an infinite series, although closed-form solutions are also possible.

  17. ProtEx: a novel technology to display exogenous proteins on the cell surface for immunomodulation.

    PubMed

    Singh, Narendra P; Yolcu, Esma S; Askenasy, Nadir; Shirwan, Haval

    2005-11-01

    Gene therapy as an immunomodulatory approach has the potential to treat various inherited and acquired immune-based human diseases. However, its clinical application has several challenges, varying from the efficiency of gene transfer, control of gene expression, cell and tissue targeting, and safety concerns associated with the introduction of exogenous DNA into cells/tissues. Gene therapy is also a time- and labor-intensive procedure. As an alternative, we recently developed a novel technology, ProtEx, that allows for rapid, efficient, and durable display of exogenous proteins on the surface of cells, tissues, and organs without detectable toxicity. This technology exploits the strong binding affinity (Kd = 10(-15) M) of streptavidin with biotin and involves generation of chimeric molecules composed of the extracellular portions of immunological proteins of interest and a modified form of streptavidin, biotinylation of biological surfaces, and decoration of the modified surface with chimeric proteins. Biotin persists on the cell surface for weeks both in vitro and in vivo, thereby providing a platform to display exogenous proteins with extended cell surface kinetics. Two chimeric proteins, rat FasL (SA-FasL) and human CD80 (CD80-SA), were generated and tested for cell surface display and immunomodulatory functions. SA-FasL and CD80-SA molecules persisted on the surface of various cell types for extended periods, varying from days to weeks in vitro and in vivo. The cell surface kinetics, however, were protein and cell type dependent. SA-FasL showed potent apoptotic activity against Fas+ cells as a soluble protein or displayed on the cell surface and effectively blocked alloreactive responses. The display of CD80-SA on the surface of tumor cells, however, converted them into antigen-presenting cells for effective stimulation of autologous and allogeneic T-cell responses. ProtEx technology, therefore, represents a practical and effective alternative to DNA

  18. The effects of poly(dimethylsiloxane) surface silanization on the mesenchymal stem cell fate.

    PubMed

    Chuah, Yon Jin; Kuddannaya, Shreyas; Lee, Min Hui Adeline; Zhang, Yilei; Kang, Yuejun

    2015-02-01

    In recent years, poly(dimethylsiloxane) (PDMS)-based microfluidic devices have become very popular for on-chip cell investigation. Maintenance of mammalian cell adhesion on the substrate surface is crucial in determining the cell viability, proliferation and differentiation. However, the inherent hydrophobicity of PDMS is unfavourable for cell culture, causing cells to eventually dislodge from the surface. Although physically adsorbed matrix proteins can promote initial cell adhesion, this effect is usually short-lived. To address this critical issue, in this study, we employed (3-aminopropyl) triethoxy silane (APTES) and cross-linker glutaraldehyde (GA) chemistry to immobilize collagen type 1 (Col1) on PDMS. These modified surfaces are highly efficient to support the adhesion of mesenchymal stem cells (MSCs) with no deterioration of their potency. Significant changes of the native PDMS surface properties were observed with the proposed surface functionalization, and MSC adhesion was improved on PDMS surfaces modified with APTES + GA + Protein. Therefore, this covalent surface modification could generate a more biocompatible platform for stabilized cell adhesion. Furthermore, this modification method facilitated long-term cell attachment, which is favourable for successful induction of osteogenesis and cell sheet formation with an increased expression of osteogenic biomarkers and comparable extracellular matrix (ECM) constituent biomarkers, respectively. The surface silanization can be applied to PDMS-based microfluidic systems for long-term study of cellular development. Similar strategies could also be applied to several other substrate materials by appropriate combinations of self-assembled monolayers (SAMs) and ECM proteins.

  19. Enhanced compatibility of chemically modified titanium surface with periodontal ligament cells

    NASA Astrophysics Data System (ADS)

    Kado, T.; Hidaka, T.; Aita, H.; Endo, K.; Furuichi, Y.

    2012-12-01

    A simple chemical modification method was developed to immobilize cell-adhesive molecules on a titanium surface to improve its compatibility with human periodontal ligament cells (HPDLCs).The polished titanium disk was immersed in 1% (v/v) p-vinylbenzoic acid solution for 2 h to introduce carboxyl groups onto the surface. After rinsing with distilled deionized water, the titanium disk was dipped into 1.47% 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution containing 0.1 mg/ml Gly-Arg-Gly-Asp-Ser (GRGDS), human plasma fibronectin (pFN), or type I collagen from calf skin (Col) to covalently immobilize the cell-adhesive molecules on the titanium surface via formation of peptide bonds. X-ray photoelectron spectroscopy analyses revealed that cell-adhesive molecules were successfully immobilized on the titanium surfaces. The Col-immobilized titanium surface revealed higher values regarding nano rough characteristics than the as-polished titanium surface under scanning probe microscopy. The number of HPDLCs attached to both the pFN- and Col-immobilized titanium surfaces was twice that attached to the as-polished titanium surfaces. The cells were larger with the cellular processes that stretched to a greater extent on the pFN- and Col-immobilized titanium surfaces than on the as-polished titanium surface (p < 0.05). HPDLCs on the Col-immobilized titanium surfaces showed more extensive expression of vinculin at the tips of cell projections and more contiguously along the cell outline than on the as-polished, GRGDS-immobilized and pFN-immobilized titanium surfaces. It was concluded that cell-adhesive molecules successfully immobilized on the titanium surface and improved the compatibility of the surface with HPDLCs. The Col-immobilized titanium surface could be used for forming ligament-like tissues around titanium dental implants.

  20. Construction of 3D micropatterned surfaces with wormlike and superhydrophilic PEG brushes to detect dysfunctional cells.

    PubMed

    Hou, Jianwen; Shi, Qiang; Ye, Wei; Fan, Qunfu; Shi, Hengchong; Wong, Shing-Chung; Xu, Xiaodong; Yin, Jinghua

    2014-12-10

    Detection of dysfunctional and apoptotic cells plays an important role in clinical diagnosis and therapy. To develop a portable and user-friendly platform for dysfunctional and aging cell detection, we present a facile method to construct 3D patterns on the surface of styrene-b-(ethylene-co-butylene)-b-styrene elastomer (SEBS) with poly(ethylene glycol) brushes. Normal red blood cells (RBCs) and lysed RBCs (dysfunctional cells) are used as model cells. The strategy is based on the fact that poly(ethylene glycol) brushes tend to interact with phosphatidylserine, which is in the inner leaflet of normal cell membranes but becomes exposed in abnormal or apoptotic cell membranes. We demonstrate that varied patterned surfaces can be obtained by selectively patterning atom transfer radical polymerization (ATRP) initiators on the SEBS surface via an aqueous-based method and growing PEG brushes through surface-initiated atom transfer radical polymerization. The relatively high initiator density and polymerization temperature facilitate formation of PEG brushes in high density, which gives brushes worm-like morphology and superhydrophilic property; the tendency of dysfunctional cells adhered on the patterned surfaces is completely different from well-defined arrays of normal cells on the patterned surfaces, providing a facile method to detect dysfunctional cells effectively. The PEG-patterned surfaces are also applicable to detect apoptotic HeLa cells. The simplicity and easy handling of the described technique shows the potential application in microdiagnostic devices.

  1. Effects of surface barrier layer in AlGaAs/GaAs solar cells

    NASA Astrophysics Data System (ADS)

    Urabe, Hiroyuki; Kuramoto, Makoto; Nakano, Tomohiro; Kawaharazuka, Atsushi; Makimoto, Toshiki; Horikoshi, Yoshiji

    2015-09-01

    In this paper, we report the effects of surface barrier layers on the characteristics of AlGaAs/GaAs solar cells. The external quantum efficiency (EQE) spectra for AlGaAs barrier samples with different barrier layer AlAs fractions and thickness of the surface barrier layer were measured to increase the solar cell efficiency. The results show that the surface barrier layer is effective to block diffusing photoexcited electrons to the surface while the thicker barrier layer absorbs higher energy photons to generate carriers which recombine at the surface. The optimal surface barrier structure is a 50 nm thick Al0.7Ga0.3As.

  2. Comparative genomics analysis of completely sequenced microbial genomes reveals the ubiquity of N-linked glycosylation in prokaryotes.

    PubMed

    Kumar, Manjeet; Balaji, Petety V

    2011-05-01

    Glycosylation of proteins in prokaryotes has been known for the last few decades. Glycan structures and/or the glycosylation pathways have been experimentally characterized in only a small number of prokaryotes. Even this has become possible only during the last decade or so, primarily due to technological and methodological developments. Glycosylated proteins are diverse in their function and localization. Glycosylation has been shown to be associated with a wide range of biological phenomena. Characterization of the various types of glycans and the glycosylation machinery is critical to understand such processes. Such studies can help in the identification of novel targets for designing drugs, diagnostics, and engineering of therapeutic proteins. In view of this, the experimentally characterized pgl system of Campylobacter jejuni, responsible for N-linked glycosylation, has been used in this study to identify glycosylation loci in 865 prokaryotes whose genomes have been completely sequenced. Results from the present study show that only a small number of organisms have homologs for all the pgl enzymes and a few others have homologs for none of the pgl enzymes. Most of the organisms have homologs for only a subset of the pgl enzymes. There is no specific pattern for the presence or absence of pgl homologs vis-à-vis the 16S rRNA sequence-based phylogenetic tree. This may be due to differences in the glycan structures, high sequence divergence, horizontal gene transfer or non-orthologous gene displacement. Overall, the presence of homologs for pgl enzymes in a large number of organisms irrespective of their habitat, pathogenicity, energy generation mechanism, etc., hints towards the ubiquity of N-linked glycosylation in prokaryotes.

  3. N-linked glycans within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance.

    PubMed

    Chion, Alain; O'Sullivan, Jamie M; Drakeford, Clive; Bergsson, Gudmundur; Dalton, Niall; Aguila, Sonia; Ward, Soracha; Fallon, Padraic G; Brophy, Teresa M; Preston, Roger J S; Brady, Lauren; Sheils, Orla; Laffan, Michael; McKinnon, Thomas A J; O'Donnell, James S

    2016-10-13

    Enhanced von Willebrand factor (VWF) clearance is important in the etiology of von Willebrand disease. However, the molecular mechanisms underlying VWF clearance remain poorly understood. In this study, we investigated the role of VWF domains and specific glycan moieties in regulating in vivo clearance. Our findings demonstrate that the A1 domain of VWF contains a receptor-recognition site that plays a key role in regulating the interaction of VWF with macrophages. In A1-A2-A3 and full-length VWF, this macrophage-binding site is cryptic but becomes exposed following exposure to shear or ristocetin. Previous studies have demonstrated that the N-linked glycans within the A2 domain play an important role in modulating susceptibility to ADAMTS13 proteolysis. We further demonstrate that