Science.gov

Sample records for cell surface n-linked

  1. Biosynthesis and role of N-linked glycosylation in cell surface structures of archaea with a focus on flagella and s layers.

    PubMed

    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

  2. Processing of N-linked oligosaccharides in soybean cultured cells.

    PubMed

    Hori, H; Elbein, A D

    1983-02-01

    Evidence, based on both in vivo and in vitro studies with suspension-cultured soybean cells, is presented to demonstrate the processing of the oligosaccharide chain of plant N-linked glycoproteins. Following a 1-h incubation of soybean cells with [2-3H]mannose, the predominant glycopeptide obtained by pronase digestion of the membrane fraction was a Man7- or Man8GlcNAc2-Asn (GlcNAc, N-acetylglucosamine). However, the major oligosaccharide isolated from the lipid-linked oligosaccharides of these cells was a Glc2- or Glc3Man9GlcNAc2. Soybean cells were incubated with [2-3H]mannose and the incorporation of mannose into Pronase-released glycopeptides was followed during a 2-h chase. During the first 10 min of labeling, the radioactivity was mostly in a large-sized glycopeptide that appeared to be a Glc1Man9GlcNAc2-peptide. During the next 60 to 90 min of chase, this radioactivity was shifted to smaller and smaller-sized glycopeptides indicating that removal of sugars (i.e., processing) had occurred. Both glucosidase and mannosidase activity was detected in membrane preparations of soybean cells. Nine different glycopeptides were isolated from Pronase digests of soybean cell membrane fractions. These glycopeptides were purified by repeated gel filtration on columns of Bio-Gel P-4. Partial characterization of these glycopeptides by endoglucosaminidase H and alpha-mannosidase digestion, and by analysis of the products, suggested the following glycopeptides: Glc1Man9GlcNAc2-Asn, Man8GlcNAc2-Asn, Man7GlcNAc2-Asn, Man6GlcNAc2-Asn, and Man5GlcNAc2-Asn. PMID:6681697

  3. 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

  4. 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.

  5. 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.

  6. Release and preparation of intact and unreduced N-linked oligosaccharides from Sf-9 insect cells.

    PubMed

    Wolff, M W; Murhammer, D W; Linhardt, R J

    1999-02-01

    Glycosylation, the addition of carbohydrates to a peptide backbone, is the most extensive cotranslational and posttranslational modification made to proteins by eukaryotic cells. The glycosylation profile of a recombinant glycoprotein can significantly affect its biological activity, which is particularly important when being used in human therapeutic applications. Therefore, defining glycan structures to ensure consistency of recombinant glycoproteins among different batches is critical. In this study we describe a method to prepare N-linked glycans derived from insect cell glycoproteins for structural analysis by capillary electrophoresis. Briefly, glycoproteins obtained from uninfected Spodoptera frugiperda Sf-9 insect cells were precipitated with ammonium sulfate and the glycans were chemically cleaved by hydrazinolysis. Following the regeneration of the glycan reducing terminal residue and the removal of contaminating proteins and peptides, the glycans were fluorescently labeled by reductive amination. Fluorescent labeling greatly enhanced the detection limit of the glycan structures determined by capillary electrophoresis. Five major glycan structures were found that migrated between tetra-mannosylated hexasaccharide and nonamannosylated undecasaccharide standards. Upon alpha-mannosidase digestion the number of glycan structures was reduced to two major structures with shorter migration times than the undigested glycans. None of the glycans were susceptible to hexosaminidase or galactosidase treatment. These results are consistent with the majority of previous results demonstrating hypermannosylated glycan structures in Sf-9 insect cells.

  7. 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.

  8. Purification and substrate specificity of beta-xylosidase from sycamore cell (Acer pseudoplatanus L.): application for structural analysis of xylose-containing N-linked oligosaccharides.

    PubMed

    Tezuka, K; Hayashi, M; Ishihara, H; Nishimura, M; Onozaki, K; Takahashi, N

    1993-06-01

    A beta-xylosidase was purified 51-fold from culture medium of sycamore (Acer pseudoplatanus L.) cells using p-nitrophenyl beta-D-xylopyranoside as a substrate. This enzyme can remove a xylose residue from asparagine-linked oligosaccharides, derivatized with 2-aminopyridine. A pentasaccharide, Xy1 beta 2Man beta 4GlcNAc beta 4(Fuc-alpha 3)GlcNAc was the favorite substrate in N-linked oligosaccharides, but a xylose residue in Xy1 beta 2(Man-alpha 3)Man beta sequence could not be removed by the enzyme. We also propose an efficient method for detection of xylose residue in N-linked oligosaccharides by a combination of the two-dimensional sugar mapping technique and the xylosidase digestion.

  9. Detection of conserved N-linked glycans and phase-variable lipooligosaccharides and capsules from campylobacter cells by mass spectrometry and high resolution magic angle spinning NMR spectroscopy.

    PubMed

    Szymanski, Christine M; Michael, Frank St; Jarrell, Harold C; Li, Jianjun; Gilbert, Michel; Larocque, Suzon; Vinogradov, Evgeny; Brisson, Jean-Robert

    2003-07-01

    Glycomics, the study of microbial polysaccharides and genes responsible for their formation, requires the continuous development of rapid and sensitive methods for the identification of glycan structures. In this study, methods for the direct analysis of sugars from 108 to 1010 cells are outlined using the human gastrointestinal pathogen, Campylobacter jejuni. Using capillary-electrophoresis coupled with sensitive electrospray mass spectrometry, we demonstrate variability in the lipid A component of C. jejuni lipooligosaccharides (LOSs). In addition, these sensitive methods have permitted the detection of phase-variable LOS core structures that were not observed previously. High resolution magic angle spinning (HR-MAS) NMR was used to examine capsular polysaccharides directly from campylobacter cells and showed profiles similar to those observed for purified polysaccharides analyzed by solution NMR. This method also exhibited the feasibility of campylobacter serotyping, mutant verification, and preliminary sugar analysis. HR-MAS NMR examination of growth from individual colonies of C. jejuni NCTC11168 indicated that the capsular glycan modifications are also phase-variable. These variants show different staining patterns on deoxycholate-PAGE and reactivity with immune sera. One of the identified modifications was a novel -OP=O(NH2)OMe phosphoramide, not observed previously in nature. In addition, HR-MAS NMR detected the N-linked glycan, GalNAc-alpha1,4-GalNAc-alpha1,4-[Glc-beta1,3-]GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac, where Bac is 2,4-diacetamido-2,4,6-trideoxy-d-glucopyranose, in C. jejuni and Campylobacter coli. The presence of this common heptasaccharide in multiple campylobacter isolates demonstrates the conservation of the N-linked protein glycosylation pathway in this organism and describes the first report of HR-MAS NMR detection of N-linked glycans on glycoproteins from intact bacterial cells.

  10. Studies on synthetic pathway of xylose-containing N-linked oligosaccharides deduced from substrate specificities of the processing enzymes in sycamore cells (Acer pseudoplatanus L.).

    PubMed

    Tezuka, K; Hayashi, M; Ishihara, H; Akazawa, T; Takahashi, N

    1992-02-01

    We measured the activities of alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase, alpha-1,6-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase, beta-1,4-mannosyl-glycoprotein beta-1,2-xylosyltransferase and glycoprotein 3-alpha-L-fucosyltransferase in the Golgi fraction of suspension-cultured cells of sycamore (Acer pseudoplatanus L.) using fluorescence-labelled oligosaccharides as acceptor substrates for these transferase reactions. The structures of the pyridylaminated oligosaccharides produced by these reactions were analyzed by two-dimensional sugar mapping using high-performance liquid chromatography. We demonstrated that (formula; see text) was processed to produce by these in vitro reactions. On the basis of these results, we discuss a biosynthetic pathway for xylose containing N-linked oligosaccharides in plant glycoproteins.

  11. Transfer of two oligosaccharides to protein in a Chinese hamster ovary cell B211 which utilizes polyprenol for its N-linked glycosylation intermediates.

    PubMed

    Kaiden, A; Rosenwald, A G; Cacan, R; Verbert, A; Krag, S S

    1998-10-15

    B211, a glycosylation mutant isolated from Chinese hamster ovary cells, synthesizes 10- to 15-fold less Glc3Man9GlcNAc2-P-P-lipid, the substrate used by the oligosaccharide transferase in the synthesis of asparagine-linked glycoproteins. B211 cells are also 10- to 15-fold deficient in the glucosylation of oligosaccharide-lipid. Despite these properties, protein glycosylation in B211 cells proceeds at a level similar to (50% of) parental cells. We asked whether the near wild-type level of glycosylation was due to the transfer of alternative oligosaccharide structures to protein in B211 cells. The aberrant size of [35S]methionine-labeled VSV G protein and the increased percentage of endoglycosidase H-resistant tryptic peptides as compared to parental cells supported this hypothesis. B211 cells were labeled with [2-3H]mannose either for 1 min or for 1 h in the presence of glycoprotein-processing inhibitors so that the oligosaccharides initially transferred to protein could be analyzed. In addition to Glc3Man9GlcNAc2, a second, endoglycosidase H-resistant oligosaccharide was transferred whose structure was determined by alpha-mannosidase digestion, gel filtration chromatography, and HPLC to be Glc0,1Man5GlcNAc2. Finally, since the synthesis of reduced amounts of Glc3Man9GlcNAc2-P-P-lipid was also a phenotype seen in another glycosylation mutant, Lec9, we analyzed the long-chain prenol in B211 cells. B211 cells synthesized and utilized polyprenol rather than dolichol for all N-linked glycosylation intermediates as determined by HPLC analysis of [3H]mevalonate-labeled lipids. Cell fusions analyzed by similar techniques indicated that B211, originally isolated as a concanavalin A-resistant cell line, is in the Lec9 complementation group.

  12. 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.

  13. Cell-free synthesis of enzymically active tissue-type plasminogen activator. Protein folding determines the extent of N-linked glycosylation.

    PubMed Central

    Bulleid, N J; Bassel-Duby, R S; Freedman, R B; Sambrook, J F; Gething, M J

    1992-01-01

    Tissue-type plasminogen activator (t-PA) is synthesized in mammalian cells as a mixture of two forms that differ in their extent of N-linked glycosylation. We have investigated the mechanism underlying this variation in glycosylation, using a cell-free system that consists of a rabbit reticulocyte lysate optimized for the formation of disulphide bonds and supplemented with dog pancreas microsomal membranes. Molecules of human t-PA synthesized in vitro are enzymically active and responsive to natural activators and inhibitors, and are glycosylated in a pattern identical with that of the protein produced in vivo. This demonstrates that t-PA synthesized in vitro folds into the same conformation as the protein synthesized in vivo. We show that the extent of glycosylation of individual t-PA molecules is dependent on the state of folding of the polypeptide chain, since the probability of addition of an oligosaccharide side chain at Asn-184 is decreased under conditions that promote the formation of enzymically active molecules. This variation in glycosylation is independent of the rate of protein synthesis. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:1520279

  14. L1CAM from human melanoma carries a novel type of N-glycan with Galβ1-4Galβ1- motif. Involvement of N-linked glycans in migratory and invasive behaviour of melanoma cells.

    PubMed

    Hoja-Łukowicz, Dorota; Link-Lenczowski, Paweł; Carpentieri, Andrea; Amoresano, Angela; Pocheć, Ewa; Artemenko, Konstantin A; Bergquist, Jonas; Lityńska, Anna

    2013-04-01

    Dramatic changes in glycan biosynthesis during oncogenic transformation result in the emergence of marker glycans on the cell surface. We analysed the N-linked glycans of L1CAM from different stages of melanoma progression, using high-performance liquid chromatography combined with exoglycosidase sequencing, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and lectin probes. L1CAM oligosaccharides are heavily sialylated, mainly digalactosylated, biantennary complex-type structures with galactose β1-4/3-linked to GlcNAc and with or without fucose α1-3/6-linked to GlcNAc. Hybrid, bisected hybrid, bisected triantennary and tetraantennary complex oligosaccharides, and β1-6-branched complex-type glycans with or without lactosamine extensions are expresses at lower abundance. We found that metastatic L1CAM possesses only α2-6-linked sialic acid and the loss of α2-3-linked sialic acid in L1CAM is a phenomenon observed during the transition of melanoma cells from VGP to a metastatic stage. Unexpectedly, we found a novel monoantennary complex-type oligosaccharide with a Galβ1-4Galβ1- epitope capped with sialic acid residues A1[3]G(4)2S2-3. To our knowledge this is the first report documenting the presence of this oligosaccharide in human cancer. The novel and unique N-glycan should be recognised as a new class of human melanoma marker. In functional tests we demonstrated that the presence of cell surface α2-3-linked sialic acid facilitates the migratory behaviour and increases the invasiveness of primary melanoma cells, and it enhances the motility of metastatic cells. The presence of cell surface α2-6-linked sialic acid enhances the invasive potential of both primary and metastatic melanoma cells. Complex-type oligosaccharides in L1CAM enhance the invasiveness of metastatic melanoma cells. PMID:22544341

  15. 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.

  16. Characterization of disease-associated N-linked glycoproteins

    PubMed Central

    Tian, Yuan; Zhang, Hui

    2013-01-01

    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. PMID:23255236

  17. α-1,6-Mannosylation of N-Linked Oligosaccharide Present on Cell Wall Proteins Is Required for Their Incorporation into the Cell Wall in the Filamentous Fungus Neurospora crassa▿†

    PubMed Central

    Maddi, Abhiram; Free, Stephen J.

    2010-01-01

    The enzyme α-1,6-mannosyltransferase (OCH-1) is required for the synthesis of galactomannans attached to the N-linked oligosaccharides of Neurospora crassa cell wall proteins. The Neurospora crassa och-1 mutant has a tight colonial phenotype and a defective cell wall. A carbohydrate analysis of the och-1 mutant cell wall revealed a 10-fold reduction in the levels of mannose and galactose and a total lack of 1,6-linked mannose residues. Analysis of the integral cell wall protein from wild-type and och-1 mutant cells showed that the mutant cell wall had reduced protein content. The och-1 mutant was found to secrete 18-fold more protein than wild-type cells. Proteomic analysis of the proteins released by the mutant into the growth medium identified seven of the major cell wall proteins. Western blot analysis of ACW-1 and GEL-1 (two glycosylphosphatidylinositol [GPI]-anchored proteins that are covalently integrated into the wild-type cell wall) showed that high levels of these proteins were being released into the medium by the och-1 mutant. High levels of ACW-1 and GEL-1 were also released from the och-1 mutant cell wall by subjecting the wall to boiling in a 1% SDS solution, indicating that these proteins are not being covalently integrated into the mutant cell wall. From these results, we conclude that N-linked mannosylation of cell wall proteins by OCH-1 is required for their efficient covalent incorporation into the cell wall. PMID:20870880

  18. 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

  19. 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

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

    PubMed

    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.

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

    PubMed

    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

  2. Functional analysis of N-linked glycosylation mutants of the measles virus fusion protein synthesized by recombinant vaccinia virus vectors.

    PubMed Central

    Alkhatib, G; Shen, S H; Briedis, D; Richardson, C; Massie, B; Weinberg, R; Smith, D; Taylor, J; Paoletti, E; Roder, J

    1994-01-01

    The role of N-linked glycosylation in the biological activity of the measles virus (MV) fusion (F) protein was analyzed by expressing glycosylation mutants with recombinant vaccinia virus vectors. There are three potential N-linked glycosylation sites located on the F2 subunit polypeptide of MV F, at asparagine residues 29, 61, and 67. Each of the three potential glycosylation sites was mutated separately as well as in combination with the other sites. Expression of mutant proteins in mammalian cells showed that all three sites are used for the addition of N-linked oligosaccharides. Cell surface expression of mutant proteins was reduced by 50% relative to the wild-type level when glycosylation at either Asn-29 or Asn-61 was abolished. Despite the similar levels of cell surface expression, the Asn-29 and Asn-61 mutant proteins had different biological activities. While the Asn-61 mutant was capable of inducing syncytium formation, the Asn-29 mutant protein did not exhibit any significant cell fusion activity. Inactivation of the Asn-67 glycosylation site also reduced cell surface transport of mutant protein but had little effect on its ability to cause cell fusion. However, when the Asn-67 mutation was combined with mutations at either of the other two sites, cleavage-dependent activation, cell surface expression, and cell fusion activity were completely abolished. Our data show that the loss of N-linked oligosaccharides markedly impaired the proteolytic cleavage, stability, and biological activity of the MV F protein. The oligosaccharide side chains in MV F are thus essential for optimum conformation of the extracellular F2 subunit that is presumed to bind cellular membranes. Images PMID:8107215

  3. Eliminating antibody polyreactivity through addition of N-linked glycosylation.

    PubMed

    Chuang, Gwo-Yu; Zhang, Baoshan; McKee, Krisha; O'Dell, Sijy; Kwon, Young Do; Zhou, Tongqing; Blinn, Julie; Lloyd, Krissey; Parks, Robert; Von Holle, Tarra; Ko, Sung-Youl; Kong, Wing-Pui; Pegu, Amarendra; Wang, Keyun; Baruah, Kavitha; Crispin, Max; Mascola, John R; Moody, M Anthony; Haynes, Barton F; Georgiev, Ivelin S; Kwong, Peter D

    2015-06-01

    Antibody polyreactivity can be an obstacle to translating a candidate antibody into a clinical product. Standard tests such as antibody binding to cardiolipin, HEp-2 cells, or nuclear antigens provide measures of polyreactivity, but its causes and the means to resolve are often unclear. Here we present a method for eliminating antibody polyreactivity through the computational design and genetic addition of N-linked glycosylation near known sites of polyreactivity. We used the HIV-1-neutralizing antibody, VRC07, as a test case, since efforts to increase VRC07 potency at three spatially distinct sites resulted in enhanced polyreactivity. The addition of N-linked glycans proximal to the polyreactivity-enhancing mutations at each of the spatially distinct sites resulted in reduced antibody polyreactivity as measured by (i) anti-cardiolipin ELISA, (ii) Luminex AtheNA Multi-Lyte ANA binding, and (iii) HEp-2 cell staining. The reduced polyreactivity trended with increased antibody concentration over time in mice, but not with improved overall protein stability as measured by differential scanning calorimetry. Moreover, glycan proximity to the site of polyreactivity appeared to be a critical factor. The results provide evidence that antibody polyreactivity can result from local, rather than global, features of an antibody and that addition of N-linked glycosylation can be an effective approach to reducing antibody polyreactivity.

  4. N-linked protein glycosylation in the ER.

    PubMed

    Aebi, Markus

    2013-11-01

    N-linked protein glycosylation in the endoplasmic reticulum (ER) is a conserved two phase process in eukaryotic cells. It involves the assembly of an oligosaccharide on a lipid carrier, dolichylpyrophosphate and the transfer of the oligosaccharide to selected asparagine residues of polypeptides that have entered the lumen of the ER. The assembly of the oligosaccharide (LLO) takes place at the ER membrane and requires the activity of several specific glycosyltransferases. The biosynthesis of the LLO initiates at the cytoplasmic side of the ER membrane and terminates in the lumen where oligosaccharyltransferase (OST) selects N-X-S/T sequons of polypeptide and generates the N-glycosidic linkage between the side chain amide of asparagine and the oligosaccharide. The N-glycosylation pathway in the ER modifies a multitude of proteins at one or more asparagine residues with a unique carbohydrate structure that is used as a signalling molecule in their folding pathway. In a later stage of glycoprotein processing, the same systemic modification is used in the Golgi compartment, but in this process, remodelling of the N-linked glycans in a protein-, cell-type and species specific manner generates the high structural diversity of N-linked glycans observed in eukaryotic organisms. This article summarizes the current knowledge of the N-glycosylation pathway in the ER that results in the covalent attachment of an oligosaccharide to asparagine residues of polypeptide chains and focuses on the model organism Saccharomyces cerevisiae. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum. PMID:23583305

  5. Molecular analysis of cell surface beta-1,4-galactosyltransferase function during cell migration.

    PubMed Central

    Appeddu, P A; Shur, B D

    1994-01-01

    Despite the identification and characterization of cell surface receptors for the extracellular matrix, it is unknown how their relative expression and cytoskeletal association regulate cell migration. Previous studies have identified beta-1,4-galactosyltransferase (GalTase; EC 2.4.1.38) on the surface of migrating cells, where it mediates cell migration on basal lamina matrices by associating with the cytoskeleton and binding to N-linked oligosaccharides in the E8 domain of laminin. In this study, the function of GalTase during cell migration was examined directly by analyzing the migration rate of stably transfected cell lines in which the relative level of surface GalTase and its ability to associate with the cytoskeleton were altered. We show here that the cytoskeleton contains a limiting, saturable, number of binding sites for surface GalTase. Furthermore, the rate of cell migration was inversely related to the ability of surface GalTase to associate with the cytoskeleton. Elevating surface GalTase in excess of the number of cytoskeleton-binding sites reduced the rate of cell migration, whereas decreasing the amount of surface GalTase available to bind the cytoskeleton increased migration rates. These results show that the rate of cell migration on basal lamina is directly dependent upon the expression of surface GalTase and the ability of this protein to associate with a limiting number of cytoskeleton-binding sites. Images PMID:8134355

  6. 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.

  7. Production of Secretory and Extracellular N-Linked Glycoproteins in Escherichia coli▿ †

    PubMed Central

    Fisher, Adam C.; Haitjema, Charles H.; Guarino, Cassandra; Çelik, Eda; Endicott, Christine E.; Reading, Craig A.; Merritt, Judith H.; Ptak, A. Celeste; Zhang, Sheng; DeLisa, Matthew P.

    2011-01-01

    The Campylobacter jejuni pgl gene cluster encodes a complete N-linked protein glycosylation pathway that can be functionally transferred into Escherichia coli. In this system, we analyzed the interplay between N-linked glycosylation, membrane translocation and folding of acceptor proteins in bacteria. We developed a recombinant N-glycan acceptor peptide tag that permits N-linked glycosylation of diverse recombinant proteins expressed in the periplasm of glycosylation-competent E. coli cells. With this “glycosylation tag,” a clear difference was observed in the glycosylation patterns found on periplasmic proteins depending on their mode of inner membrane translocation (i.e., Sec, signal recognition particle [SRP], or twin-arginine translocation [Tat] export), indicating that the mode of protein export can influence N-glycosylation efficiency. We also established that engineered substrate proteins targeted to environments beyond the periplasm, such as the outer membrane, the membrane vesicles, and the extracellular medium, could serve as substrates for N-linked glycosylation. Taken together, our results demonstrate that the C. jejuni N-glycosylation machinery is compatible with distinct secretory mechanisms in E. coli, effectively expanding the N-linked glycome of recombinant E. coli. Moreover, this simple glycosylation tag strategy expands the glycoengineering toolbox and opens the door to bacterial synthesis of a wide array of recombinant glycoprotein conjugates. PMID:21131519

  8. 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

  9. Negative feedback regulation of Wnt signaling via N-linked fucosylation in zebrafish.

    PubMed

    Feng, Lei; Jiang, Hao; Wu, Peng; Marlow, Florence L

    2014-11-15

    L-fucose, a monosaccharide widely distributed in eukaryotes and certain bacteria, is a determinant of many functional glycans that play central roles in numerous biological processes. The molecular mechanism, however, by which fucosylation mediates these processes remains largely elusive. To study how changes in fucosylation impact embryonic development, we up-regulated N-linked fucosylation via over-expression of a key GDP-Fucose transporter, Slc35c1, in zebrafish. We show that Slc35c1 overexpression causes elevated N-linked fucosylation and disrupts embryonic patterning in a transporter activity dependent manner. We demonstrate that patterning defects associated with enhanced N-linked fucosylation are due to diminished canonical Wnt signaling. Chimeric analyses demonstrate that elevated Slc35c1 expression in receiving cells decreases the signaling range of Wnt8a during zebrafish embryogenesis. Moreover, we provide biochemical evidence that this decrease is associated with reduced Wnt8 ligand and elevated Lrp6 coreceptor, which we show are both substrates for N-linked fucosylation in zebrafish embryos. Strikingly, slc35c1 expression is regulated by canonical Wnt signaling. These results suggest that Wnt limits its own signaling activity in part via up-regulation of a transporter, slc35c1 that promotes terminal fucosylation and thereby limits Wnt activity.

  10. Structure of the cell surface.

    PubMed

    Singer, S J

    1982-01-01

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

  11. Defects in the N-linked oligosaccharide biosynthetic pathway in a Trypanosoma brucei glycosylation mutant.

    PubMed

    Acosta-Serrano, Alvaro; O'Rear, Jessica; Quellhorst, George; Lee, Soo Hee; Hwa, Kuo-Yuan; Krag, Sharon S; Englund, Paul T

    2004-04-01

    Concanavalin A (ConA) kills the procyclic (insect) form of Trypanosoma brucei by binding to its major surface glycoprotein, procyclin. We previously isolated a mutant cell line, ConA 1-1, that is less agglutinated and more resistant to ConA killing than are wild-type (WT) cells. Subsequently we found that the ConA resistance phenotype in this mutant is due to the fact that the procyclin either has no N-glycan or has an N-glycan with an altered structure. Here we demonstrate that the alteration in procyclin N-glycosylation correlates with two defects in the N-linked oligosaccharide biosynthetic pathway. First, ConA 1-1 has a defect in activity of polyprenol reductase, an enzyme involved in synthesis of dolichol. Metabolic incorporation of [3H]mevalonate showed that ConA 1-1 synthesizes equal amounts of dolichol and polyprenol, whereas WT cells make predominantly dolichol. Second, we found that ConA 1-1 synthesizes and accumulates an oligosaccharide lipid (OSL) precursor that is smaller in size than that from WT cells. The glycan of OSL in WT cells is apparently Man9GlcNAc2, whereas that from ConA 1-1 is Man7GlcNAc2. The smaller OSL glycan in the ConA 1-1 explains how some procyclin polypeptides bear a Man4GlcNAc2 modified with a terminal N-acetyllactosamine group, which is poorly recognized by ConA.

  12. Contribution of leptin receptor N-linked glycans to leptin binding.

    PubMed

    Kamikubo, Yuichi; Dellas, Claudia; Loskutoff, David J; Quigley, James P; Ruggeri, Zaverio M

    2008-03-15

    The extracellular domain of the human leptin receptor (Ob-R) contains 20 potential N-glycosylation sites whose role in leptin binding remains to be elucidated. We found that a mammalian cell-expressed sOb-R (soluble Ob-R) fragment (residues 22-839 of the extracellular domain) bound leptin with a dissociation constant of 1.8 nM. This binding was inhibited by Con A (concanavalin A) or wheatgerm agglutinin. Treatment of sOb-R with peptide N-glycosidase F reduced leptin binding by approximately 80% concurrently with N-linked glycan removal. The human megakaryoblastic cell line, MEG-01, expresses two forms of the Ob-R, of approx. 170 and 130 kDa molecular mass. Endo H (endoglycosidase H) treatment and cell culture with alpha-glucosidase inhibitors demonstrated that N-linked glycans are of the complex mature type in the 170 kDa form and of the high-mannose type in the 130 kDa form. Both isoforms bound leptin, but not after peptide N-glycosidase F treatment. An insect-cell-expressed sOb-R fragment, consisting of the Ig (immunoglobulin), CRH2 (second cytokine receptor homology) and FNIII (fibronectin type III) domains, bound leptin with affinity similar to that of the entire extracellular domain, but this function was abolished after N-linked glycan removal. The same treatment had no effect on the leptin-binding activity of the isolated CRH2 domain. Our findings show that N-linked glycans within Ig and/or FNIII domains regulate Ob-R function, but are not involved in essential interactions with the ligand.

  13. The myeloid differentiation antigen CD14 is N- and O-glycosylated. Contribution of N-linked glycosylation to different soluble CD14 isoforms.

    PubMed

    Stelter, F; Pfister, M; Bernheiden, M; Jack, R S; Bufler, P; Engelmann, H; Schütt, C

    1996-03-01

    The myeloid differentiation antigen CD14 acts as the major receptor for bacterial lipopolysaccharide (LPS). A soluble form of the protein (sCD14) is present in human serum which functions as a soluble LPS receptor. We have compared the isoform patterns of soluble CD14 derived from human serum and of the recombinant proteins produced by CHO cells transfected with either the wild-type CD14 gene or with a cDNA coding for a truncated protein which lacks the C-terminal 21 amino acids [sCD14-(1-335)-peptide]. Using SDS/PAGE, two dominant isoforms (53 and 50 kDa) and two minor forms (46 and 43 kDa) can be detected in serum as well as in the supernatants of both transfectants. sCD14 is a glycoprotein which carries N- and O-linked carbohydrates. The different isoforms of sCD14-(1-335)-peptide are due to differences in the content of N-linked sugars. However after the removal of N- and O-linked carbohydrates from serum- and CHO-derived wild-type proteins, two isoforms are still present. These results indicate that N-linked glycosylation contributes to but does not fully explain the different forms of soluble CD14. We further examined whether the mutation of individual N-linked glycosylation sites influences the expression of membrane-bound and soluble CD14 forms and the ability of the membrane-bound molecule to bind LPS. As with the wild-type proteins, the different isoforms of the soluble mutants are partially due to differences in N-linked glycosylation. A truncated mutant which lacks the two N-terminal glycosylation sites {[Asp18, Asp132]CD14-(1-335)peptide} does not give rise to multiple forms on SDS gels. Like CD14-(1-335)-peptide, this mutant is not expressed on the cell surface suggesting that a smaller isoform present in the wild-type preparations results from proteolytic cleavage of the membrane-bound molecule. N-linked carbohydrates do not seem to be important for the binding of LPS to membrane-bound CD14. PMID:8612616

  14. Glucose persistence on high-mannose oligosaccharides selectively inhibits the macroautophagic sequestration of N-linked glycoproteins.

    PubMed Central

    Ogier-Denis, E; Bauvy, C; Cluzeaud, F; Vandewalle, A; Codogno, P

    2000-01-01

    The macroautophagic-lysosomal pathway is a bulk degradative process for cytosolic proteins and organelles including the endoplasmic reticulum (ER). We have previously shown that the human colonic carcinoma HT-29 cell population is characterized by a high rate of autophagic degradation of N-linked glycoproteins substituted with ER-type glycans. In the present work we demonstrate that glucosidase inhibitors [castanospermine (CST) and deoxynojirimycin] have a stabilizing effect on newly synthesized glucosylated N-linked glycoproteins and impaired their lysosomal delivery as shown by subcellular fractionation on Percoll gradients. The inhibition of macroautophagy was restricted to N-linked glycoproteins because macroautophagic parameters such as the rate of sequestration of cytosolic markers and the fractional volume occupied by autophagic vacuoles were not affected in CST-treated cells. The protection of glucosylated glycoproteins from autophagic sequestration was also observed in inhibitor-treated Chinese hamster ovary (CHO) cells and in Lec23 cells (a CHO mutant deficient in glucosidase I activity). The interaction of glucosylated glycoproteins with the ER chaperone binding protein (BiP) was prolonged in inhibitor-treated cells in comparison with untreated CHO cells. These results show that the removal of glucose from N-glycans of glycoproteins is a key event for their delivery to the autophagic pathway and that interaction with BiP could prevent or delay newly synthesized glucosylated N-linked glycoproteins from being sequestered by the autophagic pathway. PMID:10642502

  15. 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

  16. 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. PMID:27281343

  17. IFNa of black carp is an antiviral cytokine modified with N-linked glycosylation.

    PubMed

    Huang, Zhilin; Chen, Song; Liu, Jiachen; Xiao, Jun; Yan, Jun; Feng, Hao

    2015-10-01

    Type I interferons (IFNs) play an important role in the antiviral immune response in teleost fish. In this study, one type I interferon (bcIFNa) of black carp (Mylopharyngodon piceus) has been cloned and characterized. The full-length cDNA of bcIFNa gene consists of 783 nucleotides and the predicted bcIFNa protein contains 185 amino acids. Semi-quantitative RT-PCR analysis demonstrated that bcIFNa mRNA transcription level in all the selected tissues of black carp was greatly increased at 33 h post spring viremia of carp virus (SVCV) infection. The protein of bcIFNa could be detected in both the whole cell lysate and the supernatant media of HEK293T cells transfected with plasmids expressing bcIFNa through immunoblot assay. EPC cells showed greatly increased antiviral ability when the cells were treated with the bcIFNa-containing conditioned media for 24 h before SVCV infection. Mass spectrum assay and glycosidase digestion analysis determined that bcIFNa is modified with N-linked glycosylation, which occurs on the Asn (N) of 38 site of this cytokine. The un-glycosylated mutant bcIFNa-N38Q could be secreted out of the cell and showed the similar antiviral ability against SVCV as that of wild type bcIFNa, which suggested that N-linked glycosylation does not contribute directly to the antiviral property of this fish cytokine.

  18. Studying N-linked glycosylation of receptor tyrosine kinases.

    PubMed

    Itkonen, Harri M; Mills, Ian G

    2015-01-01

    Metabolic alterations have been identified as a frequent event in cancer. This is often associated with increased flux through glycolysis, and also a secondary pathway to glycolysis, hexosamine biosynthetic pathway (HBP). HBP provides substrate for N-linked glycosylation, which occurs in the endoplasmic reticulum and the Golgi apparatus. N-linked glycosylation supports protein folding and correct sorting of proteins to plasma membrane and secretion. This process generates complex glycoforms, which can be recognized by other proteins and glycosylation of receptor tyrosine kinases (RTK) can also regulate their plasma-membrane retention time. Of special interest for experimental biologists, plants produce proteins, termed lectins, which bind with high specificity to glyco-conjugates. For the purposes of molecular biology, plant lectins can be conjugated to different moieties, such as agarose beads, which enable precipitation of specifically glycosylated proteins. In this chapter, we describe in detail how to perform pull-down experiments with commercially available lectins to identify changes in the glycosylation of RTKs. PMID:25319893

  19. Functional characterization of Sporothrix schenckii glycosidases involved in the N-linked glycosylation pathway.

    PubMed

    Lopes-Bezerra, Leila M; Lozoya-Pérez, Nancy E; López-Ramírez, Luz A; Martínez-Álvarez, José A; Teixeira, Marcus M; Felipe, Maria S S; Flores-Carreón, Arturo; Mora-Montes, Héctor M

    2015-01-01

    Protein glycosylation pathways are conserved metabolic processes in eukaryotic organisms and are required for cell fitness. In fungal pathogens, the N-linked glycosylation pathway is indispensable for proper cell wall composition and virulence. In Sporothrix schenckii sensu stricto, the causative agent of sporotrichosis, little is known about this glycosylation pathway. Here, using a genome-wide screening for putative members of the glycosyl hydrolase (CAZy - GH) families 47 and 63, which group enzymes involved in the processing step during N-linked glycan maturation, we found seven homologue genes belonging to family 47 and one to family 63. The eight genes were individually expressed in C. albicans null mutants lacking either MNS1 (for members of family 47) or CWH41 (for the member of family 63). Our results indicate that SsCWH41 is the functional ortholog of CaCWH41, whereas SsMNS1 is the functional ortholog of CaMNS1. The remaining genes of family 47 encode Golgi mannosidases and endoplasmic reticulum degradation-enhancing alpha-mannosidase-like proteins (EDEMs). Since these GH families gather proteins used as target for drugs to control cell growth, identification of these genes could help in the design of antifungals that could be used to treat sporotrichosis and other fungal diseases. In addition, to our knowledge, we are the first to report that Golgi mannosidases and EDEMs are expressed and characterized in yeast cells.

  20. Functional characterization of Sporothrix schenckii glycosidases involved in the N-linked glycosylation pathway.

    PubMed

    Lopes-Bezerra, Leila M; Lozoya-Pérez, Nancy E; López-Ramírez, Luz A; Martínez-Álvarez, José A; Teixeira, Marcus M; Felipe, Maria S S; Flores-Carreón, Arturo; Mora-Montes, Héctor M

    2015-01-01

    Protein glycosylation pathways are conserved metabolic processes in eukaryotic organisms and are required for cell fitness. In fungal pathogens, the N-linked glycosylation pathway is indispensable for proper cell wall composition and virulence. In Sporothrix schenckii sensu stricto, the causative agent of sporotrichosis, little is known about this glycosylation pathway. Here, using a genome-wide screening for putative members of the glycosyl hydrolase (CAZy - GH) families 47 and 63, which group enzymes involved in the processing step during N-linked glycan maturation, we found seven homologue genes belonging to family 47 and one to family 63. The eight genes were individually expressed in C. albicans null mutants lacking either MNS1 (for members of family 47) or CWH41 (for the member of family 63). Our results indicate that SsCWH41 is the functional ortholog of CaCWH41, whereas SsMNS1 is the functional ortholog of CaMNS1. The remaining genes of family 47 encode Golgi mannosidases and endoplasmic reticulum degradation-enhancing alpha-mannosidase-like proteins (EDEMs). Since these GH families gather proteins used as target for drugs to control cell growth, identification of these genes could help in the design of antifungals that could be used to treat sporotrichosis and other fungal diseases. In addition, to our knowledge, we are the first to report that Golgi mannosidases and EDEMs are expressed and characterized in yeast cells. PMID:25526779

  1. 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.

  2. N-linked glycan characterization of heterologous proteins.

    PubMed

    Li, Huijuan; Miele, Robert G; Mitchell, Teresa I; Gerngross, Tillman U

    2007-01-01

    Our laboratory has focused on the re-engineered of the secretory pathway of Pichia pastoris to perform glycosylation reactions that mimic processing of N-glycans in humans and other higher mammals (1,2). A reporter protein with a single N-linked glycosylation site, a His-tagged Kringle 3 domain of human plasminogen (K3), was used to identify combinations of optimal leader/catalytic domain(s) to recreate human N-glycan processing in the Pichia system. In this chapter we describe detailed protocols for high-throughput purification of K3, enzymatic release of N-glycans, matrix-assisted laser desorption ionization time-of-flight and high-performance liquid chromatography analysis of the released N-glycans. The developed protocols can be adapted to the characterization of N-glycans from any purified protein expressed in P. pastoris.

  3. Pathogenesis of shigella diarrhea: evidence for an N-linked glycoprotein shigella toxin receptor and receptor modulation by beta-galactosidase.

    PubMed

    Keusch, G T; Jacewicz, M; Donohue-Rolfe, A

    1986-02-01

    Pathogenic mechanisms in infectious diseases often involve specific receptor-ligand interactions of cells and soluble molecules. To further elucidate structure-function relations for shigella toxin receptors, we studied binding of purified 125I-labeled toxin and biologic response under various conditions in an experimental model using HeLa cells. Response to toxin was reversibly inhibited by treatment of cells with trypsin or tunicamycin, an inhibitor of glycoprotein synthesis that also significantly inhibited toxin binding, a result indicating that the receptor is an N-linked glycoprotein. Removal of terminal beta-linked galactose from the HeLa cell surface with beta-galactosidase increased toxin binding and activity, and it also potentiated the effects of lysozyme and wheat-germ agglutinin, which recognize oligomeric beta 1----4-linked N-acetyl-D-glucosamine and inhibit toxin activity as well. Incubation of cells with beta-N-acetylglucosaminidase, which cleaves terminal beta-linked N-acetyl-D-glucosamine, inhibited toxin activity. Effects of beta-galactosidase were reversed by readdition of galactose to cell-surface oligosaccharide acceptors. The data demonstrate that alterations of a single sugar on cell-surface glycoproteins may have a dramatic effect on receptor activity and indicate that shigella toxin is a sugar-binding protein with specificity for beta 1----4-linked N-acetyl-D-glucosamine.

  4. Programming Surface Chemistry with Engineered Cells.

    PubMed

    Zhang, Ruihua; Heyde, Keith C; Scott, Felicia Y; Paek, Sung-Ho; Ruder, Warren C

    2016-09-16

    We have developed synthetic gene networks that enable engineered cells to selectively program surface chemistry. E. coli were engineered to upregulate biotin synthase, and therefore biotin synthesis, upon biochemical induction. Additionally, two different functionalized surfaces were developed that utilized binding between biotin and streptavidin to regulate enzyme assembly on programmable surfaces. When combined, the interactions between engineered cells and surfaces demonstrated that synthetic biology can be used to engineer cells that selectively control and modify molecular assembly by exploiting surface chemistry. Our system is highly modular and has the potential to influence fields ranging from tissue engineering to drug development and delivery. PMID:27203116

  5. Programming Surface Chemistry with Engineered Cells.

    PubMed

    Zhang, Ruihua; Heyde, Keith C; Scott, Felicia Y; Paek, Sung-Ho; Ruder, Warren C

    2016-09-16

    We have developed synthetic gene networks that enable engineered cells to selectively program surface chemistry. E. coli were engineered to upregulate biotin synthase, and therefore biotin synthesis, upon biochemical induction. Additionally, two different functionalized surfaces were developed that utilized binding between biotin and streptavidin to regulate enzyme assembly on programmable surfaces. When combined, the interactions between engineered cells and surfaces demonstrated that synthetic biology can be used to engineer cells that selectively control and modify molecular assembly by exploiting surface chemistry. Our system is highly modular and has the potential to influence fields ranging from tissue engineering to drug development and delivery.

  6. 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

  7. Diffusing colloidal probes of cell surfaces.

    PubMed

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

    2016-05-25

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

  8. Diffusing colloidal probes of cell surfaces.

    PubMed

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

    2016-05-25

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

  9. Controlled surface chemistries and quantitative cell response

    NASA Astrophysics Data System (ADS)

    Plant, Anne L.

    2002-03-01

    Living cells experience a large number of signaling cues from their extracellular matrix. As a result of these inputs, a variety of intracellular signaling pathways are apparently initiated simultaneously. The vast array of alternative responses that result from the integration of these inputs suggests that it may be reasonable to look for cellular response not as an 'on' or 'off' condition but as a distribution of responses. A difficult challenge is to determine whether variations in responses from individual cells arise from the complexity of intracellular signals or are due to variations in the cell culture environment. By controlling surface chemistry so that every cell 'sees' the same chemical and physical environment, we can begin to assess how the distribution of cell response is affected strictly by changes in the chemistry of the cell culture surface. Using the gene for green fluorescent protein linked to the gene for the promoter of the extracellular matrix protein, tenascin, we can easily probe the end product in a signaling pathway that is purported to be linked to surface protein chemistry and to cell shape. Cell response to well-controlled, well-characterized, and highly reproducible surfaces prepared using soft lithography techniques are compared with more conventional ways of preparing extracellular matrix proteins for cell culture. Using fluorescence microscopy and image analysis of populations of cells on these surfaces, we probe quantitatively the relationship between surface chemistry, cell shape and variations in gene expression endpoint.

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

    PubMed

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

    2014-02-15

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

  11. Adhesion of cells to polystyrene surfaces

    PubMed Central

    1983-01-01

    The surface treatment of polystyrene, which is required to make polystyrene suitable for cell adhesion and spreading, was investigated. Examination of surfaces treated with sulfuric acid or various oxidizing agents using (a) x-ray photoelectron and attenuated total reflection spectroscopy and (b) measurement of surface carboxyl-, hydroxyl-, and sulfur-containing groups by various radiochemical methods showed that sulfuric acid produces an insignificant number of sulfonic acid groups on polystyrene. This technique together with various oxidation techniques that render surfaces suitable for cell culture generated high surface densities of hydroxyl groups. The importance of surface hydroxyl groups for the adhesion of baby hamster kidney cells or leukocytes was demonstrated by the inhibition of adhesion when these groups were blocked: blocking of carboxyl groups did not inhibit adhesion and may raise the adhesion of a surface. These results applied to cell adhesion in the presence and absence of serum. The relative unimportance of fibronectin for the adhesion and spreading of baby hamster kidney cells to hydroxyl-rich surfaces was concluded when cells spread on such surfaces after protein synthesis was inhibited with cycloheximide, fibronectin was removed by trypsinization, and trypsin activity was stopped with leupeptin. PMID:6355120

  12. Cell-surface remodelling during mammalian erythropoiesis.

    PubMed

    Wraith, D C; Chesterton, C J

    1982-10-15

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

  13. 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

  14. Profiling of the cell surface proteome.

    PubMed

    Jang, Jun Ho; Hanash, Samir

    2003-10-01

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

  15. 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.

  16. Low-Reflectance Surfaces For Solar Cells

    NASA Technical Reports Server (NTRS)

    Bailey, Sheila G.; Landis, Geoffrey A.; Fatemi, Navid; Jenkins, Phillip P.

    1994-01-01

    Improved method for increasing solar cell efficiency has potential application for space-based and terrestrial solar power systems and optoelectronic devices. Etched low-angle grooves help recover reflected light. Light reflected from v-grooved surface trapped in cover glass and adhesive by total internal reflection. Reflected light redirected onto surface, and greater fraction of incident light absorbed, producing more electrical energy in InP solar photovoltaic cell.

  17. Enzyme Evolution by Yeast Cell Surface Engineering.

    PubMed

    Miura, Natsuko; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2015-01-01

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

  18. 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

    2016-06-01

    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/ . PMID:27111718

  19. Reactivation of cell surface transport in Reticulomyxa.

    PubMed

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

    1997-01-01

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

  20. Structure and functions of fungal cell surfaces

    NASA Technical Reports Server (NTRS)

    Nozawa, Y.

    1984-01-01

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

  1. Identification of genes involved in the biosynthesis and attachment of Methanococcus voltae N-linked glycans: insight into N-linked glycosylation pathways in Archaea.

    PubMed

    Chaban, Bonnie; Voisin, Sebastien; Kelly, John; Logan, Susan M; Jarrell, Ken F

    2006-07-01

    N-linked glycosylation is recognized as an important post-translational modification across all three domains of life. However, the understanding of the genetic pathways for the assembly and attachment of N-linked glycans in eukaryotic and bacterial systems far outweighs the knowledge of comparable processes in Archaea. The recent characterization of a novel trisaccharide [beta-ManpNAcA6Thr-(1-4)-beta-GlcpNAc3NAcA-(1-3)-beta-GlcpNAc]N-linked to asparagine residues in Methanococcus voltae flagellin and S-layer proteins affords new opportunities to investigate N-linked glycosylation pathways in Archaea. In this contribution, the insertional inactivation of several candidate genes within the M. voltae genome and their resulting effects on flagellin and S-layer glycosylation are reported. Two of the candidate genes were shown to have effects on flagellin and S-layer protein molecular mass and N-linked glycan structure. Further examination revealed inactivation of either of these two genes also had effects on flagella assembly. These genes, designated agl (archaeal glycosylation) genes, include a glycosyl transferase (aglA) involved in the attachment of the terminal sugar to the glycan and an STT3 oligosaccharyl transferase homologue (aglB) involved in the transfer of the complete glycan to the flagellin and S-layer proteins. These findings document the first experimental evidence for genes involved in any glycosylation process within the domain Archaea. PMID:16824110

  2. 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.

  3. Cell Adhesion on Surface-Functionalized Magnesium.

    PubMed

    Wagener, Victoria; Schilling, Achim; Mainka, Astrid; Hennig, Diana; Gerum, Richard; Kelch, Marie-Luise; Keim, Simon; Fabry, Ben; Virtanen, Sannakaisa

    2016-05-18

    The biocompatibility of commercially pure magnesium-based (cp Mg) biodegradable implants is compromised of strong hydrogen evolution and surface alkalization due to high initial corrosion rates of cp Mg in the physiological environment. To mitigate this problem, the addition of corrosion-retarding alloying elements or coating of implant surfaces has been suggested. In the following work, we explored the effect of organic coatings on long-term cell growth. cp Mg was coated with aminopropyltriehtoxysilane + vitamin C (AV), carbonyldiimidazole (CDI), or stearic acid (SA). All three coatings have been previously suggested to reduce initial corrosion and to enhance protein adsorption and hence cell adhesion on magnesium surfaces. Endothelial cells (DH1+/+) and osteosarcoma cells (MG63) were cultured on coated samples for up to 20 days. To quantify Mg corrosion, electrochemical impedance spectroscopy (EIS) was measured after 1, 3, and 5 days of cell culture. We also investigated the speed of initial cell spreading after seeding using fluorescently labeled fibroblasts (NIH/3T3). Hydrogen evolution after contact with cell culture medium was markedly decreased on AV- and SA-coated Mg compared to uncoated Mg. These coatings also showed improved cell adhesion and spreading after 24 h of culture comparable to tissue-treated plastic surfaces. On AV-coated cp Mg, a confluent layer of endothelial cells formed after 5 days and remained intact for up to 20 days. Together, these data demonstrate that surface coating with AV is a viable strategy for improving long-term biocompatibility of cp Mg-based implants. EIS measurements confirmed that the presence of a confluent cell layer increased the corrosion resistance. PMID:27089250

  4. HIV N-linked glycosylation site analyzer and its further usage in anchored alignment.

    PubMed

    Shaw, Timothy I; Zhang, Ming

    2013-07-01

    N-linked glycosylation is a posttranslational modification that has significantly contributed to the rapid evolution of HIV-1. In particular, enrichment of N-linked glycosylation sites can be found within Envelope variable loops, regions that play an essential role in HIV pathogenesis and immunogenicity. The web server described here, the HIV N-linked Glycosylation Site Analyzer, was developed to facilitate study of HIV diversity by tracking gp120 N-linked glycosylation sites. This server provides an automated platform for mapping and comparing variable loop N-linked glycosylation sites across populations of HIV-1 sequences. Furthermore, this server allows for refinement of HIV-1 sequence alignment by using N-linked glycosylation sites in variable loops as alignment anchors. Availability of this web server solves one of the difficult problems in HIV gp120 alignment and analysis imposed by the extraordinary HIV-1 diversity. The HIV N-linked Glycosylation Site Analyzer web server is available at http://hivtools.publichealth.uga.edu/N-Glyco/.

  5. Progenitor cells for ocular surface regenerative therapy.

    PubMed

    Casaroli-Marano, Ricardo P; Nieto-Nicolau, Nuria; Martínez-Conesa, Eva M

    2013-01-01

    The integrity and normal function of the corneal epithelium are essential for maintaining the cornea's transparency and vision. The existence of a cell population with progenitor characteristics in the limbus maintains a dynamic of constant epithelial repair and renewal. Currently, cell-based therapies for bio-replacement, such as cultured limbal epithelial transplantation and cultured oral mucosal epithelial transplantation, present very encouraging clinical results for treating limbal stem cell deficiencies. Another emerging therapeutic strategy consists of obtaining and implementing human progenitor cells of different origins using tissue engineering methods. The development of cell-based therapies using stem cells, such as human adult mesenchymal stromal cells, represents a significant breakthrough in the treatment of certain eye diseases and also offers a more rational, less invasive and more physiological approach to ocular surface regeneration. PMID:23257987

  6. Progenitor cells for ocular surface regenerative therapy.

    PubMed

    Casaroli-Marano, Ricardo P; Nieto-Nicolau, Nuria; Martínez-Conesa, Eva M

    2013-01-01

    The integrity and normal function of the corneal epithelium are essential for maintaining the cornea's transparency and vision. The existence of a cell population with progenitor characteristics in the limbus maintains a dynamic of constant epithelial repair and renewal. Currently, cell-based therapies for bio-replacement, such as cultured limbal epithelial transplantation and cultured oral mucosal epithelial transplantation, present very encouraging clinical results for treating limbal stem cell deficiencies. Another emerging therapeutic strategy consists of obtaining and implementing human progenitor cells of different origins using tissue engineering methods. The development of cell-based therapies using stem cells, such as human adult mesenchymal stromal cells, represents a significant breakthrough in the treatment of certain eye diseases and also offers a more rational, less invasive and more physiological approach to ocular surface regeneration.

  7. 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®.

  8. Roles for glycosylation of cell surface receptors involved in cellular immune recognition.

    PubMed

    Rudd, P M; Wormald, M R; Stanfield, R L; Huang, M; Mattsson, N; Speir, J A; DiGennaro, J A; Fetrow, J S; Dwek, R A; Wilson, I A

    1999-10-22

    The majority of cell surface receptors involved in antigen recognition by T cells and in the orchestration of the subsequent cell signalling events are glycoproteins. The length of a typical N-linked sugar is comparable with that of an immunoglobulin domain (30 A). Thus, by virtue of their size alone, oligosaccharides may be expected to play a significant role in the functions and properties of the cell surface proteins to which they are attached. A databank of oligosaccharide structures has been constructed from NMR and crystallographic data to aid in the interpretation of crystal structures of glycoproteins. As unambiguous electron density can usually only be assigned to the glycan cores, the remainder of the sugar is then modelled into the crystal lattice by superimposing the appropriate oligosaccharide from the database. This approach provides insights into the roles that glycosylation might play in cell surface receptors, by providing models that delineate potential close packing interactions on the cell surface. It has been proposed that the specific recognition of antigen by T cells results in the formation of an immunological synapse between the T cell and the antigen-presenting cell. The cell adhesion glycoproteins, such as CD2 and CD48, help to form a cell junction, providing a molecular spacer between opposing cells. The oligosaccharides located on the membrane proximal domains of CD2 and CD48 provide a scaffold to orient the binding faces, which leads to increased affinity. In the next step, recruitment of the peptide major histocompatibility complex (pMHC) by the T-cell receptors (TCRs) requires mobility on the membrane surface. The TCR sugars are located such that they could prevent non-specific aggregation. Importantly, the sugars limit the possible geometry and spacing of TCR/MHC clusters which precede cell signalling. We postulate that, in the final stage, the sugars could play a general role in controlling the assembly and stabilisation of the

  9. The Cell Surface Proteome of Human Mesenchymal Stromal Cells

    PubMed Central

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

    2011-01-01

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

  10. Vesicle trafficking and cell surface membrane patchiness.

    PubMed

    Tang, Q; Edidin, M

    2001-07-01

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

  11. Surface texturing and patterning in solar cells

    SciTech Connect

    Green, M.A.

    1993-11-01

    Surface texture can perform a number of functions in modern solar cell design. The most obvious function is in control of reflection from surfaces on which sunlight is incident. However, texture can also be used to influence the fate of light that is refracted into the cell. Light steering by surface texture can ensure this refracted light is absorbed in regions of the cell which are most responsive. When used with rear reflectors, surface texture can help trap weakly absorbed light into the cell, increasing the effective path length or optical thickness of the cell by factors of 30--60. Two general types of texture are considered. One involves macroscopic features of controlled shape designed to control the direction of interacting light. The other is based on the use of irregular features of size comparable to wavelength of the light. These can be very effective in scattering light into a wide range of directions. Non-optical uses of texture are also briefly described. 62 refs., 22 figs.

  12. Cell Surface Glycan Changes in the Spontaneous Epithelial-Mesenchymal Transition of Equine Amniotic Multipotent Progenitor Cells.

    PubMed

    Lange-Consiglio, Anna; Accogli, Gianluca; Cremonesi, Fausto; Desantis, Salvatore

    2014-01-01

    Amniotic epithelial cells (AECs) spontaneously transform into amniotic mesenchymal cells (AMCs) in vitro during cell culture. Glycocalyx was analyzed to identify the glycan pattern in AECs, AMCs and epithelial-mesenchymal transdifferentiated cells (EMTCs). Pure cell cultures were derived using cloned AEC and AMC cell lines obtained by the dilution technique from amniotic membranes. Mesenchymal cells generated by differentiation of clonal epithelial cells were considered transdifferentiated. Immunocytoscreen, in vitro multipotent differentiation and molecular characterization of EMTCs were performed. In combination with saponification and sialidase digestion, a panel of 12 lectins was used to analyze the glycan pattern of AEC, AMC and EMTC glycocalyx. Cytokeratin cell markers were lost in EMTCs and typical mesenchymal markers, such as vimentin, appeared. These cells retained their differentiation potential. Lectin histochemistry revealed a cell-specific glycan profile. Galactose (Gal)β1,4GlcNAc, Neu5Acα2,6Gal/GalNAc and N-acetyl neuraminic (sialic) acid (NeuNAc)α2,3Galβ1,3(±NeuNAcα2,6)GalNAc were highly expressed on the surface of all the amniotic cell cultures. AECs expressed asialoglycans with terminal GalNAc and GlcNAc. More highly mannosylated N-linked glycans and NeuNAcα2,3Galβ1,3GalNAc in O-linked glycans were expressed by EMTCs, but these cells had fewer glycans ending with fucose (Fuc), Gal, GlcNAc and GalNAc than AECs. GlcNAc- and GalNAc-terminating glycans were similarly expressed on the glycocalyx of the mesenchymal cell populations (EMTCs and AMCs). These results demonstrate for the first time that the spontaneous epithelial-mesenchymal transition (EMT) of equine amnion cells is characterized by cell surface glycan remodeling and that glycosylation changes result in a cell type-specific glycan profile. The glycopattern of equine amnion spontaneous EMTCs differs from EMT of tumoral cells.

  13. Cell Surface Glycan Changes in the Spontaneous Epithelial-Mesenchymal Transition of Equine Amniotic Multipotent Progenitor Cells.

    PubMed

    Lange-Consiglio, Anna; Accogli, Gianluca; Cremonesi, Fausto; Desantis, Salvatore

    2014-01-01

    Amniotic epithelial cells (AECs) spontaneously transform into amniotic mesenchymal cells (AMCs) in vitro during cell culture. Glycocalyx was analyzed to identify the glycan pattern in AECs, AMCs and epithelial-mesenchymal transdifferentiated cells (EMTCs). Pure cell cultures were derived using cloned AEC and AMC cell lines obtained by the dilution technique from amniotic membranes. Mesenchymal cells generated by differentiation of clonal epithelial cells were considered transdifferentiated. Immunocytoscreen, in vitro multipotent differentiation and molecular characterization of EMTCs were performed. In combination with saponification and sialidase digestion, a panel of 12 lectins was used to analyze the glycan pattern of AEC, AMC and EMTC glycocalyx. Cytokeratin cell markers were lost in EMTCs and typical mesenchymal markers, such as vimentin, appeared. These cells retained their differentiation potential. Lectin histochemistry revealed a cell-specific glycan profile. Galactose (Gal)β1,4GlcNAc, Neu5Acα2,6Gal/GalNAc and N-acetyl neuraminic (sialic) acid (NeuNAc)α2,3Galβ1,3(±NeuNAcα2,6)GalNAc were highly expressed on the surface of all the amniotic cell cultures. AECs expressed asialoglycans with terminal GalNAc and GlcNAc. More highly mannosylated N-linked glycans and NeuNAcα2,3Galβ1,3GalNAc in O-linked glycans were expressed by EMTCs, but these cells had fewer glycans ending with fucose (Fuc), Gal, GlcNAc and GalNAc than AECs. GlcNAc- and GalNAc-terminating glycans were similarly expressed on the glycocalyx of the mesenchymal cell populations (EMTCs and AMCs). These results demonstrate for the first time that the spontaneous epithelial-mesenchymal transition (EMT) of equine amnion cells is characterized by cell surface glycan remodeling and that glycosylation changes result in a cell type-specific glycan profile. The glycopattern of equine amnion spontaneous EMTCs differs from EMT of tumoral cells. PMID:26337136

  14. 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

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

    PubMed

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

    2015-01-01

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

  16. Schizosaccharomyces pombe glycosylation mutant with altered cell surface properties.

    PubMed Central

    Ballou, C E; Ballou, L; Ball, G

    1994-01-01

    Mutagenesis of Schizosaccharomyces pombe cells yielded a strain that made reduced amounts of invertase. A comparison of the O- and N-linked carbohydrate chains of the wild-type and mutant glycoproteins revealed that a single type of alpha 1-->2-linked mannose was missing in the mutant. Analysis of the wild-type galactomannoprotein showed that it contained a heterogeneous small "core" oligosaccharide fraction linked to asparagine with sugar compositions that ranged from Man9(GlcNAc)2- to Gal4Man10(GlcNAc)2-. The galactose units are in terminal positions of a Man10(GlcNAc)2- unit that is similar to the mannoprotein core of Saccharomyces cerevisiae. Attached to this core in a larger oligosaccharide fraction is an alpha 1-->6-linked polymannose chain that is substituted at position 2 with alpha-linked mannose and galactose. The O-linked sugars consist of mannose, alpha 1-->2-linked mannosylmannose and alpha 1-->2-linked galactosylmannose, along with small amounts of tri- and tetrasaccharides. The glycosylation mutant lacks alpha 1-->2-linked mannose on both the O-linked chains and the outer chain of the large N-linked chains, suggesting that it may be defective in regulation of an alpha 1,2-mannosyltransferase that adds mannose to glycoproteins in the Golgi. PMID:7937765

  17. The effect of swainsonine and castanospermine on the sulfation of the oligosaccharide chains of N-linked glycoproteins.

    PubMed

    Merkle, R K; Elbein, A D; Heifetz, A

    1985-01-25

    MDCK (Madin-Darby canine kidney) cells infected with the NWS strain of influenza virus incorporate 35SO4 into complex types of oligosaccharides of the N-linked glycoproteins. On the other hand, when these virus-infected MDCK cells are incubated in the presence of swainsonine, an inhibitor of the processing mannosidase II, approximately 40-80% of the total [35S]glycopeptides were of the hybrid types of structures. Thus, these sulfated, hybrid types of glycopeptides were completely susceptible to digestion by endoglucosaminidase H, whereas the sulfated glycopeptides from infected cells incubated without swainsonine were completely resistant to endo-beta-N-acetylglucosaminidase H. When virus-infected MDCK cells were incubated in the presence of castanospermine, an inhibitor of the processing glucosidase I, the N-linked glycopeptides contained mostly oligosaccharide chains of the Glc3Man7-9GlcNAc2 types of structures, and these oligosaccharides were devoid of sulfate. Structural analysis of these abnormally processed oligosaccharides produced in the presence of swainsonine or castanospermine indicated that they differed principally in the processing of one oligosaccharide branch as indicated by the structures shown below. They also differed in that only the swainsonine-induced structures were sulfated. These data indicate that removal of glucose units and perhaps other processing steps are necessary before sulfate residues can be added. (Formula: see text). PMID:3918027

  18. Occurrence of heterogeneity of N-linked oligosaccharides attached to sycamore (Acer pseudoplatanus L.) laccase after excretion.

    PubMed

    Tezuka, K; Hayashi, M; Ishihara, H; Onozaki, K; Nishimura, M; Takahashi, N

    1993-03-01

    The N-linked oligosaccharide moieties of sycamore (Acer pseudoplatanus L.) laccase are known to be highly heterogeneous. We confirmed that this oligosaccharide heterogeneity was caused not only during the oligosaccharide biosynthesis in Golgi apparatus, but also after the excretion of laccase protein into a culture medium. The culture medium for the sycamore cells (Acer pseudoplatanus L.) contained beta-galactosidase, alpha-L-fucosidase, beta-N-acetylglucosaminidase, alpha-mannosidase and beta-xylosidase activities. We showed that the largest sugar chain in laccase, oligosaccharide F, [formula: see text] was degraded to [formula: see text] by a crude exoglycosidase mixture in the culture medium.

  19. Femtosecond fabricated surfaces for cell biology

    NASA Astrophysics Data System (ADS)

    Day, Daniel; Gu, Min

    2010-08-01

    Microfabrication using femtosecond pulse lasers is enabling access to a range of structures, surfaces and materials that was not previously available for scientific and engineering applications. The ability to produce micrometre sized features directly in polymer and metal substrates is demonstrated with applications in cell biology. The size, shape and aspect ratio of the etched features can be precisely controlled through the manipulation of the fluence of the laser etching process with respect to the properties of the target material. Femtosecond laser etching of poly(methyl methacrylate) and aluminium substrates has enabled the production of micrometre resolution moulds that can be accurately replicated using soft lithography. The moulded surfaces are used in the imaging of T cells and demonstrate the improved ability to observe biological events over time periods greater than 10 h. These results indicate the great potential femtosecond pulse lasers may have in the future manufacturing of microstructured surfaces and devices.

  20. 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.

  1. Solar cell having improved front surface metallization

    SciTech Connect

    Lillington, D.R.; Mardesich, N.; Dill, H.G.; Garlick, G.F.J.

    1987-09-15

    This patent describes a solar cell comprising: a first layer of gallium arsenide semiconductor material of an N+ conductivity; a second layer of gallium arsenide semiconductor material of an N conductivity overlying the first layer; a third layer of gallium arsenide semiconductor material of a P conductivity overlying the N conductivity layer and forming a P-N junction therebetween. A layer of aluminium gallium arsenide semiconductor material of a p conductivity overlying the front major surface of the P conductivity third layer and having an exposed surface essentially parallel to the front major surface and at least one edge; a plurality of metallic contact lines made of a first metal alloy composition and being spaced apart by a first predetermined distance traversing the exposed surface and extending through the aluminium gallium arsenide layer to the front major surface and making electrical contact to the third layer; a plurality of longitudinally disposed metallic grid lines made of a second metal alloy composition and being spaced apart by a second predetermined distance located on the exposed surface of the aluminium gallium arsenide layer and which cross the metallic contact lines and make electrical contact to the metallic lines; a flat metallic strip disposed on the aluminium gallium arsenide layer exposed surface near the edge, the strip electrically coupling the metallic grid lines to one another; and a back contact located on the back major surface.

  2. 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

  3. 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

  4. 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.

  5. Mass Spectrometric Quantification of N-Linked Glycans by Reference to Exogenous Standards.

    PubMed

    Mehta, Nickita; Porterfield, Mindy; Struwe, Weston B; Heiss, Christian; Azadi, Parastoo; Rudd, Pauline M; Tiemeyer, Michael; Aoki, Kazuhiro

    2016-09-01

    Environmental and metabolic processes shape the profile of glycoprotein glycans expressed by cells, whether in culture, developing tissues, or mature organisms. Quantitative characterization of glycomic changes associated with these conditions has been achieved historically by reductive coupling of oligosaccharides to various fluorophores following release from glycoprotein and subsequent HPLC or capillary electrophoretic separation. Such labeling-based approaches provide a robust means of quantifying glycan amount based on fluorescence yield. Mass spectrometry, on the other hand, has generally been limited to relative quantification in which the contribution of the signal intensity for an individual glycan is expressed as a percent of the signal intensity summed over the total profile. Relative quantification has been valuable for highlighting changes in glycan expression between samples; sensitivity is high, and structural information can be derived by fragmentation. We have investigated whether MS-based glycomics is amenable to absolute quantification by referencing signal intensities to well-characterized oligosaccharide standards. We report the qualification of a set of N-linked oligosaccharide standards by NMR, HPLC, and MS. We also demonstrate the dynamic range, sensitivity, and recovery from complex biological matrices for these standards in their permethylated form. Our results indicate that absolute quantification for MS-based glycomic analysis is reproducible and robust utilizing currently available glycan standards. PMID:27432553

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

    PubMed Central

    1986-01-01

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

  8. The role of N-linked glycosylation in the protection of human and bovine lactoferrin against tryptic proteolysis.

    PubMed

    van Veen, Harrie A; Geerts, Marlieke E J; van Berkel, Patrick H C; Nuijens, Jan H

    2004-02-01

    Lactoferrin (LF) is an iron-binding glycoprotein of the innate host defence system. To elucidate the role of N-linked glycosylation in protection of LF against proteolysis, we compared the tryptic susceptibility of human LF (hLF) variants from human milk, expressed in human 293(S) cells or in the milk of transgenic mice and cows. The analysis revealed that recombinant hLF (rhLF) with mutations Ile130-->Thr and Gly404-->Cys was about twofold more susceptible than glycosylated and unglycosylated variants with the naturally occurring Ile130 and Gly404. Hence, N-linked glycosylation is not involved in protection of hLF against tryptic proteolysis. Apparently, the previously reported protection by N-linked glycosylation of hLF [van Berkel, P.H.C., Geerts, M.E.J., van Veen, H.A., Kooiman, P.M., Pieper, F., de Boer, H.A. & Nuijens, J.H. (1995) Biochem. J. 312, 107-114] is restricted to rhLF containing the Thr130 and Cys404. Comparison of the tryptic proteolysis of hLF and bovine LF (bLF) revealed that hLF is about 100-fold more resistant than bLF. Glycosylation variants A and B of bLF differed by about 10-fold in susceptibility to trypsin. This difference is due to glycosylation at Asn281 in bLF-A. Hence, glycosylation at Asn281 protects bLF against cleavage by trypsin at Lys282. PMID:14764083

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

    PubMed Central

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

    2015-01-01

    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. PMID:26100877

  10. 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

    2015-07-01

    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. PMID:26100877

  11. 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

  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. Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes.

    PubMed

    Mallanna, Sunil K; Cayo, Max A; Twaroski, Kirk; Gundry, Rebekah L; Duncan, Stephen A

    2016-09-13

    When comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS). Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly enriched during the final stages of hepatocyte differentiation. FACS purification of hepatocyte-like cells expressing SLC10A1, CLRN3, or AADAC demonstrated enrichment of cells with hepatocyte characteristics. Moreover, transcriptome analyses revealed that cells expressing the liver gene regulatory network were enriched while cells expressing a pluripotent stem cell network were depleted. In conclusion, we report an extensive catalog of cell-surface N-linked glycoproteins expressed in primary hepatocytes and identify cell-surface proteins that facilitate the purification of homogeneous populations of iPSC-derived hepatocyte-like cells.

  14. Nanoparticle energy transfer on the cell surface.

    PubMed

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

    2005-01-01

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

  15. Metabolic Labeling and Imaging of N-Linked Glycans in Arabidopsis Thaliana.

    PubMed

    Zhu, Yuntao; Wu, Jie; Chen, Xing

    2016-08-01

    Molecular imaging of glycans has been actively pursued in animal systems for the past decades. However, visualization of plant glycans remains underdeveloped, despite that glycosylation is essential for the life cycle of plants. Metabolic glycan labeling in Arabidopsis thaliana by using N-azidoacetylglucosamine (GlcNAz) as the chemical reporter is reported. GlcNAz is metabolized through the salvage pathway of N-acetylglucosamine (GlcNAc) and incorporated into N-linked glycans, and possibly intracellular O-GlcNAc. Click-labeling with fluorescent probes enables visualization of newly synthesized N-linked glycans. N-glycosylation in the root tissue was discovered to possess distinct distribution patterns in different developmental zones, suggesting that N-glycosylation is regulated in a developmental stage-dependent manner. This work shows the utility of metabolic glycan labeling in elucidating the function of N-linked glycosylation in plants.

  16. Involvement of N-linked carbohydrate chains of pig zona pellucida in sperm-egg binding.

    PubMed

    Yonezawa, N; Aoki, H; Hatanaka, Y; Nakano, M

    1995-10-01

    The sperm receptor activity of pig zona pellucida has been previously shown to exist in one of the components, pig zona protein 3 alpha (PZP3 alpha), that can be purified after the removal of sialylated and/or sulfated N-acetylpoly(lactosamine) by digestion with endo-beta-galactosidase. In this study, we examined whether N-linked or O-linked carbohydrate chains are involved in the sperm receptor activity of pig zona pellucida. The elimination of N-linked carbohydrate chains from endo-beta-galactosidase-digested PZP3 alpha by digestion with N-glycanase markedly reduced its inhibitory effect on sperm-egg binding in an in vitro competition assay, whereas the elimination of O-linked carbohydrate chains by alkali treatment hardly reduced the inhibitory effect. These results indicate that N-linked carbohydrate chains of PZP3 alpha play a major role in mediating the sperm binding of zona pellucida in pig.

  17. CZTSSe thin film solar cells: Surface treatments

    NASA Astrophysics Data System (ADS)

    Joglekar, Chinmay Sunil

    Chalcopyrite semiconducting materials, specifically CZTS, are a promising alternative to traditional silicon solar cell technology. Because of the high absorption coefficient; films of the order of 1 micrometer thickness are sufficient for the fabrication of solar cells. Liquid based synthesis methods are advantageous because they are easily scalable using the roll to roll manufacturing techniques. Various treatments are explored in this study to enhance the performance of the selenized CZTS film based solar cells. Thiourea can be used as a sulfur source and can be used to tune band gap of CZTSSe. Bromine etching can be used to manipulate the thickness of sintered CZTSSe film. The etching treatment creates recombination centers which lead to poor device performance. Various after treatments were used to improve the performance of the devices. It was observed that the performance of the solar cell devices could not be improved by any of the after treatment steps. Other surface treatment processes are explored including KCN etching and gaseous H2S treatments. Hybrid solar cells which included use of CIGS nanoparticles at the interface between CZTSSe and CdS are also explored.

  18. 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.

  19. 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

  20. Supplemental Analysis for N-linked Sugars in Adult Pig Islets.

    PubMed

    Eguchi, H; Kawamura, T; Kashiyama, N; Matsuura, R; Sakai, R; Nakahata, K; Lo, P-C; Asada, M; Maeda, A; Goto, M; Toyoda, M; Okuyama, H; Miyagawa, S

    2016-05-01

    The pig pancreas is considered to be one of the most suitable sources of islets for clinical xenotransplantation. However, after producing α1-3galactosyltransferase knockout pigs, most of the organs of these pigs showed less antigenicity to the human body. Wild-type adult pig islets (APIs) that originally produced negligible levels of α-Gal, different from neonatal porcine islet-like cell clusters, showed a clear antigenicity to human serum. Concerning the so-called non-Gal epitopes, many studies related to glycoproteins and glycolipids are ongoing in efforts to identify them. However, our knowledge of non-Gal glycoantigens remains incomplete. In our previous study, N-glycans were isolated from APIs, and the structures of 28 of the N-glycans were detected. In this study, to identify additional structures, further analyses were performed by liquid chromatography-mass spectrometry (LC-MS). N-glycans were isolated from APIs by the method described by O'Neil et al with minor modifications and LC-MS-based structural analyses were then performed. The detected N-glycan peaks in the LC-MS spectra were selected using the FLexAnalysis software program and the structures of the glycans were predicted using the GlyocoMod Tool. The API preparation contained 11 peaks and 16 structures were then nominated as containing N-linked sugars. Among them, 5 sulfated glycans were estimated, confirming the existence of sulfate structures in N-glycans in API. In addition, these data may supplement several N-glycan structures that contain two deoxyhexose units, such as fucose, to our previous report. The data herein will be helpful for future studies of antigenicity associated with API. PMID:27320609

  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. Neurite outgrowth of neuroblastoma cells: dependence on adhesion surface--cell surface interactions

    PubMed Central

    1984-01-01

    Neurite outgrowth of C 1300 neuroblastoma cells, which were dispersed from adherent cultures or grown in suspension, was studied on different protein-coated surfaces. Of 29 different surface structures studied, including surfaces treated with various fibronectins, lectins, glycosidases, or glycosyltransferases capable of stimulating fibroblast spreading, only the surfaces coated with plasma fibronectin or with a protein mixture secreted by C6 glioma cells displayed an extensive activity in the sprouting assay. Neurite outgrowth was inhibited by brain gangliosides and by colominic acid (a sialic acid polymer). A 50% inhibition of neurite outgrowth of N18 neuroblasts induced by the glioma cell proteins was observed at the following approximate concentration: 100 microM (0.2 mg/ml) GD1A ganglioside, 20 microM (0.04 mg/ml) GT1B ganglioside, and 5 mg/ml colominic acid. Specificity of inhibition was suggested by the finding that a few polyanionic substances tested were not inhibitory in the sprouting assay, and that the type of gangliosides inhibiting sprouting were found to be major sialoglycolipids of the neuroblasts. A hypothesis is discussed, according to which neurite outgrowth of neuroblasts is stimulated by adhesion involving interactions of the adhesion-mediating protein with cell surface carbohydrates characteristic of brain gangliosides. PMID:6699078

  4. 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. PMID:26610891

  5. 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

  6. Mice lacking N-acetylglucosaminyltransferase I activity die at mid-gestation, revealing an essential role for complex or hybrid N-linked carbohydrates.

    PubMed Central

    Ioffe, E; Stanley, P

    1994-01-01

    Eukaryotic cells require N-linked carbohydrates for survival. However, the biosynthetic intermediate Man5GlcNAc2Asn, in place of mature N-linked structures, allows glycoprotein synthesis and somatic cell growth to proceed normally. To determine whether the same would be true in a complex biological situation, the gene Mgat-1 was disrupted by homologous recombination in embryonic stem cells and transmitted to the germ line. The Mgat-1 gene encodes N-acetylglucosaminyltransferase I [GlcNAc-TI; alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase; UDP-N-acetyl-D-glucosamine:glycoprotein (N-acetyl-D-glucosamine to alpha-D-mannosyl-1,3-(R1)-beta-D-mannosyl-R2) beta-1,2-N-acetyl-D-glucosaminyltransferase, EC 2.4.1.101], the transferase that initiates synthesis of hybrid and complex N-linked carbohydrates from Man5GlcNAc2Asn. Mice lacking GlcNAc-TI activity did not survive to term. Biochemical and morphological analyses of embryos from 8.5 to 13.5 days of gestation showed that Mgat-1-/-embryos are developmentally retarded, most noticeably in neural tissue, and die between 9.5 and 10.5 days of development. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8290590

  7. Calculation of cell volumes and surface areas in MCNP

    SciTech Connect

    Hendricks, J.S.

    1980-01-01

    MCNP is a general Monte Carlo neutron-photon particle transport code which treats an arbitrary three-dimensional configuration of materials in geometric cells bounded by first- and second-degree surfaces, and some special fourth-degree surfaces. It is necessary to calculate cell volumes and surface areas so that cell masses, fluxes, and other important information can be determined. The volume/area calculation in MCNP computes cell volumes and surface areas for cells and surfaces rotationally symmetric about any arbitrary axis. 5 figures, 1 table.

  8. 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

    2015-01-01

    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.

  9. Specific asparagine-linked oligosaccharides are not required for certain neuron-neuron and neuron-Schwann cell interactions

    PubMed Central

    1986-01-01

    To determine whether specific asparagine-linked (N-linked) oligosaccharides present in cell surface glycoproteins are required for cell-cell interactions within the peripheral nervous system, we have used castanospermine to inhibit maturation of N-linked sugars in cell cultures of neurons or neurons plus Schwann cells. Maximally 10-15% of the N-linked oligosaccharides on neuronal proteins have normal structure when cells are cultured in the presence of 250 micrograms/ml castanospermine; the remaining oligosaccharides are present as immature carbohydrate chains not normally found in these glycoproteins. Although cultures were treated for 2 wk with castanospermine, cells always remained viable and appeared healthy. We have analyzed several biological responses of embryonic dorsal root ganglion neurons, with or without added purified populations of Schwann cells, in the presence of castanospermine. We have observed that a normal complement of mature, N- linked sugars are not required for neurite outgrowth, neuron-Schwann cell adhesion, neuron-induced Schwann cell proliferation, or ensheathment of neurites by Schwann cells. Treatment of neuronal cultures with castanospermine increases the propensity of neurites to fasciculate. Extracellular matrix deposition by Schwann cells and myelination of neurons by Schwann cells are greatly diminished in the presence of castanospermine as assayed by electron microscopy and immunocytochemistry, suggesting that specific N-linked oligosaccharides are required for the expression of these cellular functions. PMID:3522602

  10. Cell-surface carbohydrates of Entamoeba invadens.

    PubMed

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

    1997-01-01

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

  11. 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

  12. Facile cell patterning on an albumin-coated surface.

    PubMed

    Yamazoe, Hironori; Uemura, Toshimasa; Tanabe, Toshizumi

    2008-08-19

    Fabrication of micropatterned surfaces to organize and control cell adhesion and proliferation is an indispensable technique for cell-based technologies. Although several successful strategies for creating cellular micropatterns on substrates have been demonstrated, a complex multistep process and requirements for special and expensive equipment or materials limit their prevalence as a general experimental tool. To circumvent these problems, we describe here a novel facile fabrication method for a micropatterned surface for cell patterning by utilizing the UV-induced conversion of the cell adhesive property of albumin, which is the most abundant protein in blood plasma. An albumin-coated surface was prepared by cross-linking albumin with ethylene glycol diglycidyl ether and subsequent casting of the cross-linked albumin solution on the cell culture dish. While cells did not attach to the albumin surface prepared in this way, UV exposure renders the surface cell-adhesive. Thus, surface micropatterning was achieved simply by exposing the albumin-coated surface to UV light through a mask with the desired pattern. Mouse fibroblast L929 cells were inoculated on the patterned albumin substrates, and cells attached and spread in a highly selective manner according to the UV-irradiated pattern. Although detailed investigation of the molecular-level mechanism concerning the change in cell adhesiveness of the albumin-coated surface is required, the present results would give a novel facile method for the fabrication of cell micropatterned surfaces. PMID:18627191

  13. Versatile metal-organic framework-functionalized magnetic graphene nanoporous composites: As deft matrix for high-effective extraction and purification of the N-linked glycans.

    PubMed

    Wang, Jiaxi; Wang, Yanan; Gao, Mingxia; Zhang, Xiangmin; Yang, Pengyuan

    2016-08-17

    The highly selective enrichment of N-linked glycans from complex biological sample is still very important but challenging task due to the ultra-low abundance, complicated structures and strong ion suppress effect caused by distractors such as proteins, peptides and salts. Here, we firstly present a novel metal-organic frameworks (MOFs)-functionalized magnetic nanoporous carbon-graphene composites (C-magG@ZIF-8) synthesized through a smart process. The obtained materials enjoy the unique properties including strong magnetic responsiveness, a large sum of graphitized carbon pore, remarkable biocompatibility and large specific surface area. By virtue of these unique properties, the C-magG@ZIF-8 composites displayed excellent selectivity and sensitivity, good recyclability and incredible size exclusion ability (roughly 2000 times) in the N-linked glycans analysis. Furthermore, 48 N-linked glycans were clearly identified from the normal human serum treated with the C-magG@ZIF-8. There is reason to believe that our smart strategy offers new possibilities for preparing the MOFs-functionalized composites for large-scale characterization of glycoproteomics by mass spectrometry analysis.

  14. 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. PMID:26909529

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

    PubMed

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

    2006-12-01

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

  16. Melittin interaction with sulfated cell surface sugars.

    PubMed

    Klocek, Gabriela; Seelig, Joachim

    2008-03-01

    Melittin is a 26-residue cationic peptide with cytolytic and antimicrobial properties. Studies on the action mechanism of melittin have focused almost exclusively on the membrane-perturbing properties of this peptide, investigating in detail the melittin-lipid interaction. Here, we report physical-chemical studies on an alternative mechanism by which melittin could interact with the cell membrane. As the outer surface of many cells is decorated with anionic (sulfated) glycosaminoglycans (GAGs), a strong Coulombic interaction between the two oppositely charged molecules can be envisaged. Indeed, the present study using isothermal titration calorimetry reveals a high affinity of melittin for several GAGs, that is, heparan sulfate (HS), dermatan sulfate, and heparin. The microscopic binding constant of melittin for HS is 2.4 x 10 (5) M (-1), the reaction enthalpy is Delta H melittin (0) = -1.50 kcal/mol, and the peptide-to-HS stoichiometry is approximately 11 at 10 mM Tris, 100 mM NaCl at pH 7.4 and 28 degrees C. Delta H melittin (0) is characterized by a molar heat capacity of Delta C P (0) = -227 cal mol (-1) K (-1). The large negative heat capacity change indicates that hydrophobic interactions must also be involved in the binding of melittin to HS. Circular dichroism spectroscopy demonstrates that the binding of the peptide to HS induces a conformational change to a predominantly alpha-helical structure. A model for the melittin-HS complex is presented. Melittin binding was compared with that of magainin 2 and nisin Z to HS. Magainin 2 is known for its antimicrobial properties, but it does not cause lysis of the eukaryotic cells. Nisin Z shows activity against various Gram-positive bacteria. Isothermal titration calorimetry demonstrates that magainin 2 and nisin Z do not bind to HS (5-50 degrees C, 10 mM Tris, and 100 mM NaCl at pH 7.4). PMID:18220363

  17. 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

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

    PubMed

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

    2015-01-01

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

  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 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

  1. 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. PMID:27570558

  2. 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.

  3. Surface topography of granulosa cells accompanied by fragmented oocytes.

    PubMed

    Shinohara, H; Noda, M; Kima, M; Matsuda, T

    1983-07-15

    Scanning electron microscopy of granulosa cells (GC) and granulosa cell-like structures (GCLS) revealed that both had lacy foldings, or plicae, on the surface and were identical. The plicae did not always cover the entire surface of GC or GCLS. Both structures were interconnected by multivalent processes.

  4. Cell Surface-based Sensing with Metallic Nanoparticles

    PubMed Central

    Jiang, Ziwen; Rotello, Vincent M.

    2015-01-01

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

  5. Functional Divergence in the Role of N-Linked Glycosylation in Smoothened Signaling

    PubMed Central

    Marada, Suresh; Navarro, Gemma; Truong, Ashley; Stewart, Daniel P.; Arensdorf, Angela M.; Nachtergaele, Sigrid; Angelats, Edgar; Opferman, Joseph T.; Rohatgi, Rajat; McCormick, Peter J.; Ogden, Stacey K.

    2015-01-01

    The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice. PMID:26291458

  6. 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.

  7. Investigation of back surface fields effect on bifacial solar cells

    NASA Astrophysics Data System (ADS)

    Sepeai, Suhaila; Sulaiman, M. Y.; Sopian, Kamaruzzaman; Zaidi, Saleem H.

    2012-11-01

    A bifacial solar cell, in contrast with a conventional monofacial solar cell, produces photo-generated current from both front and back sides. Bifacial solar cell is an attractive candidate for enhancing photovoltaic (PV) market competitiveness as well as supporting the current efforts to increase efficiency and lower material costs. This paper reports on the fabrication of bifacial solar cells using phosphorus-oxytrichloride (POCl3) emitter formation on p-type, nanotextured silicon (Si) wafer. Backside surface field was formed through Al-diffusion using conventional screen-printing process. Bifacial solar cells with a structure of n+pp+ with and without back surface field (BSF) were fabricated in which silicon nitride (SiN) anti reflection and passivation films were coated on both sides, followed by screen printing of Argentum (Ag) and Argentum/Aluminum (Ag/Al) on front and back contacts, respectively. Bifacial solar cells without BSF exhibited open circuit voltage (VOC) of 535 mV for front and 480 mV for back surface. With Al-alloyed BSF bifacial solar cells, the VOC improved to 580 mV for the front surface and 560 mV for the back surface. Simulation of bifacial solar cells using PC1D and AFORS software demonstrated good agreement with experimental results. Simulations showed that best bifacial solar cells are achieved through a combination of high lifetime wafer, low recombination back surface field, reduced contact resistance, and superior surface passivation.

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

    PubMed

    Akeson, R; Herschman, H R

    1974-01-01

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

  9. Stem cell behavior on tailored porous oxide surface coatings.

    PubMed

    Lavenus, Sandrine; Poxson, David J; Ogievetsky, Nika; Dordick, Jonathan S; Siegel, Richard W

    2015-07-01

    Nanoscale surface topographies are known to have a profound influence on cell behavior, including cell guidance, migration, morphology, proliferation, and differentiation. In this study, we have observed the behavior of human mesenchymal stem cells cultured on a range of tailored porous SiO2 and TiO2 nanostructured surface coatings fabricated via glancing angle electron-beam deposition. By controlling the physical vapor deposition angle during fabrication, we could control systematically the deposited coating porosity, along with associated topographic features. Immunocytochemistry and image analysis quantitatively revealed the number of adherent cells, as well as their basic cellular morphology, on these surfaces. Signaling pathway studies showed that even with subtle changes in nanoscale surface structures, the behavior of mesenchymal stem cells was strongly influenced by the precise surface structures of these porous coatings.

  10. 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.

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

    PubMed

    Leshchyns'ka, Iryna; Sytnyk, Vladimir

    2015-01-01

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

  12. The Use of Yeast Surface Display in Biofuel Cells.

    PubMed

    Szczupak, Alon; Alfonta, Lital

    2015-01-01

    Biofuel cells are electrochemical devices which convert chemical energy to electricity using biochemical pathways and redox enzymes. In enzymatic fuel cells purified redox enzymes catalyze the reactions in the anode and cathode compartments whereas in microbial fuel cells (MFCs) the entire metabolism of the microorganisms is exploited. Here, a hybrid biofuel cell concept is presented, which is based on yeast surface display (YSD) of redox enzymes to catalyze the different cell reactions. PMID:26060081

  13. 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

    2009-04-01

    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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

    Ibáñez, Carlos F

    2010-05-21

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

  17. 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

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

    PubMed

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

    2016-01-01

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

  19. Cell multiplication following partial enzymatic removal of surface coat.

    PubMed

    Wyroba, E

    1978-08-01

    Treatment of Paramecium aurelia with trypsin or pronase (1 mg per 10(5) cells, at 0 to 4 degrees C) partially removes the surface coat and modifies significantly multiplication of cells. The division rate after 24 hours of cultivation is diminished approximately twice in the case of pronase-treated cells and 1.5 for tyrpsin-digested ciliates as compared with the control. On the second day the division rate increases rapidly and number of cell divisions exceeds the values observed in the control. After 72 hours of cultivation the division rate in both untreated and enzyme-treated cells is almost the same. It is concluded that the observed inhibition of cell fission results from the enzymatic removal of the surface coat--the integrity of this surface coat seems to be necessary in the process of cell division. The influence of environmental factors on the rate of growth is presented.

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

    NASA Astrophysics Data System (ADS)

    Nappi, Anthony J.; Silvers, Michael

    1984-09-01

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

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

    PubMed

    Nappi, A J; Silvers, M

    1984-09-14

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

  2. 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.

  3. Delineating diseases by IMS-MS profiling of serum N-linked glycans.

    PubMed

    Isailovic, Dragan; Plasencia, Manolo D; Gaye, Maissa M; Stokes, Sarah T; Kurulugama, Ruwan T; Pungpapong, Vitara; Zhang, Min; Kyselova, Zuzana; Goldman, Radoslav; Mechref, Yehia; Novotny, Milos V; Clemmer, David E

    2012-02-01

    Altered branching and aberrant expression of N-linked glycans is known to be associated with disease states such as cancer. However, the complexity of determining such variations hinders the development of specific glycomic approaches for assessing disease states. Here, we examine a combination of ion mobility spectrometry (IMS) and mass spectrometry (MS) measurements, with principal component analysis (PCA) for characterizing serum N-linked glycans from 81 individuals: 28 with cirrhosis of the liver, 25 with liver cancer, and 28 apparently healthy. Supervised PCA of combined ion-mobility profiles for several, to as many as 10 different mass-to-charge ratios for glycan ions, improves the delineation of diseased states. This extends an earlier study [J. Proteome Res.2008, 7, 1109-1117] of isomers associated with a single glycan (S(1)H(5)N(4)) in which PCA analysis of the IMS profiles appeared to differentiate the liver cancer group from the other samples. Although performed on a limited number of test subjects, the combination of IMS-MS for different combinations of ions and multivariate PCA analysis shows promise for characterizing disease states.

  4. 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

  5. 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.

  6. 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. PMID:27459246

  7. 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.

  8. 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.

  9. 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.

  10. Surface Markers for the Murine Oval Cell Response

    PubMed Central

    Dorrell, Craig; Erker, Laura; Lanxon-Cookson, Kelsea M.; Abraham, Stephanie L.; Victoroff, Tristan; Ro, Simon; Canaday, Pamela S.; Streeter, Philip R.; Grompe, Markus

    2011-01-01

    The biology of progenitor activation in the liver is of considerable medical and scientific interest. The powerful genetic tools available for the mouse make it an ideal model system to study this complex process involving many different cell types. However, reagents for the isolation and study of distinct hepatic subpopulations have been quite limited compared to those available for hematopoietic cells. To produce cell surface reactive reagents more specific for the oval cell response, we generated a new collection of monoclonal antibodies by immunization of Fischer rats with enzymatically dispersed nonparenchymal cells from the livers of adult mice treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Each of the resulting antibodies recognized a surface antigen present on a liver cell subset and permitted the viable isolation of the associated subpopulation by fluorescence-activated cell sorting. Differential activity was observed on normal liver cells and at different stages of oval cell activation, indicating potential utility for progenitor cell identification. The subdivision of liver cells using these tools should facilitate the study of the biology of ductal and periductal hepatic cell types, including progenitors. Conclusion A new panel of surface reactive monoclonal antibodies to support investigation of the murine oval cell response has been developed. PMID:18726953

  11. 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.

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

    PubMed Central

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

    2012-01-01

    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. PMID:22675164

  13. Microplicae--Specialized Surface Structure of Epithelial Cells of Wet-Surfaced Oral Mucosa.

    PubMed

    Asikainen, P; Sirviö, E; Mikkonen, J J W; Singh, S P; Schulten, E A J M; ten Bruggenkate, C M; Koistinen, A P; Kullaa, A M

    2015-01-01

    The surface structure of the superficial cells of the oral mucosa is decorated with numerous membrane ridges, termed microplicae (MPLs). The MPL structure is typical of the epithelial surfaces that are covered with protective mucus. Cell membrane MPLs are no longer seen as passive consequences of cellular activity. The interaction between MPLs and the mucins has been demonstrated, however the role of MPL structure seen on the upper surface of the oral epithelial cells is speculative. The cell surface is of potentially great significance, as it harbors many markers for refined prognosis and targets for oral mucosal diseases and cancer therapy. With these aspects in mind, we conducted the present review of the MPL structure and function in order to form the basis for further studies of MPLs of the oral epithelial cells.

  14. Modulated surface nanostructures for enhanced light trapping and reduced surface reflection of crystalline silicon solar cells

    NASA Astrophysics Data System (ADS)

    Tayagaki, Takeshi; Hoshi, Yusuke; Hirai, Yuji; Matsuo, Yasutaka; Usami, Noritaka

    2016-05-01

    We demonstrated the fabrication of modulated surface nanostructures as a new surface texture design for thin wafer solar cells. Using a combination of conventional alkali etching and colloidal lithography, we fabricated surface textures with micrometer and nanometre scales on a Si substrate. These modulated surface nanostructures exhibit reduced surface reflection in a broad spectral range, compared with conventional micrometer textures. We investigated optical absorption using a rigorous coupled wave analysis simulation, which revealed a significant reduction in surface reflection over a broad spectral range and efficient light trapping (comparable to that of conventional micrometer-scale textures) for the modulated nanostructures. We found that the modulated surface nanostructures have a high potential of improving the performance of thin wafer crystalline Si solar cells.

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

    PubMed Central

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

    1975-01-01

    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

  16. Emergence of an Apical Epithelial Cell Surface In Vivo.

    PubMed

    Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Maté; Wallingford, John B

    2016-01-11

    Epithelial sheets are crucial components of all metazoan animals, enclosing organs and protecting the animal from its environment. Epithelial homeostasis poses unique challenges, as addition of new cells and loss of old cells must be achieved without disrupting the fluid-tight barrier and apicobasal polarity of the epithelium. Several studies have identified cell biological mechanisms underlying extrusion of cells from epithelia, but far less is known of the converse mechanism by which new cells are added. Here, we combine molecular, pharmacological, and laser-dissection experiments with theoretical modeling to characterize forces driving emergence of an apical surface as single nascent cells are added to a vertebrate epithelium in vivo. We find that this process involves the interplay between cell-autonomous actin-generated pushing forces in the emerging cell and mechanical properties of neighboring cells. Our findings define the forces driving this cell behavior, contributing to a more comprehensive understanding of epithelial homeostasis.

  17. A requirement for trypsin-sensitive cell-surface components for cell-cell interactions of embryonic neural retina cells

    PubMed Central

    McClay, DR; Godding, LR; Fransen, ME

    1977-01-01

    A quantitative assay was used to measure the rate of collection of a population of embryonic neural retina cells to the surface of cell aggregates. The rate of collection of freshly trysinized cells was limited in the initial stages by the rate of replacement of trypsin-sensitive cell- surface components. When cells were preincubated, or "recovered," and then added to cell aggregates, collection occurred at a linear rate and was independent of protein and glycoprotein synthesis. The adhesion of recovered cells was temperature and energy dependent, and was reversibly inhibited by cytochalasin B. Colchicine had little effect on collection of recovered cells. Antiserum directed against recovered cell membranes was shown to bind to recovered cells by indirect immunofluorescence. The antiserum also was shown to inhibit collection of recovered cells to aggregates, suggesting that at least some of the antigens identified might be involved in the adhesion process. The inhibitory effect of the antiserum was dose dependent . Freshly trypsinized cells absorbed neither the immunofluorescence activity nor the adhesion-inhibiting activity. Recovered cells absorbed away both activities. In specificity studies, dorsal neural retina cells adhered to aggregates of ventral optic tectum in preference to aggregates of dorsal optic tectum. The adhesive specificity of the dorsal retina cells was less sensitive to trypsin than the adhesive specificity of ventral retina cells which adhered preferentially to dorsal tectal aggregates only after a period of recovery. PMID:562349

  18. 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

  19. 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.

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

    PubMed

    Tanaka, Tsutomu; Kondo, Akihiko

    2015-11-15

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

  1. 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

  2. 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.

  3. Structural characterization of the N-linked oligosaccharides from tomato fruit.

    PubMed

    Zeleny, R; Altmann, F; Praznik, W

    1999-05-01

    The primary structures of the N-linked oligosaccharides from tomato fruit (Lycopersicon esculentum) have been elucidated. For the isolation of the protein fraction, two procedures were employed alternatively: a low temperature acetone powder method and ammonium sulfate precipitation of the tomato extract. After peptic digestion, the glycopeptides were purified by cation-exchange chromatography; the oligosaccharides were released by N-glycosidase A and fluorescently labelled with 2-aminopyridine. Structural characterization was accomplished by means of two-dimensional HPLC in combination with exoglycosidase digestions and MALDI-TOF mass spectrometry. Two varieties as well as two stages of ripening were investigated. In all the samples, the same sixteen N-glycosidic structures were detected; the two most abundant glycans showed identical properties to those of the major N-linked oligosaccharides of horseradish peroxidase and pineapple stem bromelain, respectively and accounted for about 65-78% of the total glycan amount; oligomannosidic glycans occurred only in small quantities (3-9%). The majority of the N-glycans were beta 1,2-xylosylated and carried an alpha 1,3-fucose residue linked to the terminal N-acetylglucosamine. This structural element contributes to cross-reactions among non-related glycoproteins and has been shown to be an IgE-reactive determinant (Tretter, Altmann, Kubelka, März, & Becker, 1993). The presented study gives a possible structural explanation for reported immunological cross-reactivities between tomato and grass pollen extracts due to carbohydrate IgE epitopes (Petersen, Vieths, Aulepp, Schlaak, & Becker, 1996), thereby demonstrating the importance of the structural characterization of plant N-glycans for a more reliable interpretation of immunological data. PMID:10365448

  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. Characterization of N-linked oligosaccharides in chorion peroxidase of Aedes aegypti mosquito.

    PubMed

    Li, Junsuo S; Li, Jianyong

    2005-09-01

    A peroxidase is present in the chorion of Aedes aegypti eggs and catalyzes chorion protein cross-linking during chorion hardening, which is critical for egg survival in the environment. The unique chorion peroxidase (CPO) is a glycoprotein. This study deals with the N-glycosylation site, structures, and profile of CPO-associated oligosaccharides using mass spectrometric techniques and enzymatic digestion. CPO was isolated from chorion by solubilization and several chromatographic methods. Mono-saccharide composition was analyzed by HPLC with fluorescent detection. Our data revealed that carbohydrate (D-mannose, N-acetyl D-glucosamine, D-arabinose, N-acetyl D-galactosamine, and L-fucose) accounted for 2.24% of the CPO molecular weight. A single N-glycosylation site (Asn328-Cys- Thr) was identified by tryptic peptide mapping and de novo sequencing of native and PNGase A-deglycosylated CPO using matrix-assisted laser/desorption/ionization time-of-flight mass spectrometry (MALDI/TOF/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The Asn328 was proven to be a major fully glycosylated site. Potential tryptic glycopeptides and profile were first assessed by MALDI/TOF/MS and then by precursor ion scanning during LC/MS/MS. The structures of N-linked oligosaccharides were elucidated from the MS/MS spectra of glycopeptides and exoglycosidase sequencing of PNGase A-released oligosaccharides. These CPO-associated oligosaccharides had dominant Man3GlcNAc2 and Man3 (Fuc) GlcNAc2 and high mannose-type structures (Man(4-8)GlcNAc2). The truncated structures, Man2GlcNAc2 and Man2 (Fuc) GlcNAc2, were also identified. Comparison of CPO activity and Stokes radius between native and deglycosylated CPO suggests that the N-linked oligosaccharides influence the enzyme activity by stabilizing its folded state.

  6. 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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    1999-07-01

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

  10. 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

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

    PubMed

    Kumar, Amit; Ting, Yen-Peng

    2016-10-01

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

  12. A Method of Targeted Cell Isolation via Glass Surface Functionalization.

    PubMed

    Ansari, Ali; Patel, Reema; Schultheis, Kinsey; Naumovski, Vesna; Imoukhuede, P I

    2016-01-01

    One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual nuances in expression from patient to patient. Current methodologies that try to separate cells to fill this niche result in disruption of physiological expression, making the separation technique useless as a diagnostic tool. In this protocol, we describe the functionalization and optimization of a surface for the cellular capture and release. This functionalized surface integrates biotinylated antibodies with a glass surface functionalized with an aminosilane (APTES), desthiobiotin and streptavidin. Cell release is facilitated through the introduction of biotin, allowing the recollection and purification of cells captured by the surface. This release is done through the targeting of the secondary moiety desthiobiotin, which results in a much more gentle release paradigm. This reduction in harsh reagents and shear forces reduces changes in cellular expression. The functionalized surface captures up to 80% of cells in a single cell mixture and has demonstrated 50% capture in a dual-cell mixture. Applications of this technology to xenografts and cancer separation studies are investigated. Quantification techniques for surface verification such as plate reader and ImageJ analyses are described as well. PMID:27684992

  13. 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.

  14. 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.

  15. Responses of fibroblasts and glial cells to nanostructured platinum surfaces

    NASA Astrophysics Data System (ADS)

    Pennisi, C. P.; Sevcencu, C.; Dolatshahi-Pirouz, A.; Foss, M.; Lundsgaard Hansen, J.; Nylandsted Larsen, A.; Zachar, V.; Besenbacher, F.; Yoshida, K.

    2009-09-01

    The chronic performance of implantable neural prostheses is affected by the growth of encapsulation tissue onto the stimulation electrodes. Encapsulation is associated with activation of connective tissue cells at the electrode's metallic contacts, usually made of platinum. Since surface nanotopography can modulate the cellular responses to materials, the aim of the present work was to evaluate the 'in vitro' responses of connective tissue cells to platinum strictly by modulating its surface nanoroughness. Using molecular beam epitaxy combined with sputtering, we produced platinum nanostructured substrates consisting of irregularly distributed nanopyramids and investigated their effect on the proliferation, cytoskeletal organization and cellular morphology of primary fibroblasts and transformed glial cells. Cells were cultured on these substrates and their responses to surface roughness were studied. After one day in culture, the fibroblasts were more elongated and their cytoskeleton less mature when cultured on rough substrates. This effect increased as the roughness of the surface increased and was associated with reduced cell proliferation throughout the observation period (4 days). Morphological changes also occurred in glial cells, but they were triggered by a different roughness scale and did not affect cellular proliferation. In conclusion, surface nanotopography modulates the responses of fibroblasts and glial cells to platinum, which may be an important factor in optimizing the tissue response to implanted neural electrodes.

  16. 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.

  17. Osteoblastic cell behaviour on different titanium implant surfaces.

    PubMed

    Le Guehennec, Laurent; Lopez-Heredia, Marco-Antonio; Enkel, Benedicte; Weiss, Pierre; Amouriq, Yves; Layrolle, Pierre

    2008-05-01

    The osseointegration of oral implants is related to the early interactions between osteoblastic cells and titanium surfaces. The behaviour of osteoblastic MC3T3-E1 cells was compared on four different titanium surfaces: mirror-polished (Smooth-Ti), alumina grit-blasted (Alumina-Ti) or biphasic calcium phosphate ceramic grit-blasted (BCP-Ti) and a commercially available implant surface (SLA). Scanning electron microscopy and profilometry showed distinct microtopographies. The BCP-Ti group had higher average surface roughness (Ra=2.5 microm) than the other grit-blasted groups. Hydrophilicity and surfaces energies were determined on the different substrates by dynamic contact angle measurements. The most hydrophilic surface was the Alumina-Ti discs, while SLA was the most hydrophobic. The titanium surfaces were all oxidized as TiO2 and polluted by carbon contaminants, as determined by X-ray photoelectron spectroscopy. Alumina-Ti samples also exhibited aluminium peaks as a result of the blasting. The BCP-Ti discs contained traces of calcium and phosphorus. MC3T3-E1 cells attached, spread and proliferated on the substrates. For both the SLA and BCP-Ti groups, the entire surface was covered with a layer of osteoblastic cells after 2 days. At high magnification, the cells exhibited cytoplasmic extensions and filopodia. Compared with plastic, cell viability was similar with the Smooth-Ti, slightly lower with the Alumina-Ti and superior with the SLA and BCP-Ti groups. Alkaline phosphatase activity increased with the culture time whatever the substrate. This study shows that BCP-blasting produces rough titanium implants without surface contaminants. PMID:18226985

  18. 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

  19. 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

  20. Cell Surfaces in Plant-Microorganism Interactions

    PubMed Central

    Esquerré-Tugayé, Marie-Thérèse; Lamport, Derek T. A.

    1979-01-01

    Infection of muskmelon Cucumis melo seedlings by the fungus Colletotrichum lagenarium causes a 10-fold increase in the amount of cell wall hydroxyproline-rich glycoprotein. Evidence for this increase was provided by studying two specific markers of this glycoprotein, namely hydroxyproline and glycosylated serine. The lability of the O-glycosidic linkage of wall-bound glycosylated serine in the presence of hydrazine, was used to determine the amount of serine which is glycosylated. A large increase in the hydroxyproline content of infected plants is shown, but the ratios of glycosylated serine to hydroxyproline are similar in healthy and infected plants. As far as these markers are concerned, the hydroxyproline-rich glycoproteins secreted into the wall as a result of the disease are similar to those of healthy plants. In addition, the extent of glycosylation of the wall serine, in both healthy and infected plants, decreases as the plant ages. Serine- and hydroxyproline-rich (glyco)peptides were also isolated after trypsinolysis of the wall. These (glyco)peptides include the galactosyl-containing pentapeptide, serine-hydroxyproline4. This pentapeptide is characteristic of cell wall protein. PMID:16660956

  1. 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.

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

    PubMed

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

    2010-01-01

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

  3. Electroporation chip for adherent cells on photochemically modified polymer surfaces

    NASA Astrophysics Data System (ADS)

    Olbrich, Michael; Rebollar, Esther; Heitz, Johannes; Frischauf, Irene; Romanin, Christoph

    2008-01-01

    We present a polytetrafluoroethylene electroporation microchip with integrated electrodes for transfection of adherent biological cells. For fabrication, UV-surface modification was employed in combination with metal deposition. UV irradiation in reactive atmosphere resulted in introduction of polar chemical groups into the polytetrafluoroethylene surface for significant adhesion enhancement of both biological cells as well as metal electrodes thereon. Electroporation was demonstrated by transfection of human embryonic kidney cells with the enhanced green fluorescent protein. Transparent, working at low voltages, and easy to handle, this chip yields the potential to reduce the amount of sequential working steps necessary for transfection.

  4. Influence of engineered surface on cell directionality and motility.

    PubMed

    Tang, Qing Yuan; Tong, Wing Yin; Shi, Jue; Shi, Peng; Lam, Yun Wah; Pang, Stella W

    2014-03-01

    Control of cell migration is important in numerous key biological processes, and is implicated in pathological conditions such as cancer metastasis and inflammatory diseases. Many previous studies indicated that cell migration could be guided by micropatterns fabricated on cell culture surfaces. In this study, we designed a polydimethylsiloxane cell culture substrate with gratings punctuated by corners and ends, and studied its effects on the behavior of MC3T3-E1 osteoblast cells. MC3T3-E1 cells elongated and aligned with the gratings, and the migration paths of the cells appeared to be guided by the grating pattern. Interestingly, more than 88% of the cells cultured on these patterns were observed to reverse their migration directions at least once during the 16 h examination period. Most of the reversal events occurred at the corners and the ends of the pattern, suggesting these localized topographical features induce an abrupt loss in directional persistence. Moreover, the cell speed was observed to increase temporarily right after each directional reversal. Focal adhesion complexes were more well-established in cells on the angular gratings than on flat surfaces, but the formation of filipodia appeared to be imbalanced at the corners and the ends, possibly leading to the loss of directional persistence. This study describes the first engineered cell culture surface that consistently induces changes in the directional persistence of adherent cells. This will provide an experimental model for the study of this phenomenon and a valuable platform to control the cell motility and directionality, which can be used for cell screening and selection. PMID:24589941

  5. Effects of plasma etching solar cell front surfaces

    SciTech Connect

    Taylor, W.E.; Bunyan, S.M.; Olson, C.E.

    1980-01-01

    A front surface plasma etch with Freon 14+8% O/sub 2/ or sulfur hexafluoride (SF/sub 6/) was found to improve terrestrial solar cell output. SEM studies of these samples revealed surface pitting on Freon 14 etched samples. About 50% of the improvement from Freon etched samples can be attributed to the light capturing effects of surface pits. Output increases from SF/sub 6/ plasma etched cells were found to be comparable with Freon etched cells after subtraction of the light trapping effects. The excess output improvement might be attributed to reduced junction depth or removal of near surface lattice damage. Investigations attempting to identify the cause are described. 1 ref.

  6. 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.

  7. Micropatterned surfaces for controlling cell adhesion and rolling under flow.

    PubMed

    Nalayanda, Divya D; Kalukanimuttam, Mahendran; Schmidtke, David W

    2007-04-01

    Cell adhesion and rolling on the vascular wall is critical to both inflammation and thrombosis. In this study we demonstrate the feasibility of using microfluidic patterning for controlling cell adhesion and rolling under physiological flow conditions. By controlling the width of the lines (50-1000 microm) and the spacing between them (50-100 microm) we were able to fabricate surfaces with well-defined patterns of adhesion molecules. We demonstrate the versatility of this technique by patterning surfaces with 3 different adhesion molecules (P-selectin, E-selectin, and von Willebrand Factor) and controlling the adhesion and rolling of three different cell types (neutrophils, Chinese Hamster Ovary cells, and platelets). By varying the concentration of the incubating solution we could control the surface ligand density and hence the cell rolling velocity. Finally by patterning surfaces with both P-selectin and von Willebrand Factor we could control the rolling of both leukocytes and platelets simultaneously. The technique described in this paper provides and effective and inexpensive way to fabricate patterned surfaces for use in cell rolling assays under physiologic flow conditions. PMID:17160704

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

    PubMed

    MacDonald, Chris; Piper, Robert C

    2016-04-15

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

  9. Cell surface GRP78 as a biomarker and target for suppressing glioma cells

    PubMed Central

    Kang, Bo Ram; Yang, Seung-Hoon; Chung, Bo-Ryehn; Kim, Woong; Kim, YoungSoo

    2016-01-01

    High-grade glioma is a highly malignant and metastatic brain cancer, resistant to many existing anticancer treatments. In such glioma cancer cells, the glucose-regulated protein 78 kDa (GRP78) is particularly highly up-regulated. Former studies have thus targeted mutation-free GRP78 not only to detect glioma cancer cells specifically but also to enhance cytotoxic effect. We focus on cell surface-expressed GRP78 as a target for suppressing high-grade glioma cell lines. Glioblastoma multiforme (GBM) cell line, highly malignant glioma cells, was first injected into 5-week-old athymic mice to confirm and compare GRP78 expression in vivo in xenografted and normal brain tissue. Immunofluorescence and immunoblotting were utilized to detect surface-localized GRP78 in diverse high-grade glioma cell lines. By treating glioma cell lines with the polyclonal N-20 antibody against surface-localized GRP78, we subsequently studied the significance of surface GRP78 to the survival and growth of the glioma cell lines. We found that inhibiting the function of surface GRP78 suppressed cancer cell survival and growth proving that the surface-expressed GRP78 is a vital receptor involved in the proliferation of high-grade glioma. Our findings provide opportunities to target surface GRP78 as a biomarker for high-grade glioma and to develop effective cell-specific anticancer therapy. PMID:27713511

  10. 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.

  11. 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. PMID:27612825

  12. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2004-03-01

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

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

    PubMed Central

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

    2008-01-01

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

  15. Biosensing based on surface plasmon resonance and living cells.

    PubMed

    Chabot, Vincent; Cuerrier, Charles M; Escher, Emanuel; Aimez, Vincent; Grandbois, Michel; Charette, Paul G

    2009-02-15

    We propose the combination of surface plasmon resonance (SPR) with living cells as a biosensing method. Our detection scheme is based on the premise that cellular activity induced by external agents is often associated with changes in cellular morphology, which in turn should lead to a variation of the effective refractive index at the interface between the cell membrane and the metal layer. We monitored surface plasmon resonance signals originating from a gold surface coated with cells on a custom apparatus after injection of various agents known to influence cellular activity and morphology. Specifically, we evaluated three types of stimulation: response to an endotoxin (lipopolysaccharides), a chemical toxin (sodium azide) and a physiological agonist (thrombin). A comparison with phase contrast microscopy reveals that SPR signal variations are associated with the induction of cell death for lipopolysaccharides treatment and a contraction of the cell body for sodium azide. Thrombin-induced cellular response shows a rapid decrease of the measured laser reflectance over 5min followed by a return to the original value. For this treatment, phase contrast micrographs relate the first phase of the SPR variation to cell contraction and increase of the intercellular gaps, whereas the recovery phase can be associated with a spreading of the cell on the sensing surface. Hence, the SPR signal is very consistent with the cellular response normally observed for these treatments. This confirms the validity of the biosensing method, which could be applied to a large variety of cellular responses involving shape remodeling induced by external agents. PMID:18845432

  16. How cells tiptoe on adhesive surfaces before sticking.

    PubMed

    Pierres, Anne; Benoliel, Anne-Marie; Touchard, Dominique; Bongrand, Pierre

    2008-05-15

    Cell membranes are studded with protrusions that were thoroughly analyzed with electron microscopy. However, the nanometer-scale three-dimensional motions generated by cell membranes to fit the topography of foreign surfaces and initiate adhesion remain poorly understood. Here, we describe the dynamics of surface deformations displayed by monocytic cells bumping against fibronectin-coated surfaces. We observed membrane undulations with typically 5 nm amplitude and 5-10 s lifetime. Cell membranes behaved as independent units of micrometer size. Cells detected the presence of foreign surfaces at 50 nm separation, resulting in time-dependent amplification of membrane undulations. Molecular contact then ensued with apparent cell-membrane separation of 30-40 nm, and this distance steadily decreased during the following tens of seconds. Contact maturation was associated with in-plane egress of bulky molecules and robust membrane fluctuations. Thus, membrane undulations may be the major determinant of cell sensitivity to substrate topography, outcome of interaction, and initial kinetics of contact extension. PMID:18234815

  17. 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

  18. 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

  19. Enhanced cell attachment using a novel cell culture surface presenting functional domains from extracellular matrix proteins

    PubMed Central

    Cooke, M. J.; Phillips, S. R.; Shah, D. S.H.; Athey, D.; Lakey, J. H.

    2008-01-01

    Many factors contribute to the creation and maintenance of a realistic environment for cell growth in vitro, e.g. the consistency of the growth medium, the addition of supplements, and the surface on which the cells grow. The nature of the surface on which cells are cultured plays an important role in their ability to attach, proliferate, migrate and function. Components of the extracellular matrix (ECM) are often used to coat glass or plastic surfaces to enhance cell attachment in vitro. Fragments of ECM molecules can be immobilised on surfaces in order to mimic the effects seen by whole molecules. In this study we evaluate the application of a novel technology for the immobilisation of functional domains of known ECM proteins in a controlled manner on a surface. By examining the adherence of cultured PC12 cells to alternative growth surfaces, we show that surfaces coated with motifs from collagen I, collagen IV, fibronectin and laminin can mimic surfaces coated with the corresponding whole molecules. Furthermore, we show that the adherence of cells can be controlled by modifying the hydropathic properties of the surface to either enhance or inhibit cell attachment. Collectively, these data demonstrate the application of a new technology to enable optimisation of cell growth in the tissue culture laboratory. PMID:19002844

  20. 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.

  1. Cell and tissue behavior on micro-grooved surfaces.

    PubMed

    Walboomers, X F; Jansen, J A

    2001-11-01

    In this review, we discuss substrates and implant surfaces provided with micrometer-sized groove and ridge patterns. Such "microgrooves" influence cell behavior: the cells align themselves, and migrate guided by the surface grooves. This phenomenon is known as "contact guidance". First, cell structure and cell attachment behavior are described. Then techniques for the production of microgrooves are addressed, and a summary is given of a number of previous in-vitro and in-vivo experiments on this subject. Based on the knowledge of cell movement, we suggest a theory involving the dynamics of fibrous cellular components in the filopodium. Finally, future directions for this type of research, and implications for medical and dental implantology, are addressed.

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

    PubMed

    Duc, Thinh Nguyen; Huse, Morgan

    2015-11-20

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

  3. 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.

  4. Endocytosis of cell surface material mediates cell plate formation during plant cytokinesis.

    PubMed

    Dhonukshe, Pankaj; Baluska, Frantisek; Schlicht, Markus; Hlavacka, Andrej; Samaj, Jozef; Friml, Jirí; Gadella, Theodorus W J

    2006-01-01

    Dividing plant cells perform a remarkable task of building a new cell wall within the cytoplasm in a few minutes. A long-standing paradigm claims that this primordial cell wall, known as the cell plate, is generated by delivery of newly synthesized material from Golgi apparatus-originated secretory vesicles. Here, we show that, in diverse plant species, cell surface material, including plasma membrane proteins, cell wall components, and exogenously applied endocytic tracers, is rapidly delivered to the forming cell plate. Importantly, this occurs even when de novo protein synthesis is blocked. In addition, cytokinesis-specific syntaxin KNOLLE as well as plasma membrane (PM) resident proteins localize to endosomes that fuse to initiate the cell plate. The rate of endocytosis is strongly enhanced during cell plate formation, and its genetic or pharmacological inhibition leads to cytokinesis defects. Our results reveal that endocytic delivery of cell surface material significantly contributes to cell plate formation during plant cytokinesis.

  5. Reversed cell imprinting, AFM imaging and adhesion analyses of cells on patterned surfaces.

    PubMed

    Zhou, Xiongtu; Shi, Jian; Zhang, Fan; Hu, Jie; Li, Xin; Wang, Li; Ma, Xueming; Chen, Yong

    2010-05-01

    Cell adhesion and motility depend strongly on the interactions between cells and cell culture substratum. To observe the cell morphology at the interface between cells and artificial substratum or patterned surfaces, we have developed a technique named reversed cell imprinting. After culture and chemical fixation of the cells on a patterned hole array, a liquid polymer was poured on and UV cured, allowing taking off the cell-polymer assembly for a direct observation of the underside cell surface using atomic force microscopy. As expected, we observed local deformation of the cell membrane in the hole area with a penetration depth strongly dependent on the size and depth of the hole as well as the culture time. Quantitative analyses of Hela cells on patterned surfaces of polydimethylsiloxane (PDMS) revealed that the penetration was also position dependent over the cell attachment area due to the non-homogeneous distribution of the membrane stress. With the increase of the culture time, the penetration depth was reduced, in a close correlation with the increase of the cell spreading area. Nevertheless, both cell seeding and adhesion efficiency on high density hole arrays could be significantly increased comparing to that on a smooth surface. Patterned substrates are increasingly required to produce and interrogate new biomaterials for therapeutic benefit. Overall, this work suggests a strategy to endow conventional imaging methods with added functionality to enable easy observation of the underside cell morphology on topographic patterns. PMID:20390138

  6. 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.

  7. Micropatterned Azopolymer Surfaces Modulate Cell Mechanics and Cytoskeleton Structure.

    PubMed

    Rianna, Carmela; Ventre, Maurizio; Cavalli, Silvia; Radmacher, Manfred; Netti, Paolo A

    2015-09-30

    Physical and chemical characteristics of materials are important regulators of cell behavior. In particular, cell elasticity is a fundamental parameter that reflects the state of a cell. Surface topography finely modulates cell fate and function via adhesion mediated signaling and cytoskeleton generated forces. However, how topographies alter cell mechanics is still unclear. In this work we have analyzed the mechanical properties of peripheral and nuclear regions of NIH-3T3 cells on azopolymer substrates with different topographic patterns. Micrometer scale patterns in the form of parallel ridges or square lattices of surface elevations were encoded on light responsive azopolymer films by means of contactless optical methods. Cell mechanics was investigated by atomic force microscopy (AFM). Cells and consequently the cell cytoskeleton were oriented along the linear patterns affecting cytoskeletal structures, e.g., formation of actin stress fibers. Our data demonstrate that topographic substrate patterns are recognized by cells and mechanical information is transferred by the cytoskeleton. Furthermore, cytoskeleton generated forces deform the nucleus, changing its morphology that appears to be related to different mechanical properties in the nuclear region.

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

    PubMed

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

    2007-02-01

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

  9. 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

  10. Differentiation Between Intracellular and Cell Surface Glycosyl Transferases: Galactosyl Transferase Activity in Intact Cells and in Cell Homogenate

    PubMed Central

    Deppert, Wolfgang; Werchau, Hermann; Walter, Gernot

    1974-01-01

    Intact BHK (baby hamster kidney) cells catalyze the hydrolysis of UDP-galactose to free galactose. The generation of galactose from UDP-galactose and its intracellular utilization impede the detection of possible galactosyl transferases on the cell surface of intact cells. Several independent procedures have been used to distinguish between intracellular and cell surface glycosyl transferases. With these procedures, no evidence was obtained for the presence of detectable amounts of galactosyl transferase activity on the surface of BHK cells. The data suggest that galactosyl transferases do not play a general role in the phenomena of cell adhesion and contact inhibition. PMID:4528509

  11. Chemical Characterization of N-Linked Oligosaccharide As the Antigen Epitope Recognized by an Anti-Sperm Auto-Monoclonal Antibody, Ts4.

    PubMed

    Yoshitake, Hiroshi; Hashii, Noritaka; Kawasaki, Nana; Endo, Shuichiro; Takamori, Kenji; Hasegawa, Akiko; Fujiwara, Hiroshi; Araki, Yoshihiko

    2015-01-01

    Ts4, an anti-sperm auto-monoclonal antibody, possesses immunoreactivity to the acrosomal region of mouse epididymal spermatozoa. In addition, the mAb shows specific immunoreactivity to reproduction-related regions such as testicular germ cells and early embryo. Our qualitative study previously showed that the antigen epitope for Ts4 contained a N-linked common oligosaccharide (OS) chain on testicular glycoproteins as determined by Western blotting for testicular glycoproteins after treatment with several glycohydrolases. Since the distribution of the Ts4-epitope is unique, the OS chain in Ts4-epitope may have role(s) in the reproductive process. The aim of this study was to clarify the molecular structure of the Ts4-epitope, particularly its OS moiety. Using Ts4 immunoprecipitation combined with liquid chromatography and multiple-stage mass spectrometry, the candidate carbohydrate structure in the Ts4-epitope is proposed to be N-linked fucosylated agalacto-biantennary with bisecting N-acetylglucosamine (GlcNAc) or with N-acetylgalactosamine-GlcNAc motif. Further binding analyses using various lectins against the mouse testicular Ts4-immunoprecipitants revealed that Phaseolus vulgaris erythroagglutinin and Pisum sativum agglutinin showed positive staining of the bands corresponding to Ts4 reactive proteins. Moreover, the immunoreactivity of Ts4 against the testicular extract was completely abrogated after digestion with β-N-acetylglucosaminidase. These results show that the Ts4-epitope contains agalacto-biantennary N-glycan with bisecting GlcNAc carrying fucose residues. PMID:26222427

  12. Chemical Characterization of N-Linked Oligosaccharide As the Antigen Epitope Recognized by an Anti-Sperm Auto-Monoclonal Antibody, Ts4

    PubMed Central

    Yoshitake, Hiroshi; Hashii, Noritaka; Kawasaki, Nana; Endo, Shuichiro; Takamori, Kenji; Hasegawa, Akiko; Fujiwara, Hiroshi; Araki, Yoshihiko

    2015-01-01

    Ts4, an anti-sperm auto-monoclonal antibody, possesses immunoreactivity to the acrosomal region of mouse epididymal spermatozoa. In addition, the mAb shows specific immunoreactivity to reproduction-related regions such as testicular germ cells and early embryo. Our qualitative study previously showed that the antigen epitope for Ts4 contained a N-linked common oligosaccharide (OS) chain on testicular glycoproteins as determined by Western blotting for testicular glycoproteins after treatment with several glycohydrolases. Since the distribution of the Ts4-epitope is unique, the OS chain in Ts4-epitope may have role(s) in the reproductive process. The aim of this study was to clarify the molecular structure of the Ts4-epitope, particularly its OS moiety. Using Ts4 immunoprecipitation combined with liquid chromatography and multiple-stage mass spectrometry, the candidate carbohydrate structure in the Ts4-epitope is proposed to be N-linked fucosylated agalacto-biantennary with bisecting N-acetylglucosamine (GlcNAc) or with N-acetylgalactosamine-GlcNAc motif. Further binding analyses using various lectins against the mouse testicular Ts4-immunoprecipitants revealed that Phaseolus vulgaris erythroagglutinin and Pisum sativum agglutinin showed positive staining of the bands corresponding to Ts4 reactive proteins. Moreover, the immunoreactivity of Ts4 against the testicular extract was completely abrogated after digestion with β-N-acetylglucosaminidase. These results show that the Ts4-epitope contains agalacto-biantennary N-glycan with bisecting GlcNAc carrying fucose residues. PMID:26222427

  13. Photodynamic induction of a bacterial cell surface polypeptide.

    PubMed Central

    Hoober, J K

    1977-01-01

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

  14. Molecular cloning of gp42, a cell-surface molecule that is selectively induced on rat natural killer cells by interleukin 2: glycolipid membrane anchoring and capacity for transmembrane signaling

    PubMed Central

    1991-01-01

    We have previously shown that in vitro culture of rat natural killer (NK) cells in high concentrations of recombinant interleukin 2 (rIL-2) leads to the expression of a surface glycoprotein with a molecular mass of approximately 42 kD. This glycoprotein, gp42, is not induced on other lymphocytes and thus provides a lineage-specific marker for rIL-2- activated NK cells. We here present the nucleotide sequence for gp42 cDNA. The open reading frame encodes 233 amino acids with three potential sites for N-linked glycosylation. The deduced amino acid sequence lacks an apparent transmembrane domain and instead contains a hydrophobic COOH terminus that is characteristic of glycosylphosphatidylinositol (GPI)-anchored surface proteins. Consistent with this, gp42 is cleaved from the NK-like cell line, RNK- 16, by phosphatidylinositol-specific phospholipase C (PI-PLC), as is gp42 expressed on CHO cells that have been transformed with gp42 cDNA. On rIL-2-activated NK cells, gp42 is resistant to PI-PLC, though our studies suggest that gp42 on these cells is still expressed as a GPI- anchored molecule. Antibody to gp42 stimulates in RNK-16 cells an increase in inositol phosphates and in intracellular calciu, signals that are associated with the activation of lymphocytes, including NK cells. rIL-2-activated NK cells, however, lack this response to gp42 as well as to other stimuli. Thus, gp42, the only NK-specific activation antigen, is a GPI-anchored surface molecule with the capacity to stimulate transmembrane signaling. PMID:1845873

  15. Surface properties and early murine pre-osteoblastic cell responses of phosphoric acid modified titanium surface

    PubMed Central

    Osathanon, Thanaphum; Sawangmake, Chenphop; Ruangchainicom, Nanticha; Wutikornwipak, Pavitra; Kantukiti, Panisa; Nowwarote, Nunthawan; Pavasant, Prasit

    2015-01-01

    Aims The present study investigated the surface properties and murine pre-osteoblast cell (MC3T3-E1) responses of phosphoric acid (H3PO4) treated commercially pure titanium. Methods Titanium discs were treated with various concentration of H3PO4 (5%, 10%, and 20%; v/v) at 90 °C for 30 min. Surface properties were evaluated by profilometer, contact angle meter, and scanning electron microscopy (SEM) with energy dispersive X-rays. MC3T3-E1 attachment and spreading were evaluated by SEM and phalloidin immunohistochemistry staining. Results Surface roughness and wettability were not statistically difference among all experimental and control groups. Phosphate and oxygen were detected on H3PO4 treated surfaces. At 20 min, cell attachment was significantly higher in 10% and 20% H3PO4 treated groups compared to the control. Cells exhibited orientated-cytoskeleton fibers on 20% H3PO4 modified titanium surface. Though, there was no difference in cell spreading stage among all treatment groups. Conclusion H3PO4 treatment on titanium may influence early cell response, particularly on attachment and spreading. PMID:26937362

  16. 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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  19. 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.

  20. 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

  1. 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

    2016-07-01

    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.

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

    PubMed Central

    Gopal, P K; Ballou, C E

    1987-01-01

    The yeast Saccharomyces cerevisiae X2180 strain with the mnn1 mnn2 mnn9 mutations, all of which affect mannoprotein glycosylation, synthesizes N-linked oligosaccharides having the following structure: (Formula: see text) whereas the mnn1 mnn2 mutant extends the alpha 1----6-linked backbone of some of the core oligosaccharides by adding 20-30 mannose units. Membrane fractions from the mnn1 mnn2 and mnn1 mnn2 mnn9 mutants are equally effective in catalyzing transfer from GDP-[3H]mannose to add mannose in both alpha 1----2 and alpha 1----6 linkages to an oligosaccharide having the following structure: (Formula: see text) but neither membrane preparation can utilize the homologous mnn1 mnn2 mnn9 oligosaccharide as an acceptor. Thus, addition of the alpha 1----2-linked mannose side chain to the terminal 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. PMID:3321055

  3. Surface complexation of aluminum on isolated fish gill cells

    SciTech Connect

    Wilkinson, K.J. Campbell, G.C.; Bertsch, P.M.; Jagoe, C.H.

    1993-06-01

    Cells from the gills of largemouth bass (Micropterus salmoides) were isolated and exposed to dilute solutions of Al, Al in the presence of fluoride, or Al plus dissolved organic matter (DOM) to determine the cells` metal binding potential in an acidic medium. Microelectrophoresis was employed to monitor the extent of aluminum sorption to cells in the presence of added ligand. In the absence of Al, the gill cells exhibit an appreciable negative charge; Al binding to the cell surface increases the electric potential at the shear plane and leads to a reduction in the cell`s (negative) electrophoretic mobility. In the presence of both Al and F, aluminum complexation at the gill surface is only marginally reduced; the formation of a mixed ligand complex, [F-Al-L-cell], is proposed to account for the observed results. The presence of such ternary complexes was subsequently verified by {sup 19}F nuclear magnetic resonance spectroscopy and by potentiometry. Addition of DOM increased the negative electrophoretic mobility of the isolated gill cells both in the presence and absence of aluminum (7.4 {mu}M). 45 refs., 7 figs., 1 tab.

  4. CELL SURFACE ANTIGENS OF A MOUSE TESTICULAR TERATOMA

    PubMed Central

    Gooding, Linda R.; Edidin, Michael

    1974-01-01

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

  5. 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. PMID:25184988

  6. 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

    2014-11-01

    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.

  7. Comparison of biological activities of human antithrombins with high-mannose or complex-type nonfucosylated N-linked oligosaccharides.

    PubMed

    Yamada, Tsuyoshi; Kanda, Yutaka; Takayama, Makoto; Hashimoto, Akitoshi; Sugihara, Tsutomu; Satoh-Kubota, Ai; Suzuki-Takanami, Eri; Yano, Keiichi; Iida, Shigeru; Satoh, Mitsuo

    2016-05-01

    The structure of the N-linked oligosaccharides attached to antithrombin (AT) has been shown to affect its anticoagulant activity and pharmacokinetics. Human AT has biantennary complex-type oligosaccharides with the unique feature of lacking a core fucose, which affects its biological activities by changing its heparin-binding affinity. In human plasma, AT circulates as a mixture of the α-form bearing four oligosaccharides and the β-form lacking an oligosaccharide at Asn135. However, it remains unclear how the immature high-mannose-type oligosaccharides produced by mammalian cells affect biological activities of AT. Here, we succeeded in directly comparing the activities between the high-mannose and complex types. Interestingly, although there were no substantial differences in thrombin inhibitory activity, the high-mannose type showed higher heparin-binding affinity. The anticoagulant activities were increased by heparin and correlated with the heparin-binding affinity, resulting in the strongest anticoagulant activity being displayed in the β-form with the high-mannose type. In pharmacokinetic profiling, the high-mannose type showed a much shorter plasma half-life than the complex type. The β-form was found to have a prolonged plasma half-life compared with the α-form for the high-mannose type; conversely, the α-form showed a longer half-life than the β-form for the complex-type. The present study highlights that AT physiological activities are strictly controlled not only by a core fucose at the reducing end but also by the high-mannose-type structures at the nonreducing end. The β-form with the immature high-mannose type appears to function as a more potent anticoagulant than the AT typically found in human plasma, once it emerges in the blood. PMID:26747427

  8. 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

  9. Apoptotic epithelial cells control the abundance of Treg cells at barrier surfaces.

    PubMed

    Nakahashi-Oda, Chigusa; Udayanga, Kankanam Gamage Sanath; Nakamura, Yoshiyuki; Nakazawa, Yuta; Totsuka, Naoya; Miki, Haruka; Iino, Shuichi; Tahara-Hanaoka, Satoko; Honda, Shin-ichiro; Shibuya, Kazuko; Shibuya, Akira

    2016-04-01

    Epithelial tissues continually undergo apoptosis. Commensal organisms that inhabit the epithelium influence tissue homeostasis, in which regulatory T cells (Treg cells) have a central role. However, the physiological importance of epithelial cell apoptosis and how the number of Treg cells is regulated are both incompletely understood. Here we found that apoptotic epithelial cells negatively regulated the commensal-stimulated proliferation of Treg cells. Gut commensals stimulated CX3CR1(+)CD103(-)CD11b(+) dendritic cells (DCs) to produce interferon-β (IFN-β), which augmented the proliferation of Treg cells in the intestine. Conversely, phosphatidylserine exposed on apoptotic epithelial cells suppressed IFN-β production by the DCs via inhibitory signaling mediated by the cell-surface glycoprotein CD300a and thus suppressed Treg cell proliferation. Our findings reveal a regulatory role for apoptotic epithelial cells in maintaining the number of Treg cell and tissue homeostasis. PMID:26855029

  10. The C-Terminus of Human Nucleotide Receptor P2X7 Is Critical for Receptor Oligomerization and N-Linked Glycosylation

    PubMed Central

    Wickert, Lisa E.; Blanchette, Joshua B.; Waldschmidt, Noelle V.; Bertics, Paul J.; Denu, John M.; Denlinger, Loren C.; Lenertz, Lisa Y.

    2013-01-01

    Background The P2X7 receptor binds extracellular ATP to mediate numerous inflammatory responses and is considered a potential biomarker and therapeutic target for diverse inflammatory and neurological diseases. P2X7 contains many single nucleotide polymorphisms, including several mutations located within its intracellular C-terminal trafficking domain. Mutations within the trafficking domain result in attenuated receptor activity and cell surface presentation, but the mechanisms by which amino acid changes within this region promote altered P2X7 function have not been elucidated. Methods and Results We analyzed the amino acid sequence of P2X7 for any potential trafficking signals and found that P2X7 contains putative Arg-X-Arg ER retention sequences. Alanine substitutions near or within these sequences were constructed, and we determined that single mutation of R574 and R578 but not R576 or K579 attenuates P2X7-stimulated activation of ERK1/2 and induction of the transcription factors FosB and ΔFosB. We found that mutation of R578 within the trafficking domain to the naturally occurring Gln substitution disrupts P2X7 localization at the plasma membrane and results in R578Q displaying a higher apparent molecular weight in comparison to wild-type receptor. We used the glycosidase endoglycosidase H to determine that this difference in mass is due in part to the R578Q mutant possessing a larger mass of oligosaccharides, indicative of improper N-linked glycosylation addition and/or trimming. Chemical cross-linking experiments were also performed and suggest that the R578Q variant also does not form trimers as well as wild-type receptor, a function required for its full activity. Conclusions These data demonstrate the distal C-terminus of P2X7 is important for oligomerization and post-translational modification of the receptor, providing a mechanism by which mutations in the trafficking domain disrupt P2X7 activity and localization at the plasma membrane. PMID:23691096

  11. Surface-directed assembly of cell-laden microgels.

    PubMed

    Du, Yanan; Ghodousi, Majid; Lo, Edward; Vidula, Mahesh K; Emiroglu, Onur; Khademhosseini, Ali

    2010-02-15

    Cell-laden microscale hydrogels (microgels) can be used as tissue building blocks and assembled to create 3D tissue constructs with well-defined microarchitecture. In this article, we present a bottom-up approach to achieve microgel assembly on a patterned surface. Driven by surface tension, the hydrophilic microgels can be assembled into well-defined shapes on a glass surface patterned with hydrophobic and hydrophilic regions. We found that the cuboidic microgels ( approximately 100-200 microm in width) could self-assemble into defined shapes with high fidelity to the surface patterns. The microgel assembly process was improved by increasing the hydrophilicity of the microgels and reducing the surface tension of the surrounding solution. The assembled microgels were stabilized by a secondary crosslinking step. Assembled microgels containing cells stained with different dyes were fabricated to demonstrate the application of this approach for engineering microscale tissue constructs containing multiple cell types. This bottom-up approach enables rapid fabrication of cell-laden microgel assemblies with pre-defined geometrical and biological features, which is easily scalable and can be potentially used in microscale tissue engineering applications.

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

    PubMed

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

    2012-06-01

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

  13. Autonomous molecular cascades for evaluation of cell surfaces

    NASA Astrophysics Data System (ADS)

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

    2013-08-01

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

  14. 3D surface topology guides stem cell adhesion and differentiation.

    PubMed

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

    2015-06-01

    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 stabilizers. 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.

  15. 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

  16. 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.

  17. 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.

  18. The Role of Surface Receptor Density in Surface-Initiated Polymerizations for Cancer Cell Isolation.

    PubMed

    Lilly, Jacob L; Berron, Brad J

    2016-06-01

    Fluid biopsies potentially offer a minimally invasive alternative to traditional tissue biopsies for the continual monitoring of metastatic cancer. Current established technologies for isolating circulating tumor cells (CTCs) suffer from poor purity and yield and require fixatives that preclude the collection of viable cells for longitudinal analyses of biological function. Antigen specific lysis (ASL) is a rapid, high-purity method of cell isolation based on targeted protective coatings on antigen-presenting cells and lysis depletion of unprotected antigen-negative cells. In ASL, photoinitiators are specifically labeled on cell surfaces that enable subsequent surface-initiated polymerization. Critically, the significant determinants of process yield have yet to be investigated for this emerging technology. In this work, we show that the labeling density of photoinitiators is strongly correlated with the yield of intact cells during ASL by flow cytometry analysis. Results suggest ASL is capable of delivering ∼25% of targeted cells after isolation using traditional antibody labeling approaches. Monomer formulations of two molecular weights of PEG-diacrylate (Mn ∼ 575 and 3500) are examined. The gelation response during ASL polymerization is also investigated via protein microarray analogues on planar glass. Finally, a density threshold of photoinitiator labeling required for protection during lysis is determined for both monomer formulations. These results indicate ASL is a promising technology for high yield CTC isolation for rare-cell function assays and fluid biopsies. PMID:27206735

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

    PubMed Central

    1995-01-01

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

  20. 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.

  1. Monoclonal antibody to human endothelial cell surface internalization and liposome delivery in cell culture.

    PubMed

    Trubetskaya, O V; Trubetskoy, V S; Domogatsky, S P; Rudin, A V; Popov, N V; Danilov, S M; Nikolayeva, M N; Klibanov, A L; Torchilin, V P

    1988-02-01

    A monoclonal antibody (mAb), E25, is described that binds to the surface of cultured human endothelial cells. Upon binding E25 is rapidly internalized and digested intracellularly. Selective liposome targeting to the surface of the cells is performed using a biotinylated E25 antibody and an avidin-biotin system. Up to 30% of the cell-adherent liposomal lipid is internalized.

  2. 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

  3. 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.

  4. 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.

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

    PubMed

    Gerson, D F; Akit, J

    1980-11-01

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

  6. 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

  7. N-linked glycosylation of N48 is required for equilibrative nucleoside transporter 1 (ENT1) function.

    PubMed

    Bicket, Alex; Coe, Imogen R

    2016-08-01

    Human equilibrative nucleoside transporter 1 (hENT1) transports nucleosides and nucleoside analogue drugs across cellular membranes and is necessary for the uptake of many anti-cancer, anti-parasitic and anti-viral drugs. Previous work, and in silico prediction, suggest that hENT1 is glycosylated at Asn(48) in the first extracellular loop of the protein and that glycosylation plays a role in correct localization and function of hENT1. Site-directed mutagenesis of wild-type (wt) hENT1 removed potential glycosylation sites. Constructs (wt 3xFLAG-hENT1, N48Q-3xFLAG-hENT1 or N288Q-3xFLAG-hENT2) were transiently transfected into HEK293 cells and cell lysates were treated with or without peptide-N-glycosidase F (PNGase-F), followed by immunoblotting analysis. Substitution of N48 prevents hENT1 glycosylation, confirming a single N-linked glycosylation site. N48Q-hENT1 protein is found at the plasma membrane in HEK293 cells but at lower levels compared with wt hENT1 based on S-(4-nitrobenzyl)-6-thioinosine (NBTI) binding analysis (wt 3xFLAG-ENT1 Bmax, 41.5±2.9 pmol/mg protein; N48Q-3xFLAG-ENT1 Bmax, 13.5±0.45 pmol/mg protein) and immunofluorescence microscopy. Although present at the membrane, chloroadenosine transport assays suggest that N48Q-hENT1 is non-functional (wt 3xFLAG-ENT1, 170.80±44.01 pmol/mg protein; N48Q-3xFLAG-ENT1, 57.91±17.06 pmol/mg protein; mock-transfected 74.31±19.65 pmol/mg protein). Co-immunoprecipitation analyses suggest that N48Q ENT1 is unable to interact with self or with wt hENT1. Based on these data we propose that glycosylation at N48 is critical for the localization, function and oligomerization of hENT1. PMID:27480168

  8. N-linked glycosylation of N48 is required for equilibrative nucleoside transporter 1 (ENT1) function

    PubMed Central

    Bicket, Alex; Coe, Imogen R.

    2016-01-01

    Human equilibrative nucleoside transporter 1 (hENT1) transports nucleosides and nucleoside analogue drugs across cellular membranes and is necessary for the uptake of many anti-cancer, anti-parasitic and anti-viral drugs. Previous work, and in silico prediction, suggest that hENT1 is glycosylated at Asn48 in the first extracellular loop of the protein and that glycosylation plays a role in correct localization and function of hENT1. Site-directed mutagenesis of wild-type (wt) hENT1 removed potential glycosylation sites. Constructs (wt 3xFLAG-hENT1, N48Q-3xFLAG-hENT1 or N288Q-3xFLAG-hENT2) were transiently transfected into HEK293 cells and cell lysates were treated with or without peptide–N-glycosidase F (PNGase-F), followed by immunoblotting analysis. Substitution of N48 prevents hENT1 glycosylation, confirming a single N-linked glycosylation site. N48Q-hENT1 protein is found at the plasma membrane in HEK293 cells but at lower levels compared with wt hENT1 based on S-(4-nitrobenzyl)-6-thioinosine (NBTI) binding analysis (wt 3xFLAG-ENT1 Bmax, 41.5±2.9 pmol/mg protein; N48Q-3xFLAG-ENT1 Bmax, 13.5±0.45 pmol/mg protein) and immunofluorescence microscopy. Although present at the membrane, chloroadenosine transport assays suggest that N48Q-hENT1 is non-functional (wt 3xFLAG-ENT1, 170.80±44.01 pmol/mg protein; N48Q-3xFLAG-ENT1, 57.91±17.06 pmol/mg protein; mock-transfected 74.31±19.65 pmol/mg protein). Co-immunoprecipitation analyses suggest that N48Q ENT1 is unable to interact with self or with wt hENT1. Based on these data we propose that glycosylation at N48 is critical for the localization, function and oligomerization of hENT1. PMID:27480168

  9. Inhibition of N-linked oligosaccharide processing does not prevent the secretion of thyroglobulin. A study with swainsonine and deoxynojirimycin.

    PubMed

    Franc, J L; Hovsepian, S; Fayet, G; Bouchilloux, S

    1986-05-15

    The effects of two drugs, swainsonine (SW) and deoxynojirimycin (dNM), on synthesis and export of thyroglobulin were studied in folliculized porcine thyroid cells cultured in a serum-free medium. These drugs were expected to alter N-linked glycans in thyroglobulin. Newly synthesized thyroglobulin labeled with [2-3H]mannose or [4,5-3H]leucine was obtained by immunoprecipitation from the follicular contents, culture media and cell extracts; the first two compartments, containing secreted thyroglobulin, were sometimes analyzed together. Leucine incorporation was not inhibited by SW and only slightly by dNM. In contrast dNM strongly decreased mannose incorporation (by up to 50-75% at 1-3 mM). However after 16-h mannose labelings, SW and/or dNM at 2.5 microM and 3 mM respectively did not significantly modify the relative proportions of radioactive thyroglobulin in the above-mentioned compartments. Pronase glycopeptides prepared from these thyroglobulins were examined with respect to behaviour on concanavalin-A-Sepharose and position on Bio-Gel P-4. Oligosaccharides released by endoglucosaminidase H and with high affinity for the lectin, i.e. high-mannose and certain hybrids, were further characterized by various exoglycosidase treatments. Thyroglobulin from control cells displayed complex and high-mannose glycans comparable in size and proportion to those attributed to tissue-extracted porcine thyroglobulin. After treatment with SW (an inhibitor of alpha-mannosidase II), complex glycans were almost totally replaced by sialylated hybrid glycans. In contrast to this nearly total suppression, dNM (an inhibitor of the trimming glucosidases) caused only a 30% decrease in labeling of complex units and an about 50% increase in high-mannose glycans, covered to some degree by glucose. Finally a [3H]leucine pulse-chase study was performed on thyroglobulin secretion in the absence or presence of both SW and dNM. Though a slowdown was detectable in the first few hours, this study

  10. Emergence of an Apical Epithelial Cell Surface In Vivo.

    PubMed

    Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Maté; Wallingford, John B

    2016-01-11

    Epithelial sheets are crucial components of all metazoan animals, enclosing organs and protecting the animal from its environment. Epithelial homeostasis poses unique challenges, as addition of new cells and loss of old cells must be achieved without disrupting the fluid-tight barrier and apicobasal polarity of the epithelium. Several studies have identified cell biological mechanisms underlying extrusion of cells from epithelia, but far less is known of the converse mechanism by which new cells are added. Here, we combine molecular, pharmacological, and laser-dissection experiments with theoretical modeling to characterize forces driving emergence of an apical surface as single nascent cells are added to a vertebrate epithelium in vivo. We find that this process involves the interplay between cell-autonomous actin-generated pushing forces in the emerging cell and mechanical properties of neighboring cells. Our findings define the forces driving this cell behavior, contributing to a more comprehensive understanding of epithelial homeostasis. PMID:26766441

  11. 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. PMID:26863921

  12. Removal of N-linked carbohydrates decreases the infectivity of herpes simplex virus type 1.

    PubMed

    Kühn, J E; Eing, B R; Brossmer, R; Munk, K; Braun, R W

    1988-11-01

    Purified preparations of herpes simplex virus type 1 Angelotti were digested with the exoglycosidases sialidase, beta-galactosidase, N-acetyl-beta-D-glucosaminidase and alpha-mannosidase, and with the endoglycosidases Endo-H and Endo-F. It was found that treatment of virions with Endo-F specifically decreased viral infectivity by a factor of 10. This reduction in titre was not associated with any measurable differences in virus adsorption, suggesting a role of N-linked complex type oligosaccharide chains in penetration. In contrast, a reduction in titre observed upon digestion of virions with exoglycosidases could be attributed to a proteolytic contamination in these enzyme preparations. Treatment of virions with Endo-H, demonstrated to be free of proteolytic contamination, did not reduce viral infectivity. Analysis of endoglycosidase-digested virions by monospecific antibodies and immunoblotting revealed a susceptibility of all four major glycoproteins (gC, gB, gE and gD) to Endo-F, but only gB was susceptible to Endo-H treatment. In contrast, of all the exoglycosidases used only sialidase was found to be active towards native viral glycoproteins. Upon analysis of endoglycosidase-digested virions we could not find any evidence for proteolysis, degradation or altered protein composition of viral envelopes. In contrast, vigorous inhibition of glycoprotein glycosylation by tunicamycin led to the formation of physically intact virions almost completely lacking all major glycoproteins. These data show that digestion of intact virions with glycosidases allows an analysis of the functional relevance of carbohydrate residues without any obvious alterations in the virion glycoprotein composition.

  13. 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...

  14. Cell surface growth in Escherichia coli: distribution of matrix protein.

    PubMed Central

    Begg, K J

    1978-01-01

    Autoradiography of cell envelope "ghosts" from Escherichia coli was used to demonstrate that newly synthesized molecules of "matrix" protein are inserted at random locations over the entire surface of the outer membrane and that, once inserted, these molecules are not thereafter conserved in any fixed spatial location. Images PMID:355219

  15. 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.

  16. Unleashing Cancer Cells on Surfaces Exposing Motogenic IGDQ Peptides.

    PubMed

    Corvaglia, Valentina; Marega, Riccardo; De Leo, Federica; Michiels, Carine; Bonifazi, Davide

    2016-01-20

    Thiolated peptides bearing the Ile-Gly-Asp (IGD) motif, a highly conserved sequence of fibronectin, are used for the preparation of anisotropic self-assembled monolayers (SAM gradients) to study the whole-population migratory behavior of metastatic breast cancer cells (MDA-MB-231 cells). Ile-Gly-Asp-Gln-(IGDQ)-exposing SAMs sustain the adhesion of MDA-MB-231 cells by triggering focal adhesion kinase phosphorylation, similarly to the analogous Gly-Arg-Gly-Asp-(GRGD)-terminating surfaces. However, the biological responses of different cell lines interfaced with the SAM gradients show that only those exposing the IGDQ sequence induce significant migration of MDA-MB-231 cells. In particular, the observed migratory behavior suggests the presence of cell subpopulations associated with a "stationary" or a "migratory" phenotype, the latter determining a considerable cell migration at the sub-cm length scale. These findings are of great importance as they suggest for the first time an active role of biological surfaces exposing the IGD motif in the multicomponent orchestration of cellular signaling involved in the metastatic progression.

  17. Microarrays for the evaluation of cell-biomaterial surface interactions

    NASA Astrophysics Data System (ADS)

    Thissen, H.; Johnson, G.; McFarland, G.; Verbiest, B. C. H.; Gengenbach, T.; Voelcker, N. H.

    2007-01-01

    The evaluation of cell-material surface interactions is important for the design of novel biomaterials which are used in a variety of biomedical applications. While traditional in vitro test methods have routinely used samples of relatively large size, microarrays representing different biomaterials offer many advantages, including high throughput and reduced sample handling. Here, we describe the simultaneous cell-based testing of matrices of polymeric biomaterials, arrayed on glass slides with a low cell-attachment background coating. Arrays were constructed using a microarray robot at 6 fold redundancy with solid pins having a diameter of 375 μm. Printed solutions contained at least one monomer, an initiator and a bifunctional crosslinker. After subsequent UV polymerisation, the arrays were washed and characterised by X-ray photoelectron spectroscopy. Cell culture experiments were carried out over 24 hours using HeLa cells. After labelling with CellTracker ® Green for the final hour of incubation and subsequent fixation, the arrays were scanned. In addition, individual spots were also viewed by fluorescence microscopy. The evaluation of cell-surface interactions in high-throughput assays as demonstrated here is a key enabling technology for the effective development of future biomaterials.

  18. Tracking surface glycans on live cancer cells with single molecule sensitivity**

    PubMed Central

    Jiang, Hao; English, Brian P.; Hazan, Rachel B.; Wu, Peng

    2015-01-01

    Using a combination of metabolically labeled glycans, bioorthogonal Cu(I)-catalyzed azide-alkyne cycloaddition and controlled bleaching of fluorescent probes conjugated to azide or alkyne tagged glycans, we achieve a sufficiently low spatial density of dye labeled glycans enabling dynamic single-molecule tracking and super-resolution imaging of N-linked sialic acids and O-linked GalNAc on the membrane of live cells. Analysis of the trajectories of these dye labeled glycans in mammary cancer cells reveal constrained diffusion of both N- and O-linked glycans which we interpret as reflecting the mobility of the glycan rather than caused by transient immobilization due to spatial inhomogeneities on the plasma membrane. Stochastic optical reconstruction microscopy (STORM) imaging reveals the structure of dynamic membrane nanotubes. PMID:25515330

  19. 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

  20. 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

  1. Cell-surface markers for colon adenoma and adenocarcinoma

    PubMed Central

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

    2016-01-01

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

  2. Surface cell density effects on Escherichia coli gene expression during cell attachment.

    PubMed

    Mauter, Meagan S; Mauter, Meagan; Fait, Aaron; Elimelech, Menachem; Herzberg, Moshe

    2013-06-18

    Escherichia coli attachment to a surface initiates a complex series of interconnected signaling and regulation pathways that promote biofilm formation and maturation. The present work investigates the effect of deposited cell density on E. coli cell physiology, metabolic activity, and gene expression in the initial stages of biofilm development. Deposited cell density is controlled by exploiting the relationship between ionic strength and bacterial attachment efficiency in a packed bed column. Distinct differences in cell transcriptome are analyzed by comparing sessile cultures at two different cell surface densities and differentiating ionic strength effects by analyzing planktonic cultures in parallel. Our results indicate that operons regulating trypotophan production and the galactitol phosphotransferase system (including dihydroxyacetone phosphate synthesis) are strongly affected by cell density on the surface. Additional transcriptome and metabolomic impacts of cell density on succinate, proline, and pyroglutamic acid systems are also reported. These results are consistent with the hypothesis that surface cell density plays a major role in sessile cell physiology, commencing with the first stage of biofilm formation. These findings improve our understanding of biofilm formation in natural and engineered environmental systems and will contribute to future work ranging from pathogen migration in the environment to control of biofouling on engineered surfaces.

  3. "Race for the Surface": Eukaryotic Cells Can Win.

    PubMed

    Pham, Vy T H; Truong, Vi Khanh; Orlowska, Anna; Ghanaati, Shahram; Barbeck, Mike; Booms, Patrick; Fulcher, Alex J; Bhadra, Chris M; Buividas, Ričardas; Baulin, Vladimir; Kirkpatrick, C James; Doran, Pauline; Mainwaring, David E; Juodkazis, Saulius; Crawford, Russell J; Ivanova, Elena P

    2016-08-31

    With an aging population and the consequent increasing use of medical implants, managing the possible infections arising from implant surgery remains a global challenge. Here, we demonstrate for the first time that a precise nanotopology provides an effective intervention in bacterial cocolonization enabling the proliferation of eukaryotic cells on a substratum surface, preinfected by both live Gram-negative, Pseudomonas aeruginosa, and Gram-positive, Staphylococcus aureus, pathogenic bacteria. The topology of the model black silicon (bSi) substratum not only favors the proliferation of eukaryotic cells but is biocompatible, not triggering an inflammatory response in the host. The attachment behavior and development of filopodia when COS-7 fibroblast cells are placed in contact with the bSi surface are demonstrated in the dynamic study, which is based on the use of real-time sequential confocal imaging. Bactericidal nanotopology may enhance the prospect for further development of inherently responsive antibacterial nanomaterials for bionic applications such as prosthetics and implants.

  4. 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...

  5. 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.

  6. 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.

  7. EMA: a developmentally regulated cell-surface glycoprotein of CNS neurons that is concentrated at the leading edge of growth cones.

    PubMed

    Baumrind, N L; Parkinson, D; Wayne, D B; Heuser, J E; Pearlman, A L

    1992-08-01

    To identify cell-surface molecules that mediate interactions between neurons and their environment during neural development, we used monoclonal antibody techniques to define a developmentally regulated antigen in the central nervous system of the mouse. The antibody we produced (2A1) immunolabels cells throughout the central nervous system; we analyzed its distribution in the developing cerebral cortex, where it is expressed on cells very soon after they complete mitosis and leave the periventricular proliferative zone. Expression continues into adult life. The antibody also labels the epithelium of the choroid plexus and the renal proximal tubules, but does not label neurons of the peripheral nervous system in the dorsal root ganglia. In dissociated cell culture of embryonic cerebral cortex, 2A1 labels the surface of neurons but not glia. Immunolabeling of neurons in tissue culture is particularly prominent on the edge of growth cones, including filopodia and the leading edge of lamellipodia, when observed with either immunofluorescence or freeze-etch immunoelectron microscopy. Immunopurification with 2A1 of a CHAPS-extracted membrane preparation from brains of neonatal mice produces a broad (32-36 kD) electrophoretic band and a less prominent 70 kD band that are sensitive to N-glycosidase but not endoglycosidase H. Thus the 2A1 antibody recognizes a developmentally regulated, neuronal cell surface glycoprotein (or glycoproteins) with complex N-linked oligosaccharide side chains. We have termed the glycoprotein antigen EMA because of its prominence on the edge membrane of growth cones. EMA is similar to the M6 antigen (Lagenaur et al: J. Neurobiol. 23:71-88, 1992) in apparent molecular weight, distribution in tissue sections, and immunoreactivity on Western blots, suggesting that the two antigens are similar or identical. Expression of EMA is a very early manifestation of neuronal differentiation; its distribution on growth cones suggests a role in mediating the

  8. 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.

  9. Establishment of cell surface engineering and its development.

    PubMed

    Ueda, Mitsuyoshi

    2016-07-01

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

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

    SciTech Connect

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

    1981-01-01

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

  11. Fibronectin and asialoglyprotein receptor mediate hepatitis B surface antigen binding to the cell surface.

    PubMed

    Yang, Jing; Wang, Feng; Tian, Linlin; Su, Jing; Zhu, Xiangqian; Lin, Li; Ding, Xiaoran; Wang, Xuejun; Wang, Shengqi

    2010-06-01

    Both fibronectin and the asialoglycoprotein receptor (ASGPR) have been identified by some investigators as partners for hepatitis B virus (HBV) envelope proteins. Because fibronectin is a natural ligand for ASGPR, we speculated that HBV might attach to ASGPR expressed on the hepatocyte surface via fibronectin. To test this hypothesis, we first confirmed by co-immunoprecipitation that ASGPR, fibronectin and HBsAg bind to each other in HepG2.2.15 cells, and possible binding domains were identified by GST pull-down. In addition, by measuring binding of HBsAg to cells, we found that ASGPR and fibronectin enhanced the binding capability of HBsAg to HepG2 cells, and even to 293T and CHO cells, which normally do not bind HBV. In conclusion, our findings suggest that both fibronectin and ASGPR mediate HBsAg binding to the cell surface, which provides further evidence for the potential roles of these two proteins in mediating HBV binding to liver cells. PMID:20364278

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

    PubMed

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

    2010-09-15

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

  13. 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.

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

    PubMed

    Raiter, Annat; Yerushalmi, Rinat; Hardy, Britta

    2014-11-30

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

  15. 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.

  16. 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.

  17. The relationship of surface roughness and cell response of chemical surface modification of titanium

    PubMed Central

    Zareidoost, Amir; Ghaseme, Behrooz

    2012-01-01

    Implant surface topography influences osteoblastic proliferation, differentiation and extracellular matrix protein expressions. Previous researches proved that chemical surface modification of titanium implants could be used to improve Bone-to-implant contact. In this study, the surface topography, chemistry and biocompatibility of polished titanium surfaces treated with mixed solution of three acids containing HCl, HF and H3PO4 with different etched conditions for example concentration, time and addition of calcium chloride were studied. Osteoblast cells (MG-63) were cultured on different groups of titanium surfaces. In order to investigate titanium surfaces, SEM, AFM and EDS analyses were carried out. The results showed that surfaces treated with HCl–HF–H3PO4 had higher roughness, lower cytotoxicity level and better biocompatibility than controls. Moreover, addition of calcium chloride into mixed solution of three acids containing HCl, HF and H3PO4 is an important, predominant and new technique for obtaining biofunction in metals for biomedical use including dentistry. PMID:22460230

  18. 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

  19. Probing and Mapping Electrode Surfaces in Solid Oxide Fuel Cells

    PubMed Central

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

    2012-01-01

    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 conversion2. 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 degradation8-12. 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 deposition8, 10, 13, 14 ("coking") and sulfur poisoning11, 15 and the manner in which surface modifications stave off this degradation16. 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 and STM

  20. 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.

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

    PubMed

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

    2016-01-01

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

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

    PubMed

    Wang, Li-Chun; Almazan, Guillermina

    2016-06-01

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

  3. 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

  4. The mechanics of cell crawling over a flat surface

    NASA Astrophysics Data System (ADS)

    Alonso-Latorre, Baldomero; Rodriguez-Rodriguez, Javier; Aliseda, Alberto; Meili, Rudolf; Firtel, Richard; Lasheras, Juan

    2006-03-01

    The chemotaxis of different strains of the amoeba Dictyostelium Dicoideum when exposed to a wide range of concentrations and gradients of chemoattractant has been studied experimentally. First, the time evolution of the velocity as well as the shape of the cell have been measured from microscopy images for a large number of individuals. Secondly, the force that the amoebas exert over the substrate in order to propel themselves has also been measured. Some insights into the physical mechanism by which cells crawl over the surface are obtained by comparing the time evolution of those magnitudes for the different strains under study.

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

    PubMed

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

    2014-07-01

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

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

    PubMed Central

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

    2014-01-01

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

  7. 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

  8. Cell separation using tilted-angle standing surface acoustic waves.

    PubMed

    Ding, Xiaoyun; Peng, Zhangli; Lin, Sz-Chin Steven; Geri, Michela; Li, Sixing; Li, Peng; Chen, Yuchao; Dao, Ming; Suresh, Subra; Huang, Tony Jun

    2014-09-01

    Separation of cells is a critical process for studying cell properties, disease diagnostics, and therapeutics. Cell sorting by acoustic waves offers a means to separate cells on the basis of their size and physical properties in a label-free, contactless, and biocompatible manner. The separation sensitivity and efficiency of currently available acoustic-based approaches, however, are limited, thereby restricting their widespread application in research and health diagnostics. In this work, we introduce a unique configuration of tilted-angle standing surface acoustic waves (taSSAW), which are oriented at an optimally designed inclination to the flow direction in the microfluidic channel. We demonstrate that this design significantly improves the efficiency and sensitivity of acoustic separation techniques. To optimize our device design, we carried out systematic simulations of cell trajectories, matching closely with experimental results. Using numerically optimized design of taSSAW, we successfully separated 2- and 10-µm-diameter polystyrene beads with a separation efficiency of ∼ 99%, and separated 7.3- and 9.9-µm-polystyrene beads with an efficiency of ∼ 97%. We illustrate that taSSAW is capable of effectively separating particles-cells of approximately the same size and density but different compressibility. Finally, we demonstrate the effectiveness of the present technique for biological-biomedical applications by sorting MCF-7 human breast cancer cells from nonmalignant leukocytes, while preserving the integrity of the separated cells. The method introduced here thus offers a unique route for separating circulating tumor cells, and for label-free cell separation with potential applications in biological research, disease diagnostics, and clinical practice.

  9. Cell separation using tilted-angle standing surface acoustic waves

    PubMed Central

    Ding, Xiaoyun; Peng, Zhangli; Lin, Sz-Chin Steven; Geri, Michela; Li, Sixing; Li, Peng; Chen, Yuchao; Dao, Ming; Suresh, Subra; Huang, Tony Jun

    2014-01-01

    Separation of cells is a critical process for studying cell properties, disease diagnostics, and therapeutics. Cell sorting by acoustic waves offers a means to separate cells on the basis of their size and physical properties in a label-free, contactless, and biocompatible manner. The separation sensitivity and efficiency of currently available acoustic-based approaches, however, are limited, thereby restricting their widespread application in research and health diagnostics. In this work, we introduce a unique configuration of tilted-angle standing surface acoustic waves (taSSAW), which are oriented at an optimally designed inclination to the flow direction in the microfluidic channel. We demonstrate that this design significantly improves the efficiency and sensitivity of acoustic separation techniques. To optimize our device design, we carried out systematic simulations of cell trajectories, matching closely with experimental results. Using numerically optimized design of taSSAW, we successfully separated 2- and 10-µm-diameter polystyrene beads with a separation efficiency of ∼99%, and separated 7.3- and 9.9-µm-polystyrene beads with an efficiency of ∼97%. We illustrate that taSSAW is capable of effectively separating particles–cells of approximately the same size and density but different compressibility. Finally, we demonstrate the effectiveness of the present technique for biological–biomedical applications by sorting MCF-7 human breast cancer cells from nonmalignant leukocytes, while preserving the integrity of the separated cells. The method introduced here thus offers a unique route for separating circulating tumor cells, and for label-free cell separation with potential applications in biological research, disease diagnostics, and clinical practice. PMID:25157150

  10. Cell separation using tilted-angle standing surface acoustic waves.

    PubMed

    Ding, Xiaoyun; Peng, Zhangli; Lin, Sz-Chin Steven; Geri, Michela; Li, Sixing; Li, Peng; Chen, Yuchao; Dao, Ming; Suresh, Subra; Huang, Tony Jun

    2014-09-01

    Separation of cells is a critical process for studying cell properties, disease diagnostics, and therapeutics. Cell sorting by acoustic waves offers a means to separate cells on the basis of their size and physical properties in a label-free, contactless, and biocompatible manner. The separation sensitivity and efficiency of currently available acoustic-based approaches, however, are limited, thereby restricting their widespread application in research and health diagnostics. In this work, we introduce a unique configuration of tilted-angle standing surface acoustic waves (taSSAW), which are oriented at an optimally designed inclination to the flow direction in the microfluidic channel. We demonstrate that this design significantly improves the efficiency and sensitivity of acoustic separation techniques. To optimize our device design, we carried out systematic simulations of cell trajectories, matching closely with experimental results. Using numerically optimized design of taSSAW, we successfully separated 2- and 10-µm-diameter polystyrene beads with a separation efficiency of ∼ 99%, and separated 7.3- and 9.9-µm-polystyrene beads with an efficiency of ∼ 97%. We illustrate that taSSAW is capable of effectively separating particles-cells of approximately the same size and density but different compressibility. Finally, we demonstrate the effectiveness of the present technique for biological-biomedical applications by sorting MCF-7 human breast cancer cells from nonmalignant leukocytes, while preserving the integrity of the separated cells. The method introduced here thus offers a unique route for separating circulating tumor cells, and for label-free cell separation with potential applications in biological research, disease diagnostics, and clinical practice. PMID:25157150

  11. Comprehensive analysis of protein glycosylation by solid-phase extraction of N-linked glycans and glycosite-containing peptides

    PubMed Central

    Sun, Shisheng; Shah, Punit; Eshghi, Shadi Toghi; Yang, Weiming; Trikannad, Namita; Yang, Shuang; Chen, Lijun; Aiyetan, Paul; Höti, Naseruddin; Zhang, Zhen; Chan, Daniel W; Zhang, Hui

    2016-01-01

    Comprehensive characterization of protein glycosylation is critical for understanding the structure and function of glycoproteins. However, due to the complexity and heterogeneity of glycoprotein conformations, current glycoprotein analyses focus mainly on either the de-glycosylated glycosylation site (glycosite)-containing peptides or the released glycans. Here, we describe a chemoenzymatic method called solid phase extraction of N-linked glycans and glycosite-containing peptides (NGAG) for the comprehensive characterization of glycoproteins that is able to determine glycan heterogeneity for individual glycosites in addition to providing information about the total N-linked glycan, glycosite-containing peptide and glycoprotein content of complex samples. The NGAG method can also be applied to quantitatively detect glycoprotein alterations in total and site-specific glycan occupancies. PMID:26571101

  12. A selective method for sequential splitting of O- and N-linked glycans from N,O-glycoproteins.

    PubMed

    Likhosherstov, L M; Novikova, O S; Derevitskaya, V A; Kochetkov, N K

    1990-05-15

    O-Linked oligosaccharides from N,O-glycoproteins were selectively split off by treatment with alkaline sodium borohydride in the presence of cadmium salt. The side reaction of reductive cleavage of N-glycosylamide and peptide bonds, observed under standard conditions of splitting of O-linked chains (M NaBH4 and 50mM NaOH, 16 h, 50 degrees), was inhibited by addition of 50-10 mM cadium acetate and 5-10mM EDTA.Na4, as shown by treatment of model compounds and several glycoproteins (ovomucoid, group-specific glycoproteins H and B, fetuin, and asialofetuin). This treatment, in combination with the previously developed procedure for the release of the N-linked oligosaccharide chains by lithium borohydride, allows a sequential, selective cleavage of O-, and then N-linked oligosaccharides from N,O-glycoproteins by chemical methods.

  13. Cell-surface prion protein interacts with glycosaminoglycans.

    PubMed Central

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

    2002-01-01

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

  14. Comparison of actin and cell surface dynamics in motile fibroblasts

    PubMed Central

    1992-01-01

    We have investigated the dynamic behavior of actin in fibroblast lamellipodia using photoactivation of fluorescence. Activated regions of caged resorufin (CR)-labeled actin in lamellipodia of IMR 90 and MC7 3T3 fibroblasts were observed to move centripetally over time. Thus in these cells, actin filaments move centripetally relative to the substrate. Rates were characteristic for each cell type; 0.66 +/- 0.27 microns/min in IMR 90 and 0.36 +/- 0.16 microns/min in MC7 3T3 cells. In neither case was there any correlation between the rate of actin movement and the rate of lamellipodial protrusion. The half-life of the activated CR-actin filaments was approximately 1 min in IMR 90 lamellipodia, and approximately 3 min in MC7 3T3 lamellipodia. Thus continuous filament turnover accompanies centripetal movement. In both cell types, the length of time required for a section of the actin meshwork to traverse the lamellipodium was several times longer than the filament half-life. The dynamic behavior of the dorsal surface of the cell was also observed by tracking lectin-coated beads on the surface and phase-dense features within lamellipodia of MC7 3T3 cells. The movement of these dorsal features occurred at rates approximately three times faster than the rate of movement of the underlying bulk actin cytoskeleton, even when measured in the same individual cells. Thus the transport of these dorsal features must occur by some mechanism other than simple attachment to the moving bulk actin cytoskeleton. PMID:1400580

  15. 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.

  16. 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

  17. 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.

  18. Biomedical interfaces: titanium surface technology for implants and cell carriers.

    PubMed

    Schuler, Martin; Trentin, Diana; Textor, Marcus; Tosatti, Samuele G P

    2006-12-01

    Titanium and its alloys have become key materials for biomedical applications, mainly owing to their compatibility with human tissues and their mechanical strength. Effects of surface topography on cell and tissue response have been investigated extensively in the past, while (bio)chemical surface modification and its combination with designed topographies have remained largely unexplored. The following report describes some of the strategies used or intended to modify titanium surfaces, based on biological principles, with a focus on ultrathin biomimetic adlayers. One of the visions behind such approaches is to achieve improved healing and integration responses after implantation for patients, especially for those suffering from deficiencies, for example, diabetes or osteoporosis, two diseases that have increased drastically in our society during the last century.

  19. Enhancing Cell therapies from the Outside In: Cell Surface Engineering Using Synthetic Nanomaterials

    PubMed Central

    Stephan, Matthias T.; Irvine, Darrell J.

    2011-01-01

    Therapeutic treatments based on the injection of living cells are in clinical use and preclinical development for diseases ranging from cancer to cardiovascular disease to diabetes. To enhance the function of therapeutic cells, a variety of chemical and materials science strategies are being developed that engineer the surface of therapeutic cells with new molecules, artificial receptors, and multifunctional nanomaterials, synthetically endowing donor cells with new properties and functions. These approaches offer a powerful complement to traditional genetic engineering strategies for enhancing the function of living cells. PMID:21826117

  20. 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.

  1. Tetrabromobisphenol A Decreases Cell Surface Proteins Involved in Human Natural Killer (NK) Cell-Dependent Target Cell Lysis

    PubMed Central

    Hurd, Tasia; Whalen, Margaret M.

    2011-01-01

    Human natural killer (NK) lymphocytes are able to destroy tumor cells and virally-infected cells. Interference with their function can leave an individual with increased susceptibility to cancer development and/or viral infection. We have shown that the tumor destroying (lytic) function of NK cells can be dramatically decreased by exposure to the environmental contaminant tetra-bromobisphenol A (TBBPA). TBBPA is a flame retardant used in a variety of materials including circuit boards, carpeting, and upholstery and has been found in human blood samples. TBBPA interferes with NK cell lytic function, in part, by decreasing the ability of NK cells to bind to target cells. This study examines the effects of exposures to concentrations of TBBPA (i.e., that were able to decrease the binding capacity of NK cells) on the expression of cell-surface proteins (CD2, CD11a, CD16, CD18, and CD56) that are needed for NK cells to bind target cells. NK cells were exposed to TBBPA for 24 hr, 48 hr, and 6 d or for 1 hr followed by 24 hr, 48 hr, and 6 d in TBBPA-free media. Twenty-four hr exposures to 5 µM TBBPA caused decreases in four of the cell surface proteins examined. CD16 was decreased by > 35%. The decreases in cell surface proteins after a 48 hr exposure were similar to those seen after 24 hr. The results indicate that TBBPA exposures that decrease the binding function of human NK cells do so by decreasing the expression of cell surface proteins needed for attachment of NK cells to targets cells. PMID:21623697

  2. Chemical modification of the surfaces of bacterial cell walls.

    PubMed

    Neihof, R A; Echols, W H

    1978-01-01

    The surfaces of the isolated cell walls of four bacterial species were studied by microelectrophoresis following chemical treatments intended to remove specific charged groups. Acid-base titrations of the walls were used to assess specificity and extent of the modifications. Carboxyl groups were specifically and completely modified by activation with a water-soluble carbodiimide and subsequent reaction with a nucleophile, such as glycinamide, to give an uncharged pH-stable product. Aqueous media and mild reaction conditions make the method suitable for modifying carboxyl groups on cell surfaces too labile to withstand the harsh conditions required for conventional esterification reactions. Use of the carbodiimide-mediated reaction for discharging carboxyl groups, along with fluorodinitrobenzene for discharging amino groups and extraction procedures for removing constituents carrying phosphoester groups (teichoic acids), made it possible to obtain information about the spatial arrangement of charged groups on the wall surfaces. Removal of the exterior negative charge dominating wall surfaces allowed underlying amino groups to become electrokinetically effective and, in the case of E. coli, also revealed a lipophilic region with an affinity for a cationic surfactant.

  3. 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.

  4. 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.

  5. 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 .

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

    PubMed

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

    1988-10-01

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

  7. The surface of articular cartilage contains a progenitor cell population.

    PubMed

    Dowthwaite, Gary P; Bishop, Joanna C; Redman, Samantha N; Khan, Ilyas M; Rooney, Paul; Evans, Darrell J R; Haughton, Laura; Bayram, Zubeyde; Boyer, Sam; Thomson, Brian; Wolfe, Michael S; Archer, Charles W

    2004-02-29

    It is becoming increasingly apparent that articular cartilage growth is achieved by apposition from the articular surface. For such a mechanism to occur, a population of stem/progenitor cells must reside within the articular cartilage to provide transit amplifying progeny for growth. Here, we report on the isolation of an articular cartilage progenitor cell from the surface zone of articular cartilage using differential adhesion to fibronectin. This population of cells exhibits high affinity for fibronectin, possesses a high colony-forming efficiency and expresses the cell fate selector gene Notch 1. Inhibition of Notch signalling abolishes colony forming ability whilst activated Notch rescues this inhibition. The progenitor population also exhibits phenotypic plasticity in its differentiation pathway in an embryonic chick tracking system, such that chondroprogenitors can engraft into a variety of connective tissue types including bone, tendon and perimysium. The identification of a chondrocyte subpopulation with progenitor-like characteristics will allow for advances in our understanding of both cartilage growth and maintenance as well as provide novel solutions to articular cartilage repair. PMID:14762107

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

    PubMed

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

    2016-06-21

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

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

    PubMed Central

    Yao, Sheng; Zhu, Yuwen; Chen, Lieping

    2013-01-01

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

  10. 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

  11. RPE cell surface proteins in normal and dystrophic rats

    SciTech Connect

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

    1986-02-01

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

  12. Development of living cell force sensors for the interrogation of cell surface interactions

    NASA Astrophysics Data System (ADS)

    Brown, Scott Chang

    The measurement of cell surface interactions, or cell interaction forces, are critical for the early diagnosis and prevention of disease, the design of targeted drug and gene delivery vehicles, the development of next-generation implant materials, and much more. However, the technologies and devices that are currently available are highly limited with respect to the dynamic force range over which they can measure cell-cell or cell-substratum interactions, and with their ability to adequately mimic biologically relevant systems. Consequently, research efforts that involve cell surface interactions have been limited. In this dissertation, existing tools for research at the nanoscale (i.e., atomic force microscopy microcantilevers) are modified to develop living cell force sensors that allow for the highly sensitive measurement of cell-mediated interactions over the entire range of forces expected in biotechnology (and nano-biotechnology) research (from a single to millions of receptor-ligand bonds). Several force sensor motifs have been developed that can be used to measure interactions using single adherent cells, single suspension culture cell, and cell monolayers (tissues) over a wide range of interaction conditions (e.g., approach velocity, shear rate, contact time) using a conventional atomic force microscope. This new tool has been applied to study the pathogenesis of spontaneous pneumothorax and the interaction of cells with 14 man-made interfaces. Consequently, a new hypothesis of the interactions that manifest spontaneous pneumothorax has been developed. Additionally, these findings have the potential to lead to the development of tools for data mining materials and surfaces for unique cell interactions that could have an immense societal impact.

  13. Short-chain ceramides depress integrin cell surface expression and function in colorectal cancer cells.

    PubMed

    Morad, Samy A F; Bridges, Lance C; Almeida Larrea, Alex D; Mayen, Anthony L; MacDougall, Matthew R; Davis, Traci S; Kester, Mark; Cabot, Myles C

    2016-07-01

    Colorectal cancer (CRC) is highly metastatic, significantly so to liver, a characteristic that embodies one of the most challenging aspects of treatment. The integrin family of cell-cell and cell-matrix adhesion receptors plays a central role in migration and invasion, functions that underlie metastatic potential. In the present work we sought to determine the impact of ceramide, which plays a key modulatory role in cancer suppression, on integrin cell surface expression and function in CRC cells in order to reveal possible ceramide-centric effects on tumor cell motility. Human CRC cells LoVo, HT-29, and HCT-116 were employed, which represent lines established from primary and metastatic sites. A cell-permeable, short-chain analog, C6-ceramide, was used as ceramide mimic. Exposure of cells to C6-ceramide (24 h) promoted a dose-dependent (2.5-10 µM) decrease in the expression of cell surface β1 and β4 integrin subunits in all cell lines; at 10 µM C6-ceramide, the decreases ranged from 30 to 50% of the control. Expression of cell surface αVβ6 integrin, which is associated with advanced invasion in CRC, was also suppressed by C6-ceramide. Decreases in integrin expression translated to diminished cellular adhesion, 50% of the control at 5 µM C6-ceramide, and markedly reduced cellular migration, approximately 30-40% of the control in all cell lines. Physicochemical examination revealed potent efficacy of nano-formulated C6-ceramide, but inferior activity of dihydro-C6-ceramide and L-C6-ceramide, compared to the unsaturated counterpart and the natural d-enantiomer, respectively. These studies demonstrate novel actions of ceramides that may have application in suppression of tumor metastasis, in addition to their known tumor suppressor effects. PMID:27045476

  14. T lymphocytes from Sézary syndrome patients express beta1 integrins whose beta(1-6)-branched N-linked oligosaccharides reflect their adhesive capacity.

    PubMed

    Braut-Boucher, F; Font, J; Pichon, J; Paulin, Y; Boukhélifa, M; Aubery, M; Derappe, C

    1998-10-01

    Sézary syndrome (Sz), characterized by slowly progressing clonal proliferation of CD4+, CD45 RO+ T cells, has several forms that are distinguished according to the epidermotropic properties of the pathological cells. In a recent paper (Derappe C, Haentjens G, Lemaire S, Feugeas JP, Lebbe C, Pasqualetto V, Bussel A, Aubery M, Néel D. Leukemia 1996;10:138), we observed that T lymphocytes from most of the Sézary patients [Szbeta(1-6)+] expressed high levels of beta(1-6)-GlcNAc-branched N-linked oligosaccharides while T lymphocytes from other patients [Szbeta(1-6)-] did not. Because this observation suggests the possibility of two forms of Sz, distinguished according to the expression rate of these glycans, we looked for a possible relationship between this expression rate and T-cell adhesiveness. Using an original protocol (Braut-Boucher F, Pichon J, Rat P, Adolphe M, Aubery M, Font J. J Immunol Methods 1995;178:41), we observed that T lymphocytes obtained from the Szbeta(1-6)+ patients adhered less to normal keratinocyte monolayers than T lymphocytes from Szbeta(1-6)- patients and normal donors. As assessed by FACS analysis, all the integrin-subunits studied were more expressed on Szbeta(1-6)-, especially alpha4, alpha5, beta1 and beta2, than on Szbeta(1-6)+ and normal lymphocytes. Although these results suggest that beta1- and beta2-integrin expression is involved in the adhesive properties of these T-cells, other factors, such as glycosylation, may also contribute. To demonstrate this possibility, we sought the presence of beta(1-6)-GlcNAc-branched N-linked oligosaccharides on beta1 integrins expressed by T lymphocytes from Sz patients. Immunoblot experiments, performed using the specific lectin from Phaseolus vulgaris (Leukoagglutinin form), showed that only the beta1 integrin subunit expressed by T lymphocytes from Szbeta(1-6)+ patients carried these glycans, supporting the concept of the involvement of T-cell glycosylation in the evolution of Sz.

  15. 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

  16. Cell-surface arylsulfatase A and B on sinusoidal endothelial cells, hepatocytes, and Kupffer cells in mammalian livers.

    PubMed

    Mitsunaga-Nakatsubo, Keiko; Kusunoki, Shinichiro; Kawakami, Hayato; Akasaka, Koji; Akimoto, Yoshihiro

    2009-06-01

    Arylsulfatase A (ARSA) and B (ARSB) have been regarded as lysosomal enzymes because of their hydrolytic activity on synthetic aromatic substrates and the lysosomal localization of their enzymatic activity. Using sea urchin embryos, we previously demonstrated that the bulk of ARS is located on the cell surface of the epithelium, colocalizing with sulfated polysaccharides, and that it does not exhibit enzymatic activity. To examine whether ARSA and ARSB exist on the cell surface in mammalian tissues, we raised antibodies against ARSA and ARSB and examined immunohistochemically their localization in the liver using light and electron microscopy. Here we show that mammalian ARSA and ARSB exist on the cell surface of sinusoidal endothelial cells, hepatocytes, and sinusoidal macrophages (Kupffer cells), as well as in the lysosome. They are also colocalized with heparan sulfate proteoglycan. These results suggest that ARSA and ARSB also may function in the cell surface of mammals. This is the first report to show cell-surface localization of ARS in mammalian somatic cells. The extracellular localization of ARS will provide new insight for human ARS deficiency disorders, such as metachromatic leukodystrophy and mucopolysaccharidosis VI.

  17. Cell-Surface Protein Profiling Identifies Distinctive Markers of Progenitor Cells in Human Skeletal Muscle.

    PubMed

    Uezumi, Akiyoshi; Nakatani, Masashi; Ikemoto-Uezumi, Madoka; Yamamoto, Naoki; Morita, Mitsuhiro; Yamaguchi, Asami; Yamada, Harumoto; Kasai, Takehiro; Masuda, Satoru; Narita, Asako; Miyagoe-Suzuki, Yuko; Takeda, Shin'ichi; Fukada, So-Ichiro; Nishino, Ichizo; Tsuchida, Kunihiro

    2016-08-01

    Skeletal muscle contains two distinct stem/progenitor populations. One is the satellite cell, which acts as a muscle stem cell, and the other is the mesenchymal progenitor, which contributes to muscle pathogeneses such as fat infiltration and fibrosis. Detailed and accurate characterization of these progenitors in humans remains elusive. Here, we performed comprehensive cell-surface protein profiling of the two progenitor populations residing in human skeletal muscle and identified three previously unrecognized markers: CD82 and CD318 for satellite cells and CD201 for mesenchymal progenitors. These markers distinguish myogenic and mesenchymal progenitors, and enable efficient isolation of the two types of progenitors. Functional study revealed that CD82 ensures expansion and preservation of myogenic progenitors by suppressing excessive differentiation, and CD201 signaling favors adipogenesis of mesenchymal progenitors. Thus, cell-surface proteins identified here are not only useful markers but also functionally important molecules, and provide valuable insight into human muscle biology and diseases. PMID:27509136

  18. 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. PMID:26601950

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

    PubMed

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

    1997-12-01

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

  20. 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.

  1. The use of marsupial x eutherian somatic cell hybrids to study marsupial cell surface antigens.

    PubMed

    Sykes, P J; Hope, R M

    1978-12-01

    Buck and Bodmer (1976) have developed a technique for identifying an antigen on the surface of human x mouse somatic cell hybrids, specified by a gene on a particular human chromosome. We have successfully adapted this technique to a study of marsupial cell surface antigens. Somatic cell hybrids between Macropus rufus (Marsupialia) lymphocytes and the mouse cell lines PG19 and 1R were injected intraperitoneally into mice of the same inbred strain from which the above cell lines were derived (C57B16J and C3H, respectively). The only identified M. rufus chromosome present in the hybrid cells was the X chromosome. The antisera, after adsorption with PG19 or 1R, were tested using indirect immunofluorescence, against the hybrid cells, and also against sub-clones (derived from hybrids) which had apparently lost the M. rufus X chromosome, or at least its long arm. The results of these tests showed that the absorbed antisera contained reactivity against an M. rufus cell surface antigen (or antigens). The reactions of one of the antisera were most simply interpreted by supposing that it was detecting an M. rufus X-lined antigen(s).

  2. 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.

  3. Distribution of anionic groups at the cell surface of different Sporothrix schenckii cell types.

    PubMed

    Benchimol, M; de Souza, W; Travassos, L R

    1979-06-01

    The distribution of anionic groups at the cell surface of yeastlike forms, hyphae, and conidia of Sporothrix schenckii was studied by staining with colloidal iron hydroxide and cationized ferritin. By using colloidal iron hydroxide it was shown that the external cell wall layer of one strain (strain 1099.18) could be resolved into two reactive sublayers and that these layers were present in many but not all cells of the same population. In contrast, most cells of another strain (strain 1099.12) were stained by colloidal iron hydroxide, but only one reactive layer was seen. Acidic layers of the yeastlike forms of the two strains were much thicker than those of conidia and hyphae. By the cationized ferritin staining procedure it was observed that the acidic layers of yeast forms sloughed off of cells, probably due to cell-cell or cell-medium attrition in shaken submerged cultures or to a process by which the outer layers detach from cells as they are replaced by newly synthesized ones. The colloidal iron hydroxide- and cationized ferritin-reactive cell surface layers of S. schenckii correspond to the previously described (L. R. Travassos et al., Exp. Mycol. 1:293-305, 1977) concanavalin A-reactive peptidorhamnomannan complexes, and their reactivity is probably due to the presence of acidic amino acids of low pK values rather than to glucuronic acid units.

  4. 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.

  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. The MDCK epithelial cell line expresses a cell surface antigen of the kidney distal tubule

    PubMed Central

    1982-01-01

    Monoclonal antibodies were prepared against the Madin-Darby canine kidney (MDCK) cell line to identify epithelial cell surface macromolecules involved in renal function. Lymphocyte hybrids were generated by fusing P3U-1 myeloma cells with spleen cells from a C3H mouse immunized with MDCK cells. Hybridomas secreting anti-MDCK antibodies were obtained and clonal lines isolated in soft agarose. We are reporting on one hybridoma line that secretes a monoclonal antibody that binds to MDCK cells at levels 20-fold greater than background binding. Indirect immunofluorescence microscopy was utilized to study the distribution of antibody binding on MDCK cells and on frozen sections of dog kidney and several nonrenal tissues. In the kidney the fluorescence staining pattern demonstrates that the antibody recognizes an antigenic determinant that is expressed only on the epithelial cells of the thick ascending limb of Henle's loops and the distal convoluted tubule and appears to be localized on the basolateral plasma membrane. This antigen also has a unique distribution in non-renal tissues and can only be detected on cells known to be active in transepithelial ion movements. These results indicate the probable distal tubule origin of MDCK and suggest that the monoclonal antibody recognizes a cell surface antigen involved in physiological functions unique to the kidney distal tubule and transporting epithelia of nonrenal tissues. PMID:6178742

  7. Distribution of Anionic Groups at the Cell Surface of Different Sporothrix schenckii Cell Types

    PubMed Central

    Benchimol, Marlene; de Souza, W.; Travassos, L. R.

    1979-01-01

    The distribution of anionic groups at the cell surface of yeastlike forms, hyphae, and conidia of Sporothrix schenckii was studied by staining with colloidal iron hydroxide and cationized ferritin. By using colloidal iron hydroxide it was shown that the external cell wall layer of one strain (strain 1099.18) could be resolved into two reactive sublayers and that these layers were present in many but not all cells of the same population. In contrast, most cells of another strain (strain 1099.12) were stained by colloidal iron hydroxide, but only one reactive layer was seen. Acidic layers of the yeastlike forms of the two strains were much thicker than those of conidia and hyphae. By the cationized ferritin staining procedure it was observed that the acidic layers of yeast forms sloughed off of cells, probably due to cell-cell or cell-medium attrition in shaken submerged cultures or to a process by which the outer layers detach from cells as they are replaced by newly synthesized ones. The colloidal iron hydroxide- and cationized ferritin-reactive cell surface layers of S. schenckii correspond to the previously described (L. R. Travassos et al., Exp. Mycol. 1:293-305, 1977) concanavalin A-reactive peptidorhamnomannan complexes, and their reactivity is probably due to the presence of acidic amino acids of low pK values rather than to glucuronic acid units. Images PMID:89092

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

    PubMed Central

    Zhu, Jie; Xiang, Liang; Jiang, Faqin

    2015-01-01

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

  9. 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.

  10. Detecting Bacterial Surface Organelles on Single Cells Using Optical Tweezers.

    PubMed

    Zakrisson, Johan; Singh, Bhupender; Svenmarker, Pontus; Wiklund, Krister; Zhang, Hanqing; Hakobyan, Shoghik; Ramstedt, Madeleine; Andersson, Magnus

    2016-05-10

    Bacterial cells display a diverse array of surface organelles that are important for a range of processes such as intercellular communication, motility and adhesion leading to biofilm formation, infections, and bacterial spread. More specifically, attachment to host cells by Gram-negative bacteria are mediated by adhesion pili, which are nanometers wide and micrometers long fibrous organelles. Since these pili are significantly thinner than the wavelength of visible light, they cannot be detected using standard light microscopy techniques. At present, there is no fast and simple method available to investigate if a single cell expresses pili while keeping the cell alive for further studies. In this study, we present a method to determine the presence of pili on a single bacterium. The protocol involves imaging the bacterium to measure its size, followed by predicting the fluid drag based on its size using an analytical model, and thereafter oscillating the sample while a single bacterium is trapped by an optical tweezer to measure its effective fluid drag. Comparison between the predicted and the measured fluid drag thereby indicate the presence of pili. Herein, we verify the method using polymer coated silica microspheres and Escherichia coli bacteria expressing adhesion pili. Our protocol can in real time and within seconds assist single cell studies by distinguishing between piliated and nonpiliated bacteria.

  11. Surface Functionalized Graphene Biosensor on Sapphire for Cancer Cell Detection.

    PubMed

    Joe, Daniel J; Hwang, Jeonghyun; Johnson, Christelle; Cha, Ho-Young; Lee, Jo-Won; Shen, Xiling; Spencer, Michael G; Tiwari, Sandip; Kim, Moonkyung

    2016-01-01

    Graphene has several unique physical, optical and electrical properties such as a two-dimensional (2D) planar structure, high optical transparency and high carrier mobility at room temperature. These make graphene interesting for electrical biosensing. Using a catalyst-free chemical vapor deposition (CVD) method, graphene film is grown on a sapphire substrate. There is a single or a few sheets as confirmed by Raman spectroscopy and atomic force microscopy (AFM). Electrical graphene biosensors are fabricated to detect large-sized biological analytes such as cancer cells. Human colorectal carcinoma cells are sensed by the resistance change of an active bio-functionalized graphene device as the cells are captured by the immobilized antibody surface. The functionalized sensors show an increase in resistance as large as ~20% of the baseline with a small number of adhered cells. This study suggests that the bio-functionalized electrical graphene sensors on sapphire, which is a highly transparent material, can potentially detect circulating tumor cells (CTCs) and monitor cellular electrical behavior while being compatible with fluorescence-based optical-detection bioassays. PMID:27398439

  12. High cell-surface density of HER2 deforms cell membranes.

    PubMed

    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

  13. 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.

  14. 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.

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

    PubMed

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

    2015-02-20

    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.

  16. A reclaiming process for solar cell silicon wafer surfaces.

    PubMed

    Pa, P S

    2011-01-01

    The low yield of epoxy film and Si3N4 thin-film deposition is an important factor in semiconductor production. A new design system using a set of three lamination-shaped electrodes as a machining tool and micro electro-removal as a precision reclaiming process of the Si3N4 layer and epoxy film removal from silicon wafers of solar cells surface is presented. In the current experiment, the combination of the small thickness of the anode and cathodes corresponds to a higher removal rate for the thin films. The combination of the short length of the anode and cathodes combined with enough electric power produces fast electroremoval. A combination of the small edge radius of the anode and cathodes corresponds to a higher removal rate. A higher feed rate of silicon wafers of solar cells combined with enough electric power produces fast removal. A precise engineering technology constructed a clean production approach for the removal of surface microstructure layers from silicon wafers is to develop a mass production system for recycling defective or discarded silicon wafers from solar cells that can reduce pollution and lower cost. PMID:21446525

  17. "Race for the Surface": Eukaryotic Cells Can Win.

    PubMed

    Pham, Vy T H; Truong, Vi Khanh; Orlowska, Anna; Ghanaati, Shahram; Barbeck, Mike; Booms, Patrick; Fulcher, Alex J; Bhadra, Chris M; Buividas, Ričardas; Baulin, Vladimir; Kirkpatrick, C James; Doran, Pauline; Mainwaring, David E; Juodkazis, Saulius; Crawford, Russell J; Ivanova, Elena P

    2016-08-31

    With an aging population and the consequent increasing use of medical implants, managing the possible infections arising from implant surgery remains a global challenge. Here, we demonstrate for the first time that a precise nanotopology provides an effective intervention in bacterial cocolonization enabling the proliferation of eukaryotic cells on a substratum surface, preinfected by both live Gram-negative, Pseudomonas aeruginosa, and Gram-positive, Staphylococcus aureus, pathogenic bacteria. The topology of the model black silicon (bSi) substratum not only favors the proliferation of eukaryotic cells but is biocompatible, not triggering an inflammatory response in the host. The attachment behavior and development of filopodia when COS-7 fibroblast cells are placed in contact with the bSi surface are demonstrated in the dynamic study, which is based on the use of real-time sequential confocal imaging. Bactericidal nanotopology may enhance the prospect for further development of inherently responsive antibacterial nanomaterials for bionic applications such as prosthetics and implants. PMID:27494044

  18. A novel surface modification approach for protein and cell microarrays

    NASA Astrophysics Data System (ADS)

    Kurkuri, Mahaveer D.; Driever, Chantelle; Thissen, Helmut W.; Voelcker, Nicholas H.

    2007-01-01

    Tissue engineering and stem cell technologies have led to a rapidly increasing interest in the control of the behavior of mammalian cells growing on tissue culture substrates. Multifunctional polymer coatings can assist research in this area in many ways, for example, by providing low non-specific protein adsorption properties and reactive functional groups at the surface. The latter can be used for immobilization of specific biological factors that influence cell behavior. In this study, glass slides were coated with copolymers of glycidyl methacrylate (GMA) and poly(ethylene glycol) methacrylate (PEGMA). The coatings were prepared by three different methods based on dip and spin coating as well as polymer grafting procedures. Coatings were characterized by X-ray photoelectron spectroscopy, surface sensitive infrared spectroscopy, ellipsometry and contact angle measurements. A fluorescently labelled protein was deposited onto reactive coatings using a contact microarrayer. Printing of a model protein (fluorescein labeled bovine serum albumin) was performed at different protein concentrations, pH, temperature, humidity and using different micropins. The arraying of proteins was studied with a microarray scanner. Arrays printed at a protein concentration above 50 μg/mL prepared in pH 5 phosphate buffer at 10°C and 65% relative humidity gave the most favourable results in terms of the homogeneity of the printed spots and the fluorescence intensity.

  19. Miniaturized proton exchange fuel cell in micromachined silicon surface

    NASA Astrophysics Data System (ADS)

    D'Arrigo, Giuseppe; Spinella, Corrado; Rimini, Emanuele; Rubino, Loredana; Lorenti, Simona

    2004-01-01

    The increasing interest for light and movable electronic systems, cell phones and small digital devices, drives the technological research toward integrated regenerating power sources with small dimensions and great autonomy. Conventional batteries are already unable to deliver power in more and more shrunk volumes maintaining the requirements of long duration and lightweight. A possible solution to overcome these limits is the use of miniaturized fuel cell. The fuel cell offers a greater gravimetric energy density compared to conventional batteries. The micromachining technology of silicon is an important tool to reduce the fuel cell structure to micrometer sizes. The use of silicon also gives the opportunity to integrate the power source and the electronic circuits controlling the fuel cell on the same structure. This paper reports preliminary results concerning the micromachining procedure to fabricate an arrays of microchannels for a Si-based electrocatalytic membrane for miniaturized Si-based proton exchange membrane fuel cells. Several techniques are routinely used to fabricate arrays of microchannels embedded in crystalline silicon. In this paper we present an innovative microchannel formation process, entirely based on surface silicon micromachining, which allows us to produce rhomboidal microchannels embedded on (100) silicon wafers. Compared to the traditional techniques, the proposed process is extremely compatible with the standard microelectronics silicon technology. The kinetics of rhomboidal microchannel formation is monitored by cyclic voltammetry measurements and the results are compared with a detailed structural characterisation performed by scanning electron microscopy. The effectiveness of this process is discussed in view of the possible applications in the fuel cell application.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. Phospholipid polymer-based antibody immobilization for cell rolling surfaces in stem cell purification system.

    PubMed

    Mahara, Atsushi; Chen, Hao; Ishihara, Kazuhiko; Yamaoka, Tetsuji

    2014-01-01

    We previously developed an antibody-conjugated cell rolling column that successfully separates stem cell subpopulations depending on the cell surface marker density, but a large amount of the injected cells were retained in the column because of non-specific interactions. In this study, an amphiphilic copolymer, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (nBMA)-co-N-vinyl formamide (NVf)], with phospholipid polar side groups was designed as a novel antibody-immobilizing modifier. The formamide groups in NVf units were converted to active maleimide groups. A plastic flow microfluidic chamber was coated with the copolymers, and a reduced anti-CD90 antibody was immobilized. The adipose tissue-derived stem cells isolated from the rat were injected into the flow chamber, and their rolling behavior was observed under a microscope with a high-speed camera. Non-specific cell adhesion was reduced strongly by means of this immobilization method because of the MPC unit, resulting in a high percentage of rolling cells. These results demonstrate that a surface coated with phospholipid polar groups can be used in an effective stem cell separation system based on the cell rolling process.

  5. Targeting pancreatic progenitor cells in human embryonic stem cell differentiation for the identification of novel cell surface markers.

    PubMed

    Fishman, Bettina; Segev, Hanna; Kopper, Oded; Nissenbaum, Jonathan; Schulman, Margarita; Benvenisty, Nissim; Itskovitz-Eldor, Joseph; Kitsberg, Danny

    2012-09-01

    New sources of beta cells are needed in order to develop cell therapies for patients with diabetes. An alternative to forced expansion of post-mitotic beta cells is the induction of differentiation of stem-cell derived progenitor cells that have a natural self-expansion capacity into insulin-producing cells. In order to learn more about these progenitor cells at different stages along the differentiation process in which they become progressively more committed to the final beta cell fate, we took the approach of identifying, isolating and characterizing stage specific progenitor cells. We generated human embryonic stem cell (HESC) clones harboring BAC GFP reporter constructs of SOX17, a definitive endoderm marker, and PDX1, a pancreatic marker, and identified subpopulations of GFP expressing cells. Using this approach, we isolated a highly enriched population of pancreatic progenitor cells from hESCs and examined their gene expression with an emphasis on the expression of stage-specific cell surface markers. We were able to identify novel molecules that are involved in the pancreatic differentiation process, as well as stage-specific cell markers that may serve to define (alone or in combination with other markers) a specific pancreatic progenitor cell. These findings may help in optimizing conditions for ultimately generating and isolating beta cells for transplantation therapy.

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

    NASA Astrophysics Data System (ADS)

    Tanaka, Motomu; Sackmann, Erich

    2005-09-01

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

  7. Surface plasmon enhanced cell microscopy with blocked random spatial activation

    NASA Astrophysics Data System (ADS)

    Son, Taehwang; Oh, Youngjin; Lee, Wonju; Yang, Heejin; Kim, Donghyun

    2016-03-01

    We present surface plasmon enhanced fluorescence microscopy with random spatial sampling using patterned block of silver nanoislands. Rigorous coupled wave analysis was performed to confirm near-field localization on nanoislands. Random nanoislands were fabricated in silver by temperature annealing. By analyzing random near-field distribution, average size of localized fields was found to be on the order of 135 nm. Randomly localized near-fields were used to spatially sample F-actin of J774 cells (mouse macrophage cell-line). Image deconvolution algorithm based on linear imaging theory was established for stochastic estimation of fluorescent molecular distribution. The alignment between near-field distribution and raw image was performed by the patterned block. The achieved resolution is dependent upon factors including the size of localized fields and estimated to be 100-150 nm.

  8. Mechanotransduction Across the Cell Surface and Through the Cytoskeleton

    NASA Astrophysics Data System (ADS)

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

    1993-05-01

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

  9. 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.

  10. 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.

  11. Characterization of foamy epithelial surface cells in the canine endometrium.

    PubMed

    Bartel, C; Tichy, A; Walter, I

    2014-06-01

    In mature bitches, endometrial epithelial surface cells modify function and corresponding morphology during the oestrous cycle. During late metoestrous, endometrial epithelial surface cells frequently accumulate fat and thereby adopt a foamy morphology. This cyclic appearance of foamy endometrial epithelial cells (fEECs) seems to be physiological in the dog, whereas in other species, it indicates pathological changes. Function of these fEECs has not been identified until now. Therefore, the aim of the study was to characterize the fEECs by means of transmission electron microscopy and immunohistochemistry. Different manifestations of fEECs were observed and analysed with regard to proliferative activity and presence of different epithelial adhesion molecules including PLEKHA7, β-catenin and E-cadherin. PLEKHA7 was restricted to the apical regions of the fEECs, whereas E-cadherin and β-catenin were demonstrated basolateral. The immunohistochemical detection of steroid hormone receptors demonstrated the responsiveness of the fEECs to steroid hormones. Intense progesterone receptor expression was observed in the fEECs indicating a high responsiveness to this hormone. Considering a potential function of the fEECs, we hypothesized that leptin, a hormone produced by other lipid-accumulating cells and described to be involved in reproduction, in particular during implantation, might also originate from the fEECs which was confirmed by immunohistochemical methods. Moreover, leptin receptor was found in fEECs indicating the fEECs as both, source and target for leptin. Therefore, we conclude that fEECs in the canine uterus have a potential role in early pregnancy events and that the different observed manifestations might simply reflect the variations of signs of pseudopregnancy among bitches.

  12. Biocompatible functionalization of polymersome surfaces: a new approach to surface immobilization and cell targeting using polymersomes.

    PubMed

    Egli, Stefan; Nussbaumer, Martin G; Balasubramanian, Vimalkumar; Chami, Mohamed; Bruns, Nico; Palivan, Cornelia; Meier, Wolfgang

    2011-03-30

    Vesicles assembled from amphiphilic block copolymers represent promising nanomaterials for applications that include drug delivery and surface functionalization. One essential requirement to guide such polymersomes to a desired site in vivo is conjugation of active, targeting ligands to the surface of preformed self-assemblies. Such conjugation chemistry must fulfill criteria of efficiency and selectivity, stability of the resulting bond, and biocompatibility. We have here developed a new system that achieves these criteria by simple conjugation of 4-formylbenzoate (4FB) functionalized polymersomes with 6-hydrazinonicotinate acetone hydrazone (HyNic) functionalized antibodies in aqueous buffer. The number of available amino groups on the surface of polymersomes composed of poly(dimethylsiloxane)-block-poly(2-methyloxazoline) diblock copolymers was investigated by reacting hydrophilic succinimidyl-activated fluorescent dye with polymersomes and evaluating the resulting emission intensity. To prove attachment of biomolecules to polymersomes, HyNic functionalized enhanced yellow fluorescent protein (eYFP) was attached to 4FB functionalized polymersomes, resulting in an average number of 5 eYFP molecules per polymersome. Two different polymersome-antibody conjugates were produced using either antibiotin IgG or trastuzumab. They showed specific targeting toward biotin-patterned surfaces and breast cancer cells. Overall, the polymersome-ligand platform appears promising for therapeutic and diagnostic use.

  13. 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.

  14. Surface deformation and shear flow in ligand mediated cell adhesion.

    PubMed

    Sircar, Sarthok; Roberts, Anthony J

    2016-10-01

    We present a unified, multiscale model to study the attachment/detachment dynamics of two deforming, charged, near spherical cells, coated with binding ligands and subject to a slow, homogeneous shear flow in a viscous, ionic fluid medium. The binding ligands on the surface of the cells experience both attractive and repulsive forces in an ionic medium and exhibit finite resistance to rotation via bond tilting. The microscale drag forces and couples describing the fluid flow inside the small separation gap between the cells, are calculated using a combination of methods in lubrication theory and previously published numerical results. For a selected range of material and fluid parameters, a hysteretic transition of the sticking probability curves (i.e., the function [Formula: see text]) between the adhesion phase (when [Formula: see text]) and the fragmentation phase (when [Formula: see text]) is attributed to a nonlinear relation between the total nanoscale binding forces and the separation gap between the cells. We show that adhesion is favoured in highly ionic fluids, increased deformability of the cells, elastic binders and a higher fluid shear rate (until a critical threshold value of shear rate is reached). Within a selected range of critical shear rates, the continuation of the limit points (i.e., the turning points where the slope of [Formula: see text] changes sign) predict a bistable region, indicating an abrupt switching between the adhesion and the fragmentation regimes. Although, bistability in the adhesion-fragmentation phase diagram of two deformable, charged cells immersed in an ionic aqueous environment has been identified by some in vitro experiments, but until now, has not been quantified theoretically.

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

    PubMed Central

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

    1982-01-01

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

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

    PubMed Central

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

    1995-01-01

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

  17. 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.

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

    PubMed

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

    2005-03-01

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

  19. 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. PMID:27119260

  20. 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.

  1. 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.

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

    PubMed

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

    2016-10-01

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

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

    PubMed Central

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

    2011-01-01

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

  4. 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.

  5. Substrate recognition by the cell surface palmitoyl transferase DHHC5

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-12-01

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

  7. Novel eukaryotic enzymes modifying cell-surface biopolymers

    PubMed Central

    2010-01-01

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

  8. Surface charge of protoplasts and their significance in cell-cell interaction.

    PubMed

    Nagata, T; Melchers, G

    1978-01-01

    ζ-potential of mesophyll protoplasts of tobacco (Nicotiana tabacum L.), petunia (Petunia hybrida Hort.), turnip (Brassica rapa L.) and cowpea (Vigna unguiculata L. Walpers) was determined by use of a cell electrophoresis apparatus. All protoplasts examined showed a constant negative value of-10 to-35 mV. The addition of CaCl2 nullified the ζ-potential of tobacco protoplasts. This phenomenon is explained by DLVO theory of colloid science, which has been successfully applied to animal cells. Furthermore, positively charged polymers reversed the ζ-potential to positive values. Treatment of the protoplast surface with several enzymes was carried out to characterize the chemical nature of suface charges. The removal of surface charges was most conspicuous by the treatment of acid phosphatase (EC 3.1.3.2), but did not occur upon treatment with α-neuraminidase (EC 3.2.1.18) or Streptomyces griseus pronase. Thus a major part of the surface charge originates from the phosphate groups at the cell membrane. The significance of these studies for the properties of the protoplast surface in cell adhesion is discussed.

  9. Dynamic interplay between adhesion surfaces in carcinomas: Cell-cell and cell-matrix crosstalk

    PubMed Central

    Smith, Yvonne E; Vellanki, Sri HariKrishna; Hopkins, Ann M

    2016-01-01

    Cell-cell and cell-matrix signaling and communication between adhesion sites involve mechanisms which are required for cellular functions during normal development and homeostasis; however these cellular functions and mechanisms are often deregulated in cancer. Aberrant signaling at cell-cell and cell-matrix adhesion sites often involves downstream mediators including Rho GTPases and tyrosine kinases. This review discusses these molecules as putative mediators of cellular crosstalk between cell-cell and cell-matrix adhesion sites, in addition to their attractiveness as therapeutic targets in cancer. Interestingly, inter-junctional crosstalk mechanisms are frequently typified by the way in which bacterial and viral pathogens opportunistically infect or intoxicate mammalian cells. This review therefore also discusses the concept of learning from pathogen-host interaction studies to better understand coordinated communication between cell-cell and cell-matrix adhesion sites, in addition to highlighting the potential therapeutic usefulness of exploiting pathogens or their products to tap into inter-junctional crosstalk. Taken together, we feel that increased knowledge around mechanisms of cell-cell and cell-matrix adhesion site crosstalk and consequently a greater understanding of their therapeutic targeting offers a unique opportunity to contribute to the emerging molecular revolution in cancer biology. PMID:26981196

  10. 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

  11. Mouse Paneth Cell Secretory Responses to Cell Surface Glycolipids of Virulent and Attenuated Pathogenic Bacteria

    PubMed Central

    Tanabe, Hiroki; Ayabe, Tokiyoshi; Bainbridge, Brian; Guina, Tina; Ernst, Robert K.; Darveau, Richard P.; Miller, Samuel I.; Ouellette, Andre J.

    2005-01-01

    Mouse Paneth cells respond to bacteria and bacterial cell surface antigens by discharging secretory granules into the lumen of small intestinal crypts (T. Ayabe et al., Nat. Immunol. 1:113-118, 2000). To investigate mechanisms regulating these responses, purified surface glycolipid molecules with known acyl chain modifications and attenuated properties were tested for the ability to stimulate Paneth cell secretion. The antigens included lipopolysaccharide (LPS) from wild-type and msbB-null Escherichia coli and phoP-null and phoP-constitutive Salmonella enterica serovar Typhimurium strains, as well as LPS, lipid A, and lipoteichoic acid from Pseudomonas aeruginosa and Listeria monocytogenes grown in Mg2+-limited media. Measurements of total secreted protein, secreted lysozyme, and the bactericidal peptide activities of collected secretions showed that the purified antigens elicited similar secretory responses from Paneth cells in mouse crypts ex vivo, regardless of glycolipid acyl chain modification. Despite their impaired Tlr4 pathway, Paneth cells in ex vivo C3H/HeJ mouse crypts released equivalent amounts of bactericidal peptide activity in response to purified bacterial antigens, including lipid A. Thus, mouse Paneth cells respond equivalently to purified bacterial cell envelope glycolipids, regardless of functional Tlr4, the structural properties of glycolipid acyl chains, or their association with virulence in humans. PMID:15784576

  12. An SU-8 liquid cell for surface acoustic wave biosensors

    NASA Astrophysics Data System (ADS)

    Francis, Laurent A.; Friedt, Jean-Michel; Bartic, Carmen; Campitelli, Andrew

    2004-08-01

    One significant challenge facing biosensor development is packaging. For surface acoustic wave based biosensors, packaging influences the general sensing performance. The acoustic wave is generated and received thanks to interdigital transducers and the separation between the transducers defines the sensing area. Liquids used in biosensing experiments lead to an attenuation of the acoustic signal while in contact with the transducers. We have developed a liquid cell based on photodefinable epoxy SU-8 that prevents the presence of liquid on the transducers, has a small disturbance effect on the propagation of the acoustic wave, does not interfere with the biochemical sensing event, and leads to an integrated sensor system with reproducible properties. The liquid cell is achieved in two steps. In a first step, the SU-8 is precisely patterned around the transducers to define 120 μm thick walls. In a second step and after the dicing of the sensors, a glass capping is placed manually and glued on top of the SU-8 walls. This design approach is an improvement compared to the more classical solution consisting of a pre-molded cell that must be pressed against the device in order to avoid leaks, with negative consequences on the reproducibility of the experimental results. We demonstrate the effectiveness of our approach by protein adsorption monitoring. The packaging materials do not interfere with the biomolecules and have a high chemical resistance. For future developments, wafer level bonding of the quartz capping onto the SU-8 walls is envisioned.

  13. Structural analysis of the varicella-zoster virus gp98-gp62 complex: posttranslational addition of N-linked and O-linked oligosaccharide moieties.

    PubMed

    Montalvo, E A; Parmley, R T; Grose, C

    1985-03-01

    Varicella-zoster virus specifies the formation of several glycoproteins, including the preponderant gp98-gp62 glycoprotein complex in the outer membranes of virus-infected cells. These viral glycoproteins are recognized and precipitated by a previously described monoclonal antibody designated monoclone 3B3. When an immunoblot analysis was performed, only gp98 was reactive with monoclone 3B3 antibody; likewise, titration in the presence of increased concentrations of sodium dodecyl sulfate during antigen-antibody incubations caused selective precipitation of gp98 but not gp62. Further structural analyses of gp98 were performed by using the glycosidases endo-beta-N-acetylglucosaminidase H (endoglycosidase H) and neuraminidase and two inhibitors of glycosylation (tunicamycin and monensin). In addition to gp98, antibody 3B3 reacted with several intermediate products, including gp90, gp88, gp81, and a nonglycosylated polypeptide, p73. Since gp98 was completely resistant to digestion with endoglycosidase H, it contained only complex carbohydrate moieties; conversely, gp81 contained mainly high-mannose residues. Polypeptide p73 was immunodetected in the presence of tunicamycin and designated as a nascent recipient of N-linked sugars, whereas gp88 was considered to contain O-linked oligosaccharides because its synthesis was not affected by tunicamycin. The ionophore monensin inhibited production of mature gp98, but other intermediate forms, including gp90, were detected. Since the latter product was similar in molecular weight to the desialated form of gp98, one effect of monensin treatment of varicella-zoster virus-infected cells was to block the addition of N-acetylneuraminic acid. Monensin also blocked insertion of gp98 into the plasma membrane and, as determined by electron microscopy, inhibited envelopment of the nucleocapsid and its transport within the cytoplasm. On the basis of this study, we reached the following conclusions: the primary antibody 3B3-binding

  14. In situ recognition of cell-surface glycans and targeted imaging of cancer cells

    PubMed Central

    Xu, Xiao-Ding; Cheng, Han; Chen, Wei-Hai; Cheng, Si-Xue; Zhuo, Ren-Xi; Zhang, Xian-Zheng

    2013-01-01

    Fluorescent sensors capable of recognizing cancer-associated glycans, such as sialyl Lewis X (sLex) tetrasaccharide, have great potential for cancer diagnosis and therapy. Studies on water-soluble and biocompatible sensors for in situ recognition of cancer-associated glycans in live cells and targeted imaging of cancer cells are very limited at present. Here we report boronic acid-functionalized peptide-based fluorescent sensors (BPFSs) for in situ recognition and differentiation of cancer-associated glycans, as well as targeted imaging of cancer cells. By screening BPFSs with different structures, it was demonstrated that BPFS1 with a FRGDF peptide could recognize cell-surface glycan of sLex with high specificity and thereafter fluorescently label and discriminate cancer cells through the cooperation with the specific recognition between RGD and integrins. The newly developed peptide-based sensor will find great potential as a fluorescent probe for cancer diagnosis. PMID:24042097

  15. 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

  16. Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control.

    PubMed Central

    Hammond, C; Braakman, I; Helenius, A

    1994-01-01

    Using a pulse-chase approach combined with immunoprecipitation, we showed that newly synthesized influenza virus hemagglutinin (HA) and vesicular stomatitis virus G protein associate transiently during their folding with calnexin, a membrane-bound endoplasmic reticulum (ER) chaperone. Inhibitors of N-linked glycosylation (tunicamycin) and glucosidases I and II (castanospermine and 1-deoxynojirimycin) prevented the association, whereas inhibitors of ER alpha-mannosidases did not. Our results indicated that binding of these viral glycoproteins to calnexin correlated closely with the composition of their N-linked oligosaccharide side chains. Proteins with monoglucosylated oligosaccharides were the most likely binding species. On the basis of our data and existing information concerning the role of monoglucosylated oligosaccharides on glycoproteins, we propose that the ER contains a unique folding and quality control machinery in which calnexin acts as a chaperone that binds proteins with partially glucose-trimmed carbohydrate side chains. In this model glucosidases I and II serve as signal modifiers and UDP-glucose:glycoprotein glucosyltransferase, as a folding sensor. Images PMID:8302866

  17. Graphene Oxide Modulates B Cell Surface Phenotype and Impairs Immunoglobulin Secretion in Plasma Cell.

    PubMed

    Xu, Shaohai; Xu, Shengmin; Chen, Shaopeng; Fan, Huadong; Luo, Xun; Yang, Xiaoyao; Wang, Jun; Yuan, Hang; Xu, An; Wu, Lijun

    2016-04-01

    Since discovery, graphene oxide (GO) has been used in all aspects of human life and revealed promising applications in biomedicine. Nevertheless, the potential risks of GO were always being revealed. Although GO was found to induce immune cell death and innate immune response, little is known regarding its toxicity to the specific adaptive immune system that is crucial for protecting against exotic invasion. The B-cell mediated adaptive immune system, which composed of highly specialized cells (B and plasma cell) and specific immune response (antibody response) is the focus in our present study. Using diverse standard immunological techniques, we found that GO modulated B cell surface phenotype, both costimulatory molecules (CD80, CD86 and especially CD40) and antigen presenting molecules (both classical and nonclassical) under the condition without causing cell death. Meanwhile, the terminal differentiated immunoglobulin (Ig) secreting plasma cell was affected by GO, which displayed a less secretion of Ig and more severe ER stress caused by the retention of the secreted form of Ig in cell compartment. The combined data reveal that GO has a particular adverse effect to B cell and the humoral immunity, directly demonstrating the potential risk of GO to the specific adaptive immunity. PMID:27451788

  18. The role of cell surface receptors in the activation of human B cells by phosphorothioate oligonucleotides.

    PubMed

    Liang, H; Reich, C F; Pisetsky, D S; Lipsky, P E

    2000-08-01

    Phosphorothioate oligodeoxynucleotides (sODN) containing the CpG motif or TCG repeats induce T cell-independent polyclonal activation of human B cells. To elucidate the mechanism of this response, the role of cell surface receptors was investigated. Sepharose beads coated with stimulatory but not nonstimulatory sODNs induced B cell proliferation comparably with soluble sODNs. The B cell stimulatory activity of Sepharose-bound sODN did not result from free sODN released from the beads since media incubated with coated beads were inactive. Using FITC-labeled sODNs as probes, binding to human B cells could be detected by flow cytometry. Binding was rapid, saturable, initially temperature independent, but with a rapid off-rate. Competition studies indicated that both stimulatory sODNs and minimally stimulatory sODNs bound to the same receptor. By contrast, phosphodiester oligonucleotides with the same nucleotide sequence as sODNs and bacterial DNA inhibited the binding of sODNs to B cells minimally. Charge appeared to contribute to the binding of sODNs to B cells since binding of sODNs was competitively inhibited by negatively charged molecules, including fucoidan, poly I, and polyvinyl sulfate. These data indicate that human B cells bind sODNs by a receptor-mediated mechanism that is necessary but not sufficient for polyclonal activation.

  19. Galectin-1-mediated cell adhesion, invasion and cell death in human anaplastic large cell lymphoma: regulatory roles of cell surface glycans.

    PubMed

    Suzuki, Osamu; Abe, Masafumi

    2014-05-01

    Galectin-1 is known to be one of the extracellular matrix proteins. To elucidate the biological roles of galectin-1 in cell adhesion and invasion of human anaplastic large cell lymphoma, we performed cell adhesion and invasion assays using the anaplastic large cell lymphoma cell line H-ALCL, which was previously established in our laboratory. From the cell surface lectin array, treatment with neuraminidase from Arthrobacter ureafaciens which cleaves all linkage types of cell surface sialic acid enhanced Arachis hypogaea (PNA), Helix pomatia (HPA) and Phaseolus vulgaris-L (L-PHA) lectin binding reactivity to cell surface of lymphoma cells suggesting that neuraminidase removes cell surface sialic acid. In cell adhesion and invasion assays treatment with neuraminidase markedly enhanced cell adhesion to galectin-1 and decreased cell invasive capacity through galectin-1. α2,6-linked sialic acid may be involved in masking the effect of the interaction between galectin-1 and cell surface glycans. H-ALCL cells expressed the β-galactoside-α2,6-sialyltransferase ST6Gal1. On resialylation assay by recombinant ST6Gal1 with CMP-Neu5Ac, α2,6-resialylation of L-PHA reactive oligosaccharide by ST6Gal1 resulted in inhibition of H-ALCL cell adhesion to galectin-1 compared to the desialylated H-ALCL cells. On knockdown experiments, knockdown of ST6Gal1 dramatically enhanced cell adhesion to galectin-1. N-glycosylation inhibitor swainsonine treatment resulted in enhancement of cell adhesion to galectin-1. In glycomic analysis using the lectin blocking assay treatment with PNA, Artocarpus integrifolia (Jacalin), Glycine max (SBA), Helix pomatia (HPA), Vicia villosa (VVA), Ulex europaeus (UEA-1), Triticum vulgaris (WGA), Canavalia ensiformis (ConA), Phaseolus vulgaris-L (L-PHA), Phaseolus vulgaris-E4 (E-PHA), Datura stramonium (DSA) lectins resulted in modulation of lymphoma cell to galectin-1 suggesting that several types of glycans may regulate cell adhesion to galectin-1 by

  20. Galectin-1-mediated cell adhesion, invasion and cell death in human anaplastic large cell lymphoma: regulatory roles of cell surface glycans.

    PubMed

    Suzuki, Osamu; Abe, Masafumi

    2014-05-01

    Galectin-1 is known to be one of the extracellular matrix proteins. To elucidate the biological roles of galectin-1 in cell adhesion and invasion of human anaplastic large cell lymphoma, we performed cell adhesion and invasion assays using the anaplastic large cell lymphoma cell line H-ALCL, which was previously established in our laboratory. From the cell surface lectin array, treatment with neuraminidase from Arthrobacter ureafaciens which cleaves all linkage types of cell surface sialic acid enhanced Arachis hypogaea (PNA), Helix pomatia (HPA) and Phaseolus vulgaris-L (L-PHA) lectin binding reactivity to cell surface of lymphoma cells suggesting that neuraminidase removes cell surface sialic acid. In cell adhesion and invasion assays treatment with neuraminidase markedly enhanced cell adhesion to galectin-1 and decreased cell invasive capacity through galectin-1. α2,6-linked sialic acid may be involved in masking the effect of the interaction between galectin-1 and cell surface glycans. H-ALCL cells expressed the β-galactoside-α2,6-sialyltransferase ST6Gal1. On resialylation assay by recombinant ST6Gal1 with CMP-Neu5Ac, α2,6-resialylation of L-PHA reactive oligosaccharide by ST6Gal1 resulted in inhibition of H-ALCL cell adhesion to galectin-1 compared to the desialylated H-ALCL cells. On knockdown experiments, knockdown of ST6Gal1 dramatically enhanced cell adhesion to galectin-1. N-glycosylation inhibitor swainsonine treatment resulted in enhancement of cell adhesion to galectin-1. In glycomic analysis using the lectin blocking assay treatment with PNA, Artocarpus integrifolia (Jacalin), Glycine max (SBA), Helix pomatia (HPA), Vicia villosa (VVA), Ulex europaeus (UEA-1), Triticum vulgaris (WGA), Canavalia ensiformis (ConA), Phaseolus vulgaris-L (L-PHA), Phaseolus vulgaris-E4 (E-PHA), Datura stramonium (DSA) lectins resulted in modulation of lymphoma cell to galectin-1 suggesting that several types of glycans may regulate cell adhesion to galectin-1 by

  1. 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.

  2. Nanofiber-modified surface directed cell migration and orientation in microsystem

    PubMed Central

    Zhang, Xu; Gao, Xinghua; Jiang, Lei; Zhang, Xulang; Qin, Jianhua

    2011-01-01

    Cell-microscale pattern surface interactions are crucial to understand many fundamental biological questions and develop regenerative medicine and tissue engineering approaches. In this work, we demonstrated a simple method to pattern PDMS surface by sacrificing poly vinyl pyrrolidone (PVP) electrospinning nanofibers and investigated the growth profile of cells on the modified patterned surfaces using stroma cells. The stromal cells were observed to exhibit good viability on this modified surface and the patterned surface with alignment nanofibers could promote cell migration. Furthermore, the modified PDMS surface was integrated with microfluidic channels to create the microscale spatial factor and was used to explore the cell migration and orientation under this microsystem. Both spatial factor and patterned surfaces were found to contribute to the complex cell orientation under the combined dual effects. This established method is simple, fast, and easy for use, demonstrating the potential of this microsystem for applications in addressing biological questions in complex environment. PMID:22662030

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

    PubMed Central

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

    2015-01-01

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

  4. A yeast surface display system for the discovery of ligands that trigger cell activation.

    PubMed

    Cho, B K; Kieke, M C; Boder, E T; Wittrup, K D; Kranz, D M

    1998-11-01

    Opposing cells often communicate signalling events using multivalent interactions between receptors present on their cell surface. For example, T cells are typically activated when the T cell receptor (TCR) and its associated costimulatory molecules are multivalently engaged by the appropriate ligands present on an antigen presenting cell. In this report, yeast expressing high cell-surface levels of a TCR ligand (a recombinant antibody to the TCR Vbeta domain) were shown to act as 'pseudo' antigen presenting cells and induce T cell activation as monitored by increased levels of CD25 and CD69 and by downregulation of cell surface TCR. Similar levels of T cell activation could occur even when a 30-fold excess of irrelevant yeast was present, suggesting that such a yeast display system, by virtue of its ability to present ligands multivalently, may be used in highly sensitive procedures to identify novel polypeptides that interact multivalently with cell surface receptors and thereby trigger specific cellular responses.

  5. Five novel cell surface antigens of CNS neoplasms.

    PubMed

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

    1989-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  7. Tracking global patterns of N-linked glycosylation site variation in highly variable viral glycoproteins: HIV, SIV, and HCV envelopes and influenza hemagglutinin.

    PubMed

    Zhang, Ming; Gaschen, Brian; Blay, Wendy; Foley, Brian; Haigwood, Nancy; Kuiken, Carla; Korber, Bette

    2004-12-01

    Human and simian immunodeficiency viruses (HIV and SIV), influenza virus, and hepatitis C virus (HCV) have heavily glycosylated, highly variable surface proteins. Here we explore N-linked glycosylation site (sequon) variation at the population level in these viruses, using a new Web-based program developed to facilitate the sequon tracking and to define patterns (www.hiv.lanl.gov). This tool allowed rapid visualization of the two distinctive patterns of sequon variation found in HIV-1, HIV-2, and SIV CPZ. The first pattern (fixed) describes readily aligned sites that are either simply present or absent. These sites tend to be occupied by high-mannose glycans. The second pattern (shifting) refers to sites embedded in regions of extreme local length variation and is characterized by shifts in terms of the relative position and local density of sequons; these sites tend to be populated by complex carbohydrates. HIV, with its extreme variation in number and precise location of sequons, does not have a net increase in the number of sites over time at the population level. Primate lentiviral lineages have host species-dependent levels of sequon shifting, with HIV-1 in humans the most extreme. HCV E1 and E2 proteins, despite evolving extremely rapidly through point mutation, show limited sequon variation, although two shifting sites were identified. Human influenza A hemagglutinin H3 HA1 is accumulating sequons over time, but this trend is not evident in any other avian or human influenza A serotypes.

  8. Evolutionary Forces Shaping the Golgi Glycosylation Machinery: Why Cell Surface Glycans Are Universal to Living Cells

    PubMed Central

    Varki, Ajit

    2011-01-01

    Despite more than 3 billion years since the origin of life on earth, the powerful forces of biological evolution seem to have failed to generate any living cell that is devoid of a dense and complex array of cell surface glycans. Thus, cell surface glycans seem to be as essential for life as having a DNA genetic code, diverse RNAs, structural/functional proteins, lipid-based membranes, and metabolites that mediate energy flux and signaling. The likely reasons for this apparently universal law of biology are considered here, and include the fact that glycans have the greatest potential for generating diversity, and thus evading recognition by pathogens. This may also explain why in striking contrast to the genetic code, glycans show widely divergent patterns between taxa. On the other hand, glycans have also been coopted for myriad intrinsic functions, which can vary in their importance for organismal survival. In keeping with these considerations, a significant percentage of the genes in the typical genome are dedicated to the generation and/or turnover of glycans. Among eukaryotes, the Golgi is the subcellular organelle that serves to generate much of the diversity of cell surface glycans, carrying out various glycan modifications of glycoconjugates that transit through the Golgi, en route to the cell surface or extracellular destinations. Here I present an overview of general considerations regarding the selective forces shaping evolution of the Golgi glycosylation machinery, and then briefly discuss the common types of variations seen in each major class of glycans, finally focusing on sialic acids as an extreme example of evolutionary glycan diversity generated by the Golgi. Future studies need to address both the phylogenetic diversity the Golgi and the molecular mechanisms for its rapid responses to intrinsic and environmental stimuli. PMID:21525513

  9. 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.

  10. 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

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

    NASA Astrophysics Data System (ADS)

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

    2002-12-01

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

  12. Cell surface sialomucin and resistance to natural cell-mediated cytotoxicity of rat mammary tumor ascites cells.

    PubMed

    Sherblom, A P; Moody, C E

    1986-09-01

    MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma both contain sialomucin as a major cell surface component and are resistant to cytolysis by normal rat spleen lymphocytes [3 +/- 2% (SD) and 0 +/- 1%, respectively]. Susceptibility to lysis did not increase following treatment of cells with neuraminidase, fucosidase, or alpha- or beta-galactosidase. Treatment with trypsin significantly increased the susceptibility of MAT-B1 (14 +/- 3%) but not MAT-C1 (5 +/- 2%). Following 1 month in culture, the sialomucin content of MAT-B1 cells dropped from 30% to 8% (determined by glucosamine labeling) and natural cell-mediated cytolysis increased to 16 +/- 4%, whereas the sialomucin content and susceptibility of MAT-C1 cells did not change. The results indicate that the relatively minor changes associated with removal of cell surface sialic acid or fucose residues do not result in increased susceptibility of the ascites cells to cytolysis. However, susceptibility of MAT-B1 cells to lysis by normal rat spleen lymphocytes was inversely correlated with the amount of major glycoprotein (r = -0.96).

  13. 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

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

    PubMed

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

    1997-10-01

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

  15. Isolation of N-linked glycopeptides by hydrazine-functionalized magnetic particles.

    PubMed

    Sun, Shisheng; Yang, Ganglong; Wang, Ting; Wang, Qinzhe; Chen, Chao; Li, Zheng

    2010-04-01

    We introduce a novel combination of magnetic particles with hydrazine chemistry, dubbed as hydrazine-functionalized magnetic particles (HFMP) for isolation of glycopeptides. Four methods have been developed and compared for the production of HFMP by hydrazine modification of the surface of the carboxyl and epoxy-silanized magnetic particles, respectively. The evaluation of the capability and specificity of HFMP as well as the optimization of the coupling condition for capturing of glycoproteins were systematically investigated. The results showed that HFMP prepared by adipic dihydrazide functionalization from carboxyl-silanized magnetic particles (HFCA) displayed the maximum capture capacity and isolated efficiency for glycoprotein. When measured with glycoproteins, the capacity of the HFCA (1 g) for coupling bovine fetuin was 130 +/- 5.3 mg. The capability of this method was also confirmed by successful isolation of all formerly glycosylated peptides from standard glycoproteins and identification of their glycosylation sites, which demonstrated the feasibility of the HFCA as an alternative solid support for isolation of glycoproteins/glycopeptides. PMID:20169334

  16. Proteomic profiling of N-linked glycoproteins identifies ConA-binding procathepsin D as a novel serum biomarker for hepatocellular carcinoma.

    PubMed

    Qi, Yi-Jun; Ward, Douglas G; Pang, Chun; Wang, Qi-Ming; Wei, Wenbin; Ma, Jin; Zhang, Juan; Lou, Qiang; Shimwell, Neil J; Martin, Ashley; Wong, Nathalie; Chao, Wei-Xia; Wang, Ming; Ma, Yuan-Fang; Johnson, Philip J

    2014-02-01

    The aim of this study was to identify novel biomarkers for the diagnosis of, and potential therapeutic targets for, hepatocellular carcinoma (HCC). Multilectin affinity chromatography was used to enrich N-linked glycoproteins from nontumorous liver and HCC tissues followed by 2DE and protein identification by MS. Twenty-eight differentially expressed proteins were identified. Western blotting validated consistently lower concentrations of human liver carboxylesterase 1 and haptoglobin, and higher concentration of procathepsin D (pCD) in HCC tissues. Knockdown of cathepsin D (CD) expression mediated by siRNA significantly inhibited the in vitro invasion of two HCC cell lines, SNU449 and SNU473, which normally secrete high-levels of CD. Prefractionation using individual lectins demonstrated an elevation in ConA-binding glycoforms of proCD and CD in HCC tissues. In the serum of HCC patients, "ConA-binding proCD" (ConA-pCD) is significantly increased in concentration and this increase is comprised of several distinct upregulated acidic isoforms (pI 4.5-5.5). Receiver operating characteristic analysis showed that the sensitivity and specificity of serum ConA-pCD for HCC diagnosis were 85% and 80%, respectively. This is the first report that serum ConA-pCD is increased significantly in HCC and is potentially useful as a serological biomarker for diagnosis of HCC.

  17. Enhanced mechanical properties and in vitro cell response of surface mechanical attrition treated pure titanium.

    PubMed

    Zhao, Changli; Han, Pei; Ji, Weiping; Zhang, Xiaonong

    2012-08-01

    Surface mechanical attrition treatment (SMAT) was used to fabricate nanocrystalline surface layers on the commercial purity titanium. X-ray diffraction and transmission electron microscopy results indicate that the top layer contained nanograins. Enhanced strength and microhardness were achieved due to the surface nanostructure. Cell culture tests have shown a greater adhered cell density and more extensively spreading morphologies of Saos-2 cells on the SMAT substrates compared to those on the as-received Ti counterparts. Enhanced cell viability and cell cycle were also achieved on the SMAT Ti substrates. These could be attributed to the nanostructure grains with the increased surface hydrophilicity and roughness on the SMAT Ti.

  18. [Principles of treatment in ocular burns regarding the ocular surface and limbal stem cells].

    PubMed

    Potop, V; Dumitrache, Marieta

    2005-01-01

    The term ocular surface emphasizes the functional interdependence of the nonkeratinizing epithelium of cornea and conjunctiva. The limbal stem cells are responsible for replacement of corneal epithelium following ocular surface injuries. Over the past decades important advances in the management of chemical injury have occurred based on the application of theories on ocular surface and limbal stem cells. PMID:16245740

  19. 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.

  20. 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.

  1. Low Abundant N-linked Glycosylation in Hen Egg White Lysozyme Is Localized at Nonconsensus Sites.

    PubMed

    Asperger, Arndt; Marx, Kristina; Albers, Christian; Molin, Laura; Pinato, Odra

    2015-06-01

    Although wild-type hen egg white lysozyme (HEL) is lacking the consensus sequence motif NX(S/T), in 1995 Trudel et al. (Biochem. Cell Biol. 1995, 73, 307-309) proposed the existence of a low abundant N-glycosylated form of HEL; however, the identity of active glycosylation sites in HEL remained a matter of speculation. For the first time since Trudel's initial work, we report here a comprehensive characterization by means of mass spectrometry of N-glycosylation in wild-type HEL. Our analytical approach comprised ZIC-HILIC enrichment of N-glycopeptides from HEL trypsin digest, deglycosylation by (18)O/PNGase F as well as by various endoglycosidases, and LC-MS/MS analysis of both intact and deglycosylated N-glycopeptides engaging multiple techniques of ionization and fragmentation. A novel data interpretation workflow based on MS/MS spectra classification and glycan database searching enabled the straightforward identification of the asparagine-rich N-glycopeptide [34-45] FESNFNTQATNR and allowed for compositional profiling of its modifying N-glycans. The overall heterogeneity profile of N-glycans in HEL comprised at least 26 different compositions. Results obtained from deglycosylation experiments provided clear evidence of asparagine residues N44 and N39 representing active glycosylation sites in HEL. Both of these sites do not fall into any known N-glycosylation-specific sequence motif but are localized in rarely observed nonconsensus sequons (NXN, NXQ).

  2. Low Abundant N-linked Glycosylation in Hen Egg White Lysozyme Is Localized at Nonconsensus Sites.

    PubMed

    Asperger, Arndt; Marx, Kristina; Albers, Christian; Molin, Laura; Pinato, Odra

    2015-06-01

    Although wild-type hen egg white lysozyme (HEL) is lacking the consensus sequence motif NX(S/T), in 1995 Trudel et al. (Biochem. Cell Biol. 1995, 73, 307-309) proposed the existence of a low abundant N-glycosylated form of HEL; however, the identity of active glycosylation sites in HEL remained a matter of speculation. For the first time since Trudel's initial work, we report here a comprehensive characterization by means of mass spectrometry of N-glycosylation in wild-type HEL. Our analytical approach comprised ZIC-HILIC enrichment of N-glycopeptides from HEL trypsin digest, deglycosylation by (18)O/PNGase F as well as by various endoglycosidases, and LC-MS/MS analysis of both intact and deglycosylated N-glycopeptides engaging multiple techniques of ionization and fragmentation. A novel data interpretation workflow based on MS/MS spectra classification and glycan database searching enabled the straightforward identification of the asparagine-rich N-glycopeptide [34-45] FESNFNTQATNR and allowed for compositional profiling of its modifying N-glycans. The overall heterogeneity profile of N-glycans in HEL comprised at least 26 different compositions. Results obtained from deglycosylation experiments provided clear evidence of asparagine residues N44 and N39 representing active glycosylation sites in HEL. Both of these sites do not fall into any known N-glycosylation-specific sequence motif but are localized in rarely observed nonconsensus sequons (NXN, NXQ). PMID:25964011

  3. Identification of N-linked carbohydrates from severe acute respiratory syndrome (SARS) spike glycoprotein

    PubMed Central

    Ritchie, Gayle; Harvey, David J.; Feldmann, Friederike; Stroeher, Ute; Feldmann, Heinz; Royle, Louise; Dwek, Raymond A.; Rudd, Pauline M.

    2012-01-01

    N-glycans were released from the SARS coronavirus (SARS-CoV) spike glycoprotein produced in Vero E6 cells and their structures were determined by a combination of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, negative ion electrospray collision-induced dissociation time-of-flight mass spectrometry and normal-phase high-performance liquid chromatography with exoglycosidase digestion. Major glycans were high-mannose (Man5–9GlcNAc2), hybrid and bi-, tri- and tetra-antennary complex with and without bisecting GlcNAc and core fucose. Complex glycans with fewer than the full complement of galactose residues were present and sialylation was negligible. Treatment with the glucosidase inhibitor N-butyl-deoxynojirimycin (NB-DNJ) inhibited N-glycan processing as evidenced by the appearance of glycans of composition Glc3Man7–9GlcNAc2. However, some complex glycans remained suggesting the presence of an α-endomannosidase. Our data in tissue culture indicate that inhibition of N-glycan processing may be considered as a therapeutic strategy against SARS CoV infections. PMID:20129637

  4. 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.

  5. 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.

  6. 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

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

    PubMed

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

    2015-01-01

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

  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. 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.

  10. 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.

  11. 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

  12. Structure-based comparative analysis and prediction of N-linked glycosylation sites in evolutionarily distant eukaryotes.

    PubMed

    Lam, Phuc Vinh Nguyen; Goldman, Radoslav; Karagiannis, Konstantinos; Narsule, Tejas; Simonyan, Vahan; Soika, Valerii; Mazumder, Raja

    2013-04-01

    The asparagine-X-serine/threonine (NXS/T) motif, where X is any amino acid except proline, is the consensus motif for N-linked glycosylation. Significant numbers of high-resolution crystal structures of glycosylated proteins allow us to carry out structural analysis of the N-linked glycosylation sites (NGS). Our analysis shows that there is enough structural information from diverse glycoproteins to allow the development of rules which can be used to predict NGS. A Python-based tool was developed to investigate asparagines implicated in N-glycosylation in five species: Homo sapiens, Mus musculus, Drosophila melanogaster, Arabidopsis thaliana and Saccharomyces cerevisiae. Our analysis shows that 78% of all asparagines of NXS/T motif involved in N-glycosylation are localized in the loop/turn conformation in the human proteome. Similar distribution was revealed for all the other species examined. Comparative analysis of the occurrence of NXS/T motifs not known to be glycosylated and their reverse sequence (S/TXN) shows a similar distribution across the secondary structural elements, indicating that the NXS/T motif in itself is not biologically relevant. Based on our analysis, we have defined rules to determine NGS. Using machine learning methods based on these rules we can predict with 93% accuracy if a particular site will be glycosylated. If structural information is not available the tool uses structural prediction results resulting in 74% accuracy. The tool was used to identify glycosylation sites in 108 human proteins with structures and 2247 proteins without structures that have acquired NXS/T site/s due to non-synonymous variation. The tool, Structure Feature Analysis Tool (SFAT), is freely available to the public at http://hive.biochemistry.gwu.edu/tools/sfat. PMID:23459159

  13. Isolation of a mutant Arabidopsis plant that lacks N-acetyl glucosaminyl transferase I and is unable to synthesize Golgi-modified complex N-linked glycans.

    PubMed Central

    von Schaewen, A; Sturm, A; O'Neill, J; Chrispeels, M J

    1993-01-01

    The complex asparagine-linked glycans of plant glycoproteins, characterized by the presence of beta 1-->2 xylose and alpha 1-->3 fucose residues, are derived from typical mannose9(N-acetylglucosamine)2 (Man9GlcNAc2) N-linked glycans through the activity of a series of glycosidases and glycosyl transferases in the Golgi apparatus. By screening leaf extracts with an antiserum against complex glycans, we isolated a mutant of Arabidopsis thaliana that is blocked in the conversion of high-manne to complex glycans. In callus tissues derived from the mutant plants, all glycans bind to concanavalin A. These glycans can be released by treatment with endoglycosidase H, and the majority has the same size as Man5GlcNAc1 glycans. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, the mutant cells synthesize Man9GlcNAc2 and Man8GlcNAc2 glycans, suggesting that the biochemical lesion in the mutant is not in the biosynthesis of high-mannose glycans in the endoplasmic reticulum but in their modification in the Golgi. Direct enzyme assays of cell extracts show that the mutant cells lack N-acetyl glucosaminyl transferase I, the first enzyme in the pathway of complex glycan biosynthesis. The mutant plants are able to complete their development normally under several environmental conditions, suggesting that complex glycans are not essential for normal developmental processes. By crossing the complex-glycan-deficient strain of A. thaliana with a transgenic strain that expresses the glycoprotein phytohemagglutinin, we obtained a unique strain that synthesizes phytohemagglutinin with two high-mannose glycans, instead of one high-mannose and one complex glycan. PMID:8278542

  14. Isolation of a mutant Arabidopsis plant that lacks N-aetyl glucosaminyl transferase I and is unable to synthesize Golgi-modified complex N-linked glycans

    SciTech Connect

    Schaewen, A. von; O'Neill, J.; Chrispeels, M.J. ); Sturm, A. )

    1993-08-01

    The complex asparagine-linked glycans of plant glycoproteins, characterized by the presence of [beta]1[yields]2 xylose and [alpha]1[yields]3 fucose residues, are derived from typical mannose[sub 9](N-acetylglucosamine)[sub 2] (Man[sub 9]GlcNAc[sub 2]) N-linked glycans through the activity of a series of glycosidases and glycosyl transferases in the Golgi apparatus. By screening leaf extracts with an antiserum against complex glycans, we isolated a mutant of Arbidopsis thaliana that is blocked in the conversion of high-manne to complex glycans. In callus tissues derived from the mutant plants, all glycans bind to concanavalin A. These glycans can be released by treatment with endoglycosidase H, and the majority has the same size as Man[sub 5]GlcNAc[sub 1] glycans. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, the mutant cells synthesize Man[sub 9]GlcNAc[sub 2] and Man[sub 8]GlcNAc[sub 2] glycans, suggesting that the biochemical lesion in the mutant is not in the biosynthesis of high-mannose glycans in the endoplasmic reticulum but in their modification in the Golgi. Direct enzyme assays of cell extracts show that the mutant cells lack N-acetyl glucosaminyl transferase I, the first enzyme in the pathway of complex glycan biosynthesis. The mutant plants are able to complete their development normally under several environmental conditions, suggesting that complex glycans are not essential for normal developmental processes. By crossing the complex-glycan-deficient strain of A. thaliana with a transgenic strain that expresses the glycoprotein phytohemagglutinin, a unique strain was obtained that synthesizes phytohemagglutinin with two high-mannose glycans, instead of one high-mannose and one complex glycan. 42 refs., 8 figs., 1 tab.

  15. 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.

  16. Micropatterned Hydrogel Surface with High-Aspect-Ratio Features for Cell Guidance and Tissue Growth.

    PubMed

    Hu, Yuhang; You, Jin-Oh; Aizenberg, Joanna

    2016-08-31

    Surface topography has been introduced as a new tool to coordinate cell selection, growth, morphology, and differentiation. The materials explored so far for making such structural surfaces are mostly rigid and impermeable. Hydrogel, on the other hand, was proved a better synthetic media for cell culture because of its biocompatibility, softness, and high permeability. Herein, we fabricated a poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogel substrate with high-aspect-ratio surface microfeatures. Such structural surface could effectively guide the orientation and shape of human mesenchymal stem cells (HMSCs). Notably, on the flat hydrogel surface, cells rounded up, whereas on the microplate patterned hydrogel surface, cells elongated and aligned along the direction parallel to the plates. The microplates were 2 μm thick, 20 μm tall, and 10-50 μm wide. The interplate spacing was 5-15 μm, and the intercolumn spacing was 5 μm. The elongation of cell body was more pronounced on the patterns with narrower interplate spacing and wider plates. The cells behaved like soft solid. The competition between surface energy and elastic energy defined the shape of the cells on the structured surfaces. The soft permeable hydrogel scaffold with surface structures was also demonstrated as being viable for long-term cell culture, and could be used to generate interconnected tissues with finely tuned cell morphology and alignment across a few centimeter sizes. PMID:27089518

  17. Micropatterned Hydrogel Surface with High-Aspect-Ratio Features for Cell Guidance and Tissue Growth.

    PubMed

    Hu, Yuhang; You, Jin-Oh; Aizenberg, Joanna

    2016-08-31

    Surface topography has been introduced as a new tool to coordinate cell selection, growth, morphology, and differentiation. The materials explored so far for making such structural surfaces are mostly rigid and impermeable. Hydrogel, on the other hand, was proved a better synthetic media for cell culture because of its biocompatibility, softness, and high permeability. Herein, we fabricated a poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogel substrate with high-aspect-ratio surface microfeatures. Such structural surface could effectively guide the orientation and shape of human mesenchymal stem cells (HMSCs). Notably, on the flat hydrogel surface, cells rounded up, whereas on the microplate patterned hydrogel surface, cells elongated and aligned along the direction parallel to the plates. The microplates were 2 μm thick, 20 μm tall, and 10-50 μm wide. The interplate spacing was 5-15 μm, and the intercolumn spacing was 5 μm. The elongation of cell body was more pronounced on the patterns with narrower interplate spacing and wider plates. The cells behaved like soft solid. The competition between surface energy and elastic energy defined the shape of the cells on the structured surfaces. The soft permeable hydrogel scaffold with surface structures was also demonstrated as being viable for long-term cell culture, and could be used to generate interconnected tissues with finely tuned cell morphology and alignment across a few centimeter sizes.

  18. Nano-patterned SU-8 surface using nanosphere-lithography for enhanced neuronal cell growth

    NASA Astrophysics Data System (ADS)

    Kim, Eunhee; Yoo, Seung-Jun; Kim, Eunjung; Kwon, Tae-Hwan; Zhang, Li; Moon, Cheil; Choi, Hongsoo

    2016-04-01

    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.

  19. 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.

  20. 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.

  1. Cell Surface Human Airway Trypsin-Like Protease Is Lost During Squamous Cell Carcinogenesis.

    PubMed

    Duhaime, Michael J; Page, Khaliph O; Varela, Fausto A; Murray, Andrew S; Silverman, Michael E; Zoratti, Gina L; List, Karin

    2016-07-01

    Cancer progression is accompanied by increased levels of extracellular proteases that are capable of remodeling the extracellular matrix, as well as cleaving and activating growth factors and receptors that are involved in pro-cancerous signaling pathways. Several members of the type II transmembrane serine protease (TTSP) family have been shown to play critical roles in cancer progression, however, the expression or function of the TTSP Human Airway Trypsin-like protease (HAT) in carcinogenesis has not been examined. In the present study we aimed to determine the expression of HAT during squamous cell carcinogenesis. HAT transcript is present in several tissues containing stratified squamous epithelium and decreased expression is observed in carcinomas. We determined that HAT protein is consistently expressed on the cell surface in suprabasal/apical layers of squamous cells in healthy cervical and esophageal epithelia. To assess whether HAT protein is differentially expressed in normal tissue versus tissue in different stages of carcinogenesis, we performed a comprehensive immunohistochemical analysis of HAT protein expression levels and localization in arrays of paraffin embedded human cervical and esophageal carcinomas compared to the corresponding normal tissue. We found that HAT protein is expressed in the non-proliferating, differentiated cellular strata and is lost during the dedifferentiation of epithelial cells, a hallmark of squamous cell carcinogenesis. Thus, HAT expression may potentially be useful as a marker for clinical grading and assessment of patient prognosis in squamous cell carcinomas.

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

    PubMed

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

    2011-01-21

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

  3. Role of the Cell Surface of Methanosarcina mazei in Cell Aggregation †

    PubMed Central

    Robinson, Ralph W.; Aldrich, H. C.; Hurst, Steven F.; Bleiweis, A. S.

    1985-01-01

    Colonial aggregates of Methanosarcina (= Methanococcus) mazei were examined with scanning and transmission electron microscopy. Cells are irregular and grouped into multicellular sarcinal colonies, which may disaggregate in older cultures. The protoplast is bounded by a typical trilaminar plasma membrane, outside of which is a matrix of loose fibrils. The presence and compactness of matrix material are responsible for the close packing of cells, and colony disaggregation seems to be the result of matrix shedding and degradation. The cell envelope contains complex hetero polysaccharides of N-acetylgalactosamine and galacturonic and glucuronic acids. Polymers extruded by M. mazei are likely quite adhesive in nature, accounting for its strong adherence to surfaces and hardiness compared with many other methanogens. Images PMID:16346718

  4. Synthetic surfaces as artificial antigen presenting cells in the study of T cell receptor triggering and immunological synapse formation.

    PubMed

    Irvine, Darrell J; Doh, Junsang

    2007-08-01

    T cell activation occurs when T cell receptors engage peptide-major histocompatibility complex (pMHC) molecules displayed on the surface of antigen presenting cells (APCs). Clustering of TCRs and other receptors in physical patterns at the T-APC interface forms a structure known as an immunological synapse (IS). Studies of the IS are challenging due to the cell-cell contact context of the governing interactions. Model surfaces as synthetic APCs have thus been developed, where the type, quantity, and physical arrangement of ligands displayed to T cells are precisely controlled. These model systems have provided important insights into the structure and function of the IS. PMID:17398113

  5. Global transcriptomic analysis of model human cell lines exposed to surface-modified gold nanoparticles: the effect of surface chemistry.

    PubMed

    Grzincic, E M; Yang, J A; Drnevich, J; Falagan-Lotsch, P; Murphy, C J

    2015-01-28

    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.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

    Arnal-Estapé, Anna; Nguyen, Don X.

    2015-01-01

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

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

    PubMed

    Cuthbertson, Leslie; Kos, Veronica; Whitfield, Chris

    2010-09-01

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

  9. 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.

  10. Influence of anode surface chemistry on microbial fuel cell operation.

    PubMed

    Santoro, Carlo; Babanova, Sofia; Artyushkova, Kateryna; Cornejo, Jose A; Ista, Linnea; Bretschger, Orianna; Marsili, Enrico; Atanassov, Plamen; Schuler, Andrew J

    2015-12-01

    Self-assembled monolayers (SAMs) modified gold anodes are used in single chamber microbial fuel cells for organic removal and electricity generation. Hydrophilic (N(CH3)3(+), OH, COOH) and hydrophobic (CH3) SAMs are examined for their effect on bacterial attachment, current and power output. The different substratum chemistry affects the community composition of the electrochemically active biofilm formed and thus the current and power output. Of the four SAM-modified anodes tested, N(CH3)3(+) results in the shortest start up time (15 days), highest current achieved (225 μA cm(-2)) and highest MFC power density (40 μW cm(-2)), followed by COOH (150 μA cm(-2) and 37 μW cm(-2)) and OH (83 μA cm(-2) and 27 μW cm(-2)) SAMs. Hydrophobic SAM decreases electrochemically active bacteria attachment and anode performance in comparison to hydrophilic SAMs (CH3 modified anodes 7 μA cm(-2) anodic current and 1.2 μW cm(-2) MFC's power density). A consortium of Clostridia and δ-Proteobacteria is found on all the anode surfaces, suggesting a synergistic cooperation under anodic conditions.

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

    PubMed

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

    2015-05-01

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

  12. ABC Transporters Involved in Export of Cell Surface Glycoconjugates

    PubMed Central

    Cuthbertson, Leslie; Kos, Veronica; Whitfield, Chris

    2010-01-01

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

  13. Activity-dependent mobilization of the adhesion molecule polysialic NCAM to the cell surface of neurons and endocrine cells.

    PubMed Central

    Kiss, J Z; Wang, C; Olive, S; Rougon, G; Lang, J; Baetens, D; Harry, D; Pralong, W F

    1994-01-01

    The alpha-2,8-linked sialic acid polymer (PSA) on the neural cell adhesion molecule (NCAM) is an important regulator of cell surface interactions. We have examined the translocation of PSA-NCAM to the surface of cultured cortical neurons and insulin secreting beta cells under different conditions of cell activity. Endoneuraminidase N, an enzyme that specifically cleaves PSA chains, was used to remove pre-existing PSA from the plasma membrane and the re-expression of the molecule was monitored by immunocytochemistry. Punctate PSA immunostaining was restored on the surface of 68% of neurons within 1 h. This recovery was almost completely prevented by tetrodotoxin, suggesting that spontaneous electrical activity is required. K+ depolarization (50 mM) allowed recovery of PSA surface staining in the presence of tetrodotoxin and this effect required the presence of extracellular Ca2+. Rapid redistribution of PSA-NCAM to the surface of beta cells was observed under conditions that stimulate insulin secretion. Ca2+ channel inhibition decreased both PSA-NCAM expression and insulin secretion to control, non-stimulated levels. Finally, subcellular fractionation of an insulin-secreting cell line showed that the secretory vesicle fraction is highly enriched in PSA-NCAM. These results suggest that PSA-NCAM can be translocated to the cell surface via regulated exocytosis. Taken together, our results provide unprecedented evidence linking cell activity and PSA-NCAM expression, and suggest a mechanism for rapid modulation of cell surface interactions. Images PMID:7957094

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

    PubMed

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

    2010-01-01

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

  15. Controlling cell adhesion via replication of laser micro/nano-textured surfaces on polymers.

    PubMed

    Koufaki, Niki; Ranella, Anthi; Aifantis, Katerina E; Barberoglou, Marios; Psycharakis, Stylianos; Fotakis, Costas; Stratakis, Emmanuel

    2011-12-01

    The aim of this study is to investigate cell adhesion and viability on highly rough polymeric surfaces with gradient roughness ratios and wettabilities prepared by microreplication of laser micro/nano-textured Si surfaces. Negative replicas on polydimethylsiloxane as well as positive ones on a photocurable (organically modified ceramic) and a biodegradable (poly(lactide-co-glycolide)) polymer have been successfully reproduced. The final culture substrates comprised from forests of micron-sized conical spikes exhibiting a range of roughness ratios and wettabilities, was achieved by changing the laser fluence used to fabricate the original template surfaces. Cell culture experiments were performed with the fibroblast NIH/3T3 and PC12 neuronal cell lines in order to investigate how these surfaces are capable of modulating different types of cellular responses including, viability, adhesion and morphology. The results showed a preferential adhesion of both cell types on the microstructured surfaces compared to the unstructured ones. In particular, the fibroblast NIH/3T3 cells show optimal adhesion for small roughness ratios, independent of the surface wettability and polymer type, indicating a non-monotonic dependence of cell adhesion on surface energy. In contrast, the PC12 cells were observed to adhere well to the patterned surfaces independent of the roughness ratio and wettability. These experimental findings are correlated with micromechanical measurements performed on the unstructured and replicated surfaces and discussed on the basis of previous observations describing the relation of cell response to surface energy and rigidity.

  16. Morphological investigations of cells that adhered to the irregular patterned polydimethylsiloxane (PDMS) surface without reagents.

    PubMed

    Chung, Sung Hee; Min, Junhong

    2009-07-01

    Polydimethylsiloxane (PDMS) surface consisting irregular pattern was investigated to develop cell-based biochip using PDMS. PDMS surface was modified with nano- and micro-combined patterns using surface deformation technology. Hydrophobicity of nano-patterned PDMS surface was sustained. Nevertheless it has irregular patterns consisting of micro- and nano-patterns. According to atomic force microscopy (AFM), scanning electron microscopy (SEM) and confocal microscopy results by immunostaining method, human mammary epithelial cells (HMEC) adhered well on irregularly patterned surface without any reagents such as gelatin and collagen, compared to commercial culture dish. It implies PDMS material can be utilized as template for cell-based biochip without any reagents. PMID:19427124

  17. SURFACE SPECIALIZATIONS OF FUNDULUS CELLS AND THEIR RELATION TO CELL MOVEMENTS DURING GASTRULATION

    PubMed Central

    Trinkaus, J. P.; Lentz, Thomas L.

    1967-01-01

    Cell movements in Fundulus blastoderms during gastrulation were studied utilizing time-lapse cinemicrography and electron microscopy. Time-lapse films reveal that cells of the enveloping layer undulate and sometimes separate briefly but remain together in a cohesive layer. During epiboly, the marginal enveloping layer cells move over the periblast as it expands over the yolk sphere. Movement occurs as a result of ruffled membrane activity of the free borders of the marginal cells. Deep blastomeres become increasingly active during blastula and gastrula stages. Lobopodia project from the blastomeres in blastulae and adhere to other cells in gastrulae, giving the cells traction for movement. Contact specializations are formed by the lateral adjacent plasma membranes of enveloping layer cells. An apical junction is characterized by an intercellular gap of 60–75 A. Below this contact, the plasma membranes are separated by 120 A or more. In mid-gastrulae, cytoplasmic fibrils occur adjacent to some apical junctions, and small desmosomes appear below the apical junction. Septate desmosomes also appear at this time. A junction with an intercellular gap of 60 A occurs between marginal enveloping layer cells and periblast. Contacts between deep blastomeres become numerous in gastrulae and consist of contacts at the crests of surface undulations, short areas of contact in which the plasma membranes are 60 or 120 A apart, and long regions characterized by a 200-A intercellular gap. Lobopodia contact other blastomeres only in gastrulae. These junctions contain a 200-A intercellular space. Some deep blastomeres are in contact with the tips of periblast microvilli. The mechanism of epiboly in Fundulus is discussed and reevaluated in terms of these observations. The enveloping layer is adherent to the margin of the periblast and moves over it as a coherent cellular sheet. Periblast epiboly involves a controlled flow of cytoplasm from the thicker periblast into the thinner yolk

  18. Acid base properties of cyanobacterial surfaces I: Influences of growth phase and nitrogen metabolism on cell surface reactivity

    NASA Astrophysics Data System (ADS)

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

    2008-03-01

    Significant efforts have been made to elucidate the chemical properties of bacterial surfaces for the purposes of refining surface complexation models that can account for their metal sorptive behavior under diverse conditions. However, the influence of culturing conditions on surface chemical parameters that are modeled from the potentiometric titration of bacterial surfaces has received little regard. While culture age and metabolic pathway have been considered as factors potentially influencing cell surface reactivity, statistical treatments have been incomplete and variability has remained unconfirmed. In this study, we employ potentiometric titrations to evaluate variations in bacterial surface ligand distributions using live cells of the sheathless cyanobacterium Anabaena sp. strain PCC 7120, grown under a variety of batch culture conditions. We evaluate the ability for a single set of modeled parameters, describing acid-base surface properties averaged over all culture conditions tested, to accurately account for the ligand distributions modeled for each individual culture condition. In addition to considering growth phase, we assess the role of the various assimilatory nitrogen metabolisms available to this organism as potential determinants of surface reactivity. We observe statistically significant variability in site distribution between the majority of conditions assessed. By employing post hoc Tukey-Kramer analysis for all possible pair-wise condition comparisons, we conclude that the average parameters are inadequate for the accurate chemical description of this cyanobacterial surface. It was determined that for this Gram-negative bacterium in batch culture, ligand distributions were influenced to a greater extent by nitrogen assimilation pathway than by growth phase.

  19. Simulation of Red Blood Cell Interaction with the Endothelial Cell Surface

    NASA Astrophysics Data System (ADS)

    Aidun, Cyrus; Jia, Xinli; Morris, Jeff; McLaughlin, John

    2005-11-01

    It is hypothesized that the stress field on the EC surface felt through hydrodynamic interaction at the extracellular layer, known as the glycocalyx, is the pathophysiological link between the hemodynamics and the cell function. Simulations revealing the general stress distribution on the EC surface, and in particular the mechanical interactions of red blood cells (RBC's) with the EC's will be presented. The current focus is to investigate the drag force and the bending moment on the core proteins in the EC glycocalyx. The glycocalyx has been modeled as a quasiperiodic array of cylinders (Weinbaum et al., PNAS 100, 1988-7995, 2003). The height and diameter of the cylinders were assumed to be 150 nm and 6 nm, respectively, and the gap between cylinders was 8 nm. Weinbaum et al. computed the average velocity profile by treating the glycocalyx as a porous medium. The focus of the work to be presented is on the effects upon the EC by close encounters with RBC's over a long period of time. We will present results for the flow in and above a model glycocalyx caused by the motion of a nearby surface. The flow was computed using the lattice Boltzmann method (LBM). The results of the LBM for the mean flow and the bending moment and drag on a model protein fiber will be compared with the predictions obtained from the model of Weinbaum.

  20. Binding of Recombinant Feline Immunodeficiency Virus Surface Glycoprotein to Feline Cells: Role of CXCR4, Cell-Surface Heparans, and an Unidentified Non-CXCR4 Receptor

    PubMed Central

    de Parseval, Aymeric; Elder, John H.

    2001-01-01

    To address the role of CXCR4 in the cell-surface attachment of the feline immunodeficency virus (FIV), a soluble fusion protein, gp95-Fc, consisting of the surface glycoprotein (SU, gp95) of either a primary (PPR) or cell line-adapted (34TF10) FIV strain was fused in frame with the Fc domain of human immunoglobulin G1. The recombinant SU-immunoadhesins were used as probes to investigate the cellular binding of FIV SU. In agreement with the host cell range properties of both viruses, binding of 34TF10 gp95-Fc was observed for all cell lines tested, whereas PPR gp95-Fc bound only to primary feline T cells. 34TF10 gp95-Fc also bound to Jurkat and HeLa cells, consistent with the ability of FIV to use human CXCR4 as a fusion receptor. As expected, 34TF10 gp95-Fc binding to Jurkat cells was blocked by addition of stromal cell-derived factor 1α (SDF-1α), as was binding to the 3201 feline lymphoma cell line. However, SDF-1α, RANTES, macrophage inflammatory protein 1β, and heparin all failed to inhibit the binding of either gp95-Fc to primary T cells, suggesting that a non-CXCR4 receptor is involved in the binding of FIV SU. In this regard, an unidentified 40-kDa protein species from the surface of primary T cells but not Jurkat and 3201 cells specifically coprecipitated with both gp95-Fc. Yet another type of binding of 34TF10 gp95-Fc to adherent kidney cells was noted. SDF-1α failed to block the binding of 34TF10 gp95-Fc to either HeLa, Crandel feline leukemia, or G355-5 cells. However, binding was severely impaired in the presence of soluble heparin, as well as after enzymatic removal of surface heparans or on cells deficient in heparan expression. These overall findings suggest that in addition to CXCR4, a non-CXCR4 receptor and cell-surface heparans also play an important role in FIV gp95 cell surface interactions on specific target cells. PMID:11312323

  1. 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.

  2. Usp12 stabilizes the T-cell receptor complex at the cell surface during signaling

    PubMed Central

    Jahan, Akhee S.; Lestra, Maxime; Swee, Lee Kim; Fan, Ying; Lamers, Mart M.; Tafesse, Fikadu G.; Theile, Christopher S.; Spooner, Eric; Bruzzone, Roberto; Ploegh, Hidde L.; Sanyal, Sumana

    2016-01-01

    Posttranslational modifications are central to the spatial and temporal regulation of protein function. Among others, phosphorylation and ubiquitylation are known to regulate proximal T-cell receptor (TCR) signaling. Here we used a systematic and unbiased approach to uncover deubiquitylating enzymes (DUBs) that participate during TCR signaling in primary mouse T lymphocytes. Using a C-terminally modified vinyl methyl ester variant of ubiquitin (HA-Ub-VME), we captured DUBs that are differentially recruited to the cytosol on TCR activation. We identified ubiquitin-specific peptidase (Usp) 12 and Usp46, which had not been previously described in this pathway. Stimulation with anti-CD3 resulted in phosphorylation and time-dependent translocation of Usp12 from the nucleus to the cytosol. Usp12−/− Jurkat cells displayed defective NFκB, NFAT, and MAPK activities owing to attenuated surface expression of TCR, which were rescued on reconstitution of wild type Usp12. Proximity-based labeling with BirA-Usp12 revealed several TCR adaptor proteins acting as interactors in stimulated cells, of which LAT and Trat1 displayed reduced expression in Usp12−/− cells. We demonstrate that Usp12 deubiquitylates and prevents lysosomal degradation of LAT and Trat1 to maintain the proximal TCR complex for the duration of signaling. Our approach benefits from the use of activity-based probes in primary cells without any previous genome modification, and underscores the importance of ubiquitin-mediated regulation to refine signaling cascades. PMID:26811477

  3. An Update on Ocular Surface Epithelial Stem Cells: Cornea and Conjunctiva

    PubMed Central

    Ramos, Tiago; Scott, Deborah; Ahmad, Sajjad

    2015-01-01

    The human ocular surface (front surface of the eye) is formed by two different types of epithelia: the corneal epithelium centrally and the conjunctival epithelium that surrounds this. These two epithelia are maintained by different stem cell populations (limbal stem cells for the corneal epithelium and the conjunctival epithelial stem cells). In this review, we provide an update on our understanding of these epithelia and their stem cells systems, including embryology, new markers, and controversy around the location of these stem cells. We also provide an update on the translation of this understanding into clinical applications for the treatment of debilitating ocular surface diseases. PMID:26146504

  4. An Update on Ocular Surface Epithelial Stem Cells: Cornea and Conjunctiva.

    PubMed

    Ramos, Tiago; Scott, Deborah; Ahmad, Sajjad

    2015-01-01

    The human ocular surface (front surface of the eye) is formed by two different types of epithelia: the corneal epithelium centrally and the conjunctival epithelium that surrounds this. These two epithelia are maintained by different stem cell populations (limbal stem cells for the corneal epithelium and the conjunctival epithelial stem cells). In this review, we provide an update on our understanding of these epithelia and their stem cells systems, including embryology, new markers, and controversy around the location of these stem cells. We also provide an update on the translation of this understanding into clinical applications for the treatment of debilitating ocular surface diseases. PMID:26146504

  5. Particles induced surface nanoroughness of titanium surface and its influence on adhesion of osteoblast-like MG-63 cells

    NASA Astrophysics Data System (ADS)

    Solař, P.; Kylián, O.; Marek, A.; Vandrovcová, M.; Bačáková, L.; Hanuš, J.; Vyskočil, J.; Slavínská, D.; Biederman, H.

    2015-01-01

    Titanium is one of the most common materials employed for production of implants, which is due to its good biocompatibility. However, the colonization of titanium surface by osteoblast cells may be influenced by its roughness and therefore precise control of roughness of titanium surface as well as identification of its optimal value for growth of cells is of high importance. In this study the nanorough titanium surfaces were prepared on polished disks of TiAlV by two step method of deposition. In the first step TiAlV were coated by nanoparticles generated by gas aggregation sources. Such prepared films of nanoparticles were subsequently covered with a titanium overlayer. Different values of surface roughness in the range 1-100 nm were achieved by variation of the size and number of the nanoparticles. Such prepared surfaces were subsequently used for investigation of influence of roughness of titanium surfaces on the adhesion of human osteoblast-like MG-63 cells. It was found out that 7 days after seeding the highest number of adhering cells was observed for samples with root-mean-square roughness of 30 nm.

  6. Functionalized titanium oxide surfaces with phosphated carboxymethyl cellulose: characterization and bonelike cell behavior.

    PubMed

    Pasqui, Daniela; Rossi, Antonella; Di Cintio, Federica; Barbucci, Rolando

    2007-12-01

    The performance of dental or orthopedic implants is closely dependent on surface properties in terms of topography and chemistry. A phosphated carboxymethylcellulose containing one phosphate group for each disaccharide unit was synthesized and used to functionalize titanium oxide surfaces with the aim to improve osseointegration with the host tissue. The modified surfaces were chemically characterized by means of X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. The investigation of the surface topography was performed by atomic force microscopy measurements before and after the polysaccharide coating. In vitro biological tests using osteoblastlike cells demonstrated that functionalized TiO(2) surfaces modulated cell response, in terms of adhesion, proliferation,and morphology. Phosphated carboxymethylcellulose promoted better cell adhesion and significantly enhanced their proliferation. The morphology of cells was polygonal and more spread on this type of modified surface.These findings suggest that the presence of a phosphate polysaccharide coating promotes osteoblast growth on the surface potentially improving biomaterial osseointegration.

  7. Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

    SciTech Connect

    Li, Ying; Huang, Xiaohua; An, Yue; Ren, Feng; Yang, Zara Zhuyun; Zhu, Hongmei; Zhou, Lei; He, Xiaowen; Schachner, Melitta; Xiao, Zhicheng; Ma, Keli; Li, Yali

    2013-10-25

    Highlights: •Down-regulating FUT9 and ST3Gal4 expression blocks L1-induced neuronal differentiation of ESCs. •Up-regulating FUT9 and ST3Gal4 expression in L1-ESCs depends on the activation of PLCγ. •L1 promotes ESCs to differentiate into neuron through regulating cell surface glycosylation. -- Abstract: Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study, we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression.

  8. Hydrophobic fractal surface from glycerol tripalmitate and the effects on C6 glioma cell growth.

    PubMed

    Zhang, Shanshan; Chen, Xuerui; Yu, Jing; Hong, Biyuan; Lei, Qunfang; Fang, Wenjun

    2016-06-01

    To provide a biomimic environment for glial cell culture, glycerol tripalmitate (PPP) has been used as a raw material to prepare fractal surfaces with different degrees of hydrophobicity. The spontaneous formation of the hydrophobic fractal surfaces was monitored by differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The surface morphologies were observed by a scanning electron microscope (SEM), and then the fractal dimension (FD) values of the surfaces were determined with the box-counting method. C6 glioma cells were cultured and compared on different hydrophobic PPP surfaces and poly-L-lysine (PLL)-coated surface. The cell numbers as a function of incubation time on different surfaces during the cell proliferation process were measured, and the cell morphologies were observed under a fluorescence microscope. Influences of hydrophobic fractal surfaces on the cell number and morphology were analyzed. The experimental results show that the cell proliferation rates decrease while the cell morphology complexities increase with the growth of the fractal dimensions of the PPP surfaces. PMID:26970826

  9. Tracking traction force changes of single cells on the liquid crystal surface.

    PubMed

    Soon, Chin Fhong; Tee, Kian Sek; Youseffi, Mansour; Denyer, Morgan C T

    2015-03-01

    Cell migration is a key contributor to wound repair. This study presents findings indicating that the liquid crystal based cell traction force transducer (LCTFT) system can be used in conjunction with a bespoke cell traction force mapping (CTFM) software to monitor cell/surface traction forces from quiescent state in real time. In this study, time-lapse photo microscopy allowed cell induced deformations in liquid crystal coated substrates to be monitored and analyzed. The results indicated that the system could be used to monitor the generation of cell/surface forces in an initially quiescent cell, as it migrated over the culture substrate, via multiple points of contact between the cell and the surface. Future application of this system is the real-time assaying of the pharmacological effects of cytokines on the mechanics of cell migration. PMID:25808839

  10. Tracking Traction Force Changes of Single Cells on the Liquid Crystal Surface

    PubMed Central

    Soon, Chin Fhong; Tee, Kian Sek; Youseffi, Mansour; Denyer, Morgan C. T.

    2015-01-01

    Cell migration is a key contributor to wound repair. This study presents findings indicating that the liquid crystal based cell traction force transducer (LCTFT) system can be used in conjunction with a bespoke cell traction force mapping (CTFM) software to monitor cell/surface traction forces from quiescent state in real time. In this study, time-lapse photo microscopy allowed cell induced deformations in liquid crystal coated substrates to be monitored and analyzed. The results indicated that the system could be used to monitor the generation of cell/surface forces in an initially quiescent cell, as it migrated over the culture substrate, via multiple points of contact between the cell and the surface. Future application of this system is the real-time assaying of the pharmacological effects of cytokines on the mechanics of cell migration. PMID:25808839

  11. Clonal evolution of myeloma cells leads to quantitative changes in immunoglobulin secretion and surface antigen expression.

    PubMed Central

    Leibson, P J; Loken, M R; Panem, S; Schreiber, H

    1979-01-01

    We report that a cloned population of tumor cells can rapidly produce variants that differ in their quantitative expression of surface proteins and in their rate of immunoglobulin secretion. A fresh clonal isolate of S107 myeloma cells possessing large amounts of surface IgA was continuously passaged in vitro for 2 years. During this period, fluorescence-activated cell sorter analysis indicated the development of subpopulations possessing decreased amounts of surface IgA. Cells from these variant subpopulations were isolated by first using the cell sorter to enrich for cells with decreased amounts of surface IgA and then cloning the selected population in soft agar. The 50 sublines that were isolated showed heritable differences in their levels of surface IgA and H-2 antigens and in their rates of myeloma protein secretion. Sublines having either large amounts, intermediate amounts, or absence of surface IgA also had corresponding large amounts, intermediate amounts, or absence of myeloma protein secretion. In contrast, a decrease or loss of surface Ig did not correlate with a decrease or loss of viral envelope glycoprotein gp71 and H-2 antigens. The variants did not resemble the phenotypes of less-differentiated normal lymphocyte populations of the B-cell lineage. The isolation and characterization of these variants allows us to explore the mechanisms and pathways of tumor cell differentiation as well as to study the regulation and function of cell surface proteins. PMID:288078

  12. Characterization of the Cell Surface Properties of Drinking Water Pathogens by Microbial Adhesion to Hydrocarbon and Electrophoretic Mobility Measurements

    EPA Science Inventory

    The surface characteristics of microbial cells directly influence their mobility and behavior within aqueous environments. The cell surface hydrophobicity (CSH) and electrophoretic mobility (EPM) of microbial cells impact a number of interactions and processes including aggregati...

  13. Effects of Polymer Surfaces on Proliferation and Differentiation of Embryonic Stem Cells and Bone Marrow Stem Cells

    NASA Astrophysics Data System (ADS)

    Qin, Sisi; Liao, Wenbin; Ma, Yupo; Simon, Marcia; Rafailovich, Miriam; Stony Brook Medical Center Collaboration; Stony Brook Dental Schoo Collaboration

    2013-03-01

    Currently, proliferation and differentiation of stem cell is usually accomplished either in vivo, or on chemical coated tissue culture petri dish with the presence of feeder cells. Here we investigated whether they can be directly cultured on polymeric substrates, in the absence of additional factors. We found that mouse embryonic stem cells did not require gelatin and could remain in the undifferentiated state without feeder cells at least for four passages on partially sulfonated polystyrene. The modulii of cells was measured and found to be higher for cells plated directly on the polymer surface than for those on the same surface covered with gelatin and feeder cells. When plated with feeder cells, the modulii was not sensitive to gelatin. Whereas the differentiation properties of human bone marrow stem cells, which are not adherent, are less dependent on either chemical or mechanical properties of the substrate. However, they behave differently on different toughness hydrogels as oppose to on polymer coated thin films.

  14. Hsc70 regulates cell surface ASIC2 expression and vascular smooth muscle cell migration.

    PubMed

    Grifoni, Samira C; McKey, Susan E; Drummond, Heather A

    2008-05-01

    Recent studies suggest members of the degenerin (DEG)/epithelial Na(+) channel (ENaC)/acid-sensing ion channel (ASIC) protein family play an important role in vascular smooth muscle cell (VSMC) migration. In a previous investigation, we found suppression of a certain DEG/ENaC/ASIC member, ASIC2, increased VSMC chemotactic migration, raising the possibility that ASIC2 may play an inhibitory role. Because ASIC2 protein was retained in the cytoplasm, we reasoned increasing surface expression of ASIC2 might unmask the inhibitory role of ASIC2 in VSMC migration so we could test the hypothesis that ASIC2 inhibits VSMC migration. Therefore, we used the chemical chaperone glycerol to enhance ASIC2 expression. Glycerol 1) increased cytoplasm ASIC2 expression, 2) permitted detection of ASIC2 at the cell surface, and 3) inhibited platelet-derived growth factor (PDGF)-bb mediated VSMC migration. Furthermore, ASIC2 silencing completely abolished the inhibitory effect of glycerol on migration, suggesting upregulation of ASIC2 is responsible for glycerol-induced inhibition of VSMC migration. Because other investigators have shown that glycerol regulates ENaC/ASIC via interactions with a certain heat shock protein, heat shock protein 70 (Hsc70), we wanted to determine the importance of Hsc70 on ASIC2 expression in VSMCs. We found that Hsc70 silencing increases ASIC2 cell surface expression and inhibits VSMC migration, which is abolished by cosilencing ASIC2. These data demonstrate that Hsc70 inhibits ASIC2 expression, and, when the inhibitory effect of Hsc70 is removed, ASIC2 expression increases, resulting in reduced VSMC migration. Because VSMC migration contributes to vasculogenesis and remodeling following vascular injury, our findings raise the possibility that ASIC2-Hsc70 interactions may play a role in these processes. PMID:18310515

  15. Surface reactivity and cell responses to chrysotile asbestos nanofibers.

    PubMed

    Turci, Francesco; Colonna, Massimiliano; Tomatis, Maura; Mantegna, Stefano; Cravotto, Giancarlo; Gulino, Giulia; Aldieri, Elisabetta; Ghigo, Dario; Fubini, Bice

    2012-04-16

    High aspect-ratio nanomaterials (HARNs) have recently attracted great attention from nanotoxicologists because of their similarity to asbestos. However, the actual risk associated with the exposure to nanosized asbestos, which escapes most regulations worldwide, is still unknown. Nanometric fibers of chrysotile asbestos have been prepared from two natural sources to investigate whether nanosize may modulate asbestos toxicity and gain insight on the hazard posed by naturally occurring asbestos, which may be defined as HARNs because of their dimensions. Power ultrasound was used to obtain nanofibers from two different chrysotile specimens, one from the dismissed asbestos mine in Balangero (Italian Western Alps) and the other from a serpentine outcrop in the Italian Central Alps. Electron microscopy, X-ray diffraction, and fluorescence spectroscopy revealed that the procedure does not affect mineralogical and chemical composition. Surface reactions related to oxidative stress, free radical generation, bioavailability of iron, and antioxidant depletion, revealed a consistent reduction in reactivity upon reduction in size. When tested on A549 human epithelial cells, the pristine but not the nanosized fibers proved cytotoxic (LDH release), induced NO production, and caused lipid peroxidation. However, nanofibers still induced some toxicity relevant oxidative stress activity (ROS production) in a dose-dependent fashion. The reduction in length and a lack of poorly coordinated bioavailable iron in nanochrysotile may explain this behavior. The present study provides a one-step procedure for the preparation of a homogeneous batch of natural asbestos nanofibers and shows how a well-known toxic material might not necessarily become more toxic than its micrometric counterpart when reduced to the nanoscale.