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Sample records for core fucosylated n-glycans

  1. Divergent Chemoenzymatic Synthesis of Asymmetrical-Core-Fucosylated and Core-Unmodified N-Glycans

    NARCIS (Netherlands)

    Li, Tiehai; Huang, Min; Liu, Lin; Wang, Shuo; Moremen, Kelley W; Boons, Geert-Jan|info:eu-repo/dai/nl/088245489

    2016-01-01

    A divergent chemoenzymaytic approach for the preparation of core-fucosylated and core-unmodified asymmetrical N-glycans from a common advances precursor is described. An undecasaccharide was synthesized by sequential chemical glycosylations of an orthogonally protected core fucosylated

  2. Divergent Chemoenzymatic Synthesis of Asymmetrical-Core-Fucosylated and Core-Unmodified N-Glycans

    NARCIS (Netherlands)

    Li, Tiehai; Huang, Min; Liu, Lin; Wang, Shuo; Moremen, Kelley W; Boons, Geert-Jan

    2016-01-01

    A divergent chemoenzymaytic approach for the preparation of core-fucosylated and core-unmodified asymmetrical N-glycans from a common advances precursor is described. An undecasaccharide was synthesized by sequential chemical glycosylations of an orthogonally protected core fucosylated hexasaccharid

  3. Divergent Chemoenzymatic Synthesis of Asymmetrical-Core-Fucosylated and Core-Unmodified N-Glycans.

    Science.gov (United States)

    Li, Tiehai; Huang, Min; Liu, Lin; Wang, Shuo; Moremen, Kelley W; Boons, Geert-Jan

    2016-12-23

    A divergent chemoenzymaytic approach for the preparation of core-fucosylated and core-unmodified asymmetrical N-glycans from a common advances precursor is described. An undecasaccharide was synthesized by sequential chemical glycosylations of an orthogonally protected core fucosylated hexasaccharide that is common to all mammalian core fucosylated N-glycans. Antennae-selective enzymatic extension of the undecasaccharide using a panel of glycosyl transferases afforded core fucosylated asymmetrical triantennary N-glycan isomers, which are potential biomarkers for breast cancer. A unique aspect of our approach is that a fucosidase (FucA1) has been identified that selectively can cleave a core-fucoside without affecting the fucoside of a sialyl Lewis(X) epitope to give easy access to core-unmodified compounds.

  4. Exquisite specificity of mitogenic lectin from Cephalosporium curvulum to core fucosylated N-glycans.

    Science.gov (United States)

    Inamdar, Shashikala R; Eligar, Sachin M; Ballal, Suhas; Belur, Shivakumar; Kalraiya, Rajiv D; Swamy, Bale M

    2016-02-01

    Lectins are carbohydrate binding proteins that are gaining attention as important tools for the identification of specific glycan markers expressed during different stages of the cancer. We earlier reported the purification of a mitogenic lectin from human pathogenic fungus Cephalosporium curvulum (CSL) that has complex sugar specificity when analysed by hapten inhibition assay. In the present study, we report the fine sugar specificity of CSL as determined by glycan array analysis. The results revealed that CSL has exquisite specificity towards core fucosylated N-glycans. Fucosylated trimannosyl core is the basic structure required for the binding of CSL. The presence of fucose in the side chain further enhances the avidity of CSL towards such glycans. The affinity of CSL is drastically reduced towards the non-core fucosylated glycans, in spite of their side chain fucosylation. CSL showed no binding to the tested O-glycans and monosaccharides. These observations suggest the unique specificity of CSL towards core fucosylated N-glycans, which was further validated by binding of CSL to human colon cancer epithelial and hepatocarcinoma cell lines namely HT29 and HepG2, respectively, that are known to express core fucosylated N-glycans, using AOL and LCA as positive controls. LCA and AOL are fucose specific lectins that are currently being used clinically for the diagnosis of hepatocellular carcinomas. Most of the gastrointestinal markers express core fucosylated N-glycans. The high affinity and exclusive specificity of CSL towards α1-6 linkage of core fucosylated glycans compared to other fucose specific lectins, makes it a promising molecule that needs to be further explored for its application in the diagnosis of gastrointestinal cancer.

  5. Profiling of core fucosylated N-glycans using a novel bacterial lectin that specifically recognizes α1,6 fucosylated chitobiose

    Science.gov (United States)

    Vainauskas, Saulius; Duke, Rebecca M.; McFarland, James; McClung, Colleen; Ruse, Cristian; Taron, Christopher H.

    2016-01-01

    A novel fucose-binding lectin (SL2-1) from the bacterium Streptomyces rapamycinicus was identified by analysis of metagenomic DNA sequences. SL2-1 belongs to a new group of bacterial fucose-specific lectins that have no similarity to known bacterial fucose-binding proteins, but are related to certain eukaryotic fucose-binding lectins. The 17 kDa protein was expressed recombinantly in E. coli and purified by affinity chromatography. Glycan microarray analysis with fluorescently labeled recombinant SL2-1 demonstrated its ability to bind to core α1-6 fucosylated N-glycans, but not to core α1-3 fucosylated N-glycans, or other α1-2, α1-3 and α1-4 fucosylated oligosaccharides. The minimal high affinity binding epitope of SL2-1 was α1-6 fucosylated di-n-acetylchitobiose. The recombinant lectin was efficient in detection of N-glycan core fucosylation using lectin blotting and lectin ELISA assays. Finally, a workflow using SL2-1 for selective and quantitative profiling of core fucosylated N-glycans using UPLC-HILIC-FLR analysis was established. The approach was validated for selective capture and analysis of core fucosylated N-glycans present in complex glycan mixtures derived from mammalian serum IgG. PMID:27678371

  6. An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins

    DEFF Research Database (Denmark)

    Hägglund, Per; Matthiesen, R.; Elortza, F.;

    2007-01-01

    between the two N-acetylglucosamine (GlcNAc) residues in the conserved N-glycan core structure, leaving single GlcNAc residues with putative fucosyl side chains attached to the peptide. To enable digestion of complex and hybrid type N-glycans, a number of exoglycosidases (β-galactosidase, neuraminidase...

  7. Specific N-glycans of Hepatocellular Carcinoma Cell Surface and the Abnormal Increase of Core-α-1, 6-fucosylated Triantennary Glycan via N-acetylglucosaminyltransferases-IVa Regulation.

    Science.gov (United States)

    Nie, Huan; Liu, Xia; Zhang, Yubao; Li, Tingting; Zhan, Chao; Huo, Wenjuan; He, Anshun; Yao, Yuanfei; Jin, Yu; Qu, Youpeng; Sun, Xue-Long; Li, Yu

    2015-11-05

    Glycosylation alterations of cell surface proteins are often observed during the progression of malignancies. The specific cell surface N-glycans were profiled in hepatocellular carcinoma (HCC) with clinical tissues (88 tumor and adjacent normal tissues) and the corresponding serum samples of HCC patients. The level of core-α-1,6-fucosylated triantennary glycan (NA3Fb) increased both on the cell surface and in the serum samples of HCC patients (p IVa (GnT-IVa), which was related to the synthesis of the NA3Fb, was substantially increased in HCC tissues. Knockdown of GnT-IVa leads to a decreased level of NA3Fb and decreased ability of invasion and migration in HCC cells. NA3Fb can be regarded as a specific cell surface N-glycan of HCC. The high expression of GnT-IVa is the cause of the abnormal increase of NA3Fb on the HCC cell surface, which regulates cell migration. This study demonstrated the specific N-glycans of the cell surface and the mechanisms of altered glycoform related with HCC. These findings lead to better understanding of the function of glycan and glycosyltransferase in the tumorigenesis, progression and metastasis of HCC.

  8. Revisiting the substrate specificity of mammalian α1,6-fucosyltransferase reveals that it catalyzes core fucosylation of N-glycans lacking α1,3-arm GlcNAc.

    Science.gov (United States)

    Yang, Qiang; Zhang, Roushu; Cai, Hui; Wang, Lai-Xi

    2017-09-08

    The mammalian α1,6-fucosyltransferase (FUT8) catalyzes the core fucosylation of N-glycans in the biosynthesis of glycoproteins. Previously, intensive in vitro studies with crude extract or purified enzyme concluded that the attachment of a GlcNAc on the α1,3 mannose arm of N-glycan is essential for FUT8-catalyzed core fucosylation. In contrast, we have recently shown that expression of erythropoietin in a GnTI knock-out, FUT8-overexpressing cell line results in the production of fully core-fucosylated glycoforms of the oligomannose substrate Man5GlcNAc2, suggesting that FUT8 can catalyze core fucosylation of N-glycans lacking an α1,3-arm GlcNAc in cells. Here, we revisited the substrate specificity of FUT8 by examining its in vitro activity toward an array of selected N-glycans, glycopeptides, and glycoproteins. Consistent with previous studies, we found that free N-glycans lacking an unmasked α1,3-arm GlcNAc moiety are not FUT8 substrates. However, Man5GlcNAc2 glycan could be efficiently core-fucosylated by FUT8 in an appropriate protein/peptide context, such as with the erythropoietin protein, a V3 polypeptide derived from HIV-1 gp120, or a simple 9-fluorenylmethyl chloroformate-protected Asn moiety. Interestingly, when placed in the V3 polypeptide context, a mature bi-antennary complex-type N-glycan also could be core-fucosylated by FUT8, albeit at much lower efficiency than the Man5GlcNAc2 peptide. This study represents the first report of in vitro FUT8-catalyzed core fucosylation of N-glycans lacking the α1,3-arm GlcNAc moiety. Our results suggest that an appropriate polypeptide context or other adequate structural elements in the acceptor substrate could facilitate the core fucosylation by FUT8. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Synthesis and microarray-assisted binding studies of core xylose and fucose containing N-glycans.

    Science.gov (United States)

    Brzezicka, Katarzyna; Echeverria, Begoña; Serna, Sonia; van Diepen, Angela; Hokke, Cornelis H; Reichardt, Niels-Christian

    2015-05-15

    The synthesis of a collection of 33 xylosylated and core-fucosylated N-glycans found only in nonmammalian organisms such as plants and parasitic helminths has been achieved by employing a highly convergent chemo-enzymatic approach. The influence of these core modifications on the interaction with plant lectins, with the human lectin DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Nonintegrin), and with serum antibodies from schistosome-infected individuals was studied. Core xylosylation markedly reduced or completely abolished binding to several mannose-binding plant lectins and to DC-SIGN, a C-type lectin receptor present on antigen presenting cells. Employing the synthetic collection of core-fucosylated and core-xylosylated N-glycans in the context of a larger glycan array including structures lacking these core modifications, we were able to dissect core xylose and core fucose specific antiglycan antibody responses in S. mansoni infection sera, and we observed clear and immunologically relevant differences between children and adult groups infected with this parasite. The work presented here suggests that, quite similar to bisecting N-acetylglucosamine, core xylose distorts the conformation of the unsubstituted glycan, with important implications for the immunogenicity and protein binding properties of complex N-glycans.

  10. Fucosyltransferases produce N-glycans containing core l-galactose.

    Science.gov (United States)

    Ohashi, Hiroyuki; Ohashi, Takao; Kajiura, Hiroyuki; Misaki, Ryo; Kitamura, Shinichi; Fujiyama, Kazuhito

    2017-01-29

    l-Galactose (l-Gal) containing N-glycans and cell wall polysaccharides have been detected in the l-Fuc deficient mur1 mutant of Arabidopsis thaliana. The l-Gal residue is thought to be transferred from GDP-l-Gal, which is a structurally related analog of GDP-l-Fuc, but in vitrol-galactosylation activity has never been detected. In this study, we carried out preparative scale GDP-l-Gal synthesis using recombinant A. thaliana GDP-mannose-3',5'-epimerase. We also demonstrated the l-galactosylation assay of mouse α1,6-fucosyltransferase (MmFUT8) and A. thaliana α1,3-fucosyltransferase (AtFucTA). Both fucosyltransferases showed l-galactosylation activity from GDP-l-Gal to asparagine-linked N-acetyl-β-d-glucosamine of asialo-agalacto-bi-antennary N-glycan instead of l-fucosylation. In addition, the apparent Km values of MmFUT8 and AtFucTA suggest that l-Fuc was preferentially transferred to N-glycan compared with l-Gal by fucosyltransferases. Our results clearly demonstrate that MmFUT8 and AtFucTA transfer l-Gal residues from GDP-l-Gal and synthesize l-Gal containing N-glycan in vitro.

  11. Increased outer arm and core fucose residues on the N-glycans of mutated alpha-1 antitrypsin protein from alpha-1 antitrypsin deficient individuals.

    Science.gov (United States)

    McCarthy, Cormac; Saldova, Radka; O'Brien, M Emmet; Bergin, David A; Carroll, Tomás P; Keenan, Joanne; Meleady, Paula; Henry, Michael; Clynes, Martin; Rudd, Pauline M; Reeves, Emer P; McElvaney, Noel G

    2014-02-01

    Alpha-1 antitrypsin (AAT) is the major physiological inhibitor of a range of serine proteases, and in the lung, it maintains a protease-antiprotease balance. AAT deficiency (AATD) is an autosomal co-dominant condition with the Z mutation being the most common cause. Individuals homozygous for Z (PiZZ) have low levels of circulating mutant Z-AAT protein leading to premature emphysematous lung disease. Extensive glycoanalysis has been performed on normal AAT (M-AAT) from healthy individuals and the importance of glycosylation in affecting the immune modulatory roles of AAT is documented. However, no glycoanalysis has been carried out on Z-AAT from deficient individuals to date. In this study, we investigate whether the glycans present on Z-AAT differ to those found on M-AAT from healthy controls. Plasma AAT was purified from 10 individuals: 5 AATD donors with the PiZZ phenotype and 5 PiMM healthy controls. Glycoanalysis was performed employing N-glycan release, exoglycosidase digestion and UPLC analysis. No difference in branched glycans was identified between AATD and healthy controls. However, a significant increase in both outer arm (α1-3) (p = 0.04) and core (α1-6) fucosylated glycans (p < 0.0001) was found on Z-AAT compared to M-AAT. This study has identified increased fucosylation on N-glycans of Z-AAT indicative of ongoing inflammation in AATD individuals with implications for early therapeutic intervention.

  12. Presence of galactosylated core fucose on N-glycans in the planaria Dugesia japonica.

    Science.gov (United States)

    Paschinger, Katharina; Razzazi-Fazeli, Ebrahim; Furukawa, Kiyoshi; Wilson, Iain B H

    2011-06-01

    Planarial species are of especial interest to biologists due to the phenomenon of pluripotency and, in comparison to other developmental processes, it can be hypothesised that glycan-lectin interactions may play a role. In order to examine the N-glycans of one of these organisms, Dugesia japonica, peptide:N-glycosidase A was employed and the released glycans were subject to pyridylamination, HPLC and mass spectrometric analysis. A range of oligomannosidic glycans was observed with a trimethylated Man(5) GlcNAc(2) structure being the dominant species. Three glycans were also observed to contain deoxyhexose; in particular, a glycan with the composition Hex(4) HexNAc(2) Fuc(1) Me(2) was revealed by exoglycosidase digestion, in combination with MS/MS, to contain a galactosylated core α1,6-fucose residue, whereas this core modification was found to be capped with a methylhexose residue in the case of a Hex(5) HexNAc(2) Fuc(1) Me(3) structure. This is the first report of these types of structures in a platyhelminth and indicates that the 'GalFuc' modification of N-glycans is not just restricted to molluscs and nematodes.

  13. Reduced immunogenicity of Arabidopsis hgl1 mutant N-glycans caused by altered accessibility of xylose and core fucose epitopes.

    Science.gov (United States)

    Kaulfürst-Soboll, Heidi; Rips, Stephan; Koiwa, Hisashi; Kajiura, Hiroyuki; Fujiyama, Kazuhito; von Schaewen, Antje

    2011-07-01

    Arabidopsis N-glycosylation mutants with enhanced salt sensitivity show reduced immunoreactivity of complex N-glycans. Among them, hybrid glycosylation 1 (hgl1) alleles lacking Golgi α-mannosidase II are unique, because their glycoprotein N-glycans are hardly labeled by anti-complex glycan antibodies, even though they carry β1,2-xylose and α1,3-fucose epitopes. To dissect the contribution of xylose and core fucose residues to plant stress responses and immunogenic potential, we prepared Arabidopsis hgl1 xylT double and hgl1 fucTa fucTb triple mutants by crossing previously established T-DNA insertion lines and verified them by mass spectrometry analyses. Root growth assays revealed that hgl1 fucTa fucTb but not hgl1 xylT plants are more salt-sensitive than hgl1, hinting at the importance of core fucose modification and masking of xylose residues. Detailed immunoblot analyses with anti-β1,2-xylose and anti-α1,3-fucose rabbit immunoglobulin G antibodies as well as cross-reactive carbohydrate determinant-specific human immunoglobulin E antibodies (present in sera of allergy patients) showed that xylose-specific reactivity of hgl1 N-glycans is indeed reduced. Based on three-dimensional modeling of plant N-glycans, we propose that xylose residues are tilted by 30° because of untrimmed mannoses in hgl1 mutants. Glycosidase treatments of protein extracts restored immunoreactivity of hgl1 N-glycans supporting these models. Furthermore, among allergy patient sera, untrimmed mannoses persisting on the α1,6-arm of hgl1 N-glycans were inhibitory to immunoreaction with core fucoses to various degrees. In summary, incompletely trimmed glycoprotein N-glycans conformationally prevent xylose and, to lesser extent, core fucose accessibility. Thus, in addition to N-acetylglucosaminyltransferase I, Golgi α-mannosidase II emerges as a so far unrecognized target for lowering the immunogenic potential of plant-derived glycoproteins.

  14. THE N-GLYCANS OF TRICHOMONAS VAGINALIS CONTAIN VARIABLE CORE AND ANTENNAL MODIFICATIONS

    Science.gov (United States)

    Paschinger, Katharina; Hykollari, Alba; Razzazi-Fazeli, Ebrahim; Greenwell, Pamela; Leitsch, David; Walochnik, Julia; Wilson, Iain B. H.

    2012-01-01

    Trichomonad species are widespread unicellular flagellated parasites of vertebrates which interact with their hosts through carbohydrate-lectin interactions. In the past, some data has been accumulated regarding their lipo(phospho)glycans, a major glycoconjugate on their cell surfaces; on the other hand, other than biosynthetic aspects, few details about their N-linked oligosaccharides are known. In this study, we present both mass spectrometric and HPLC data about the N-glycans of different strains of Trichomonas vaginalis, a parasite of the human reproductive tract. The major structure in all strains examined is a truncated oligomannose form (Man5GlcNAc2) with α1,2-mannose residues, compatible with a previous bioinformatic examination of the glycogenomic potential of T. vaginalis. In addition, dependent on the strain, N-glycans modified by pentose residues, phosphate or phosphoethanolamine and terminal N-acetyllactosamine (Galβ1,4GlcNAc) units were found. The modification of N-glycans by N-acetyllactosamine in at least some strains is shared with the lipo(phospho)glycan and may represent a further interaction partner for host galectins, thereby playing a role in binding of the parasite to host epithelia. On the other hand, the variation in glycosylation between strains may be the result of genetic diversity within this species. PMID:21983210

  15. The identification and characterization of novel N-glycan-based biomarkers in gastric cancer.

    Directory of Open Access Journals (Sweden)

    Long Liu

    Full Text Available BACKGROUND AND AIMS: To identify and validate N-glycan biomarkers in gastric cancer (GC and to elucidate their underlying molecular mechanism of action. METHODS: In total, 347 individuals, including patients with GC (gastric cancer or atrophic gastritis and healthy controls, were randomly divided into a training group (n=287 and a retrospective validation group (n=60. Serum N-glycan profiling was achieved with DNA sequencer-assisted/fluorophore-assisted carbohydrate electrophoresis (DSA-FACE. Two diagnostic models were constructed based on the N-glycan profiles using logistic stepwise regression. The diagnostic performance of each model was assessed in retrospective, prospective (n=60, and follow-up (n=40 cohorts. Lectin blotting was performed to determine total core-fucosylation, and the expression of genes involved in core-fucosylation in GC was analyzed by reverse transcriptase-polymerase chain reaction. RESULTS: We identified at least 9 N-glycan structures (peaks and the levels of core fucose residues and fucosyltransferase were significantly decreased in GC. Two diagnostic models, designated GCglycoA and GCglycoB, were constructed to differentiate GC from control and atrophic gastritis. The areas under the receiver operating characteristic (ROC curves (AUC for both GCglycoA and GCglycoB were higher than those for CEA, CA19-9, CA125 and CA72-4. Compared with CEA, CA19-9, CA125 and CA72-4, the sensitivity of GCglycoA increased 29.66%, 37.28%, 56.78% and 61.86%, respectively, and the accuracy increased 10.62%, 16.82%, 25.67% and 28.76%, respectively. For GCglycoB, the sensitivity increased 27.97%, 35.59%, 55.09% and 60.17% and the accuracy increased 21.26%, 24.64%, 31.40% and 34.30% compared with CEA, CA19-9, CA125 and CA72-4, respectively. After curative surgery, the core fucosylated peak (peak 3 and the total core fucosylated N-glycans (sumfuc were reversed. CONCLUSIONS: The results indicated that the diagnostic models based on N-glycan

  16. Two types of galactosylated fucose motifs are present on N-glycans of Haemonchus contortus.

    Science.gov (United States)

    Paschinger, Katharina; Wilson, Iain B H

    2015-06-01

    N-Glycans from the nematode Haemonchus contortus (barber pole worm), a parasite of sheep and cattle, were the first to be described to possess up to three fucose residues associated with the N,N'-diacetylchitobiosyl core, two being on the reducing-terminal proximal GlcNAc and one on the distal core GlcNAc residue. The assumption was that truncated glycans from this organism with three hexose residues have the composition Man3GlcNAc2Fuc1-3. In this study, we have performed HPLC and MALDI-TOF MS/MS in combination with selected digestions of N-glycans from Haemonchus. A dominant trifucosylated Hex3HexNAc2Fuc3 glycan was modified not only with α1,6-fucose but also with a proximal core α1,3-fucose and a galactosylated distal α1,3-fucose; thereby, only two of the hexose residues were mannose. Other N-glycans displayed galactosylation of the core α1,6-fucose, antennal fucosylation or modification with phosphorylcholine. Thus, the N-glycans of Haemonchus contain a number of potentially immunogenic glycan epitopes also found in other parasites and our proposed structures are in line with the previously defined specificity of nematode glycosyltransferases as we show that distal fucosylation and the presence of an α1,6-mannose are apparently mutually exclusive. These data are thereby of importance for engineering cell lines capable of mimicking Haemonchus-type N-glycans in the preparation of recombinant proteins as vaccine candidates.

  17. Orthogonal Assessment of Biotherapeutic Glycosylation: A Case Study Correlating N-Glycan Core Afucosylation of Herceptin with Mechanism of Action.

    Science.gov (United States)

    Upton, Rosie; Bell, Leonard; Guy, Colin; Caldwell, Paul; Estdale, Sian; Barran, Perdita E; Firth, David

    2016-10-18

    In the development of therapeutic antibodies and biosimilars, an appropriate biopharmaceutical CMC control strategy that connects critical quality attributes with mechanism of action should enable product assessment at an early stage of development in order to mitigate risk. Here we demonstrate a new analytical workflow using trastuzumab which comprises "middle-up" analysis using a combination of IdeS and the endoglycosidases EndoS and EndoS2 to comprehensively map the glycan content. Enzymatic cleavage between the two N-acetyl glucosamine residues of the chitobiose core of N-glycans significantly simplifies the oligosaccharide component enabling facile distinction of GlcNAc from GlcNAc with core fucose. This approach facilitates quantitative determination of total Fc-glycan core-afucosylation, which was in turn correlated with receptor binding affinity by surface plasmon resonance and in vitro ADCC potency with a cell based bioassay. The strategy also quantifies Fc-glycan occupancy and the relative contribution from high mannose glycans.

  18. N-glycan containing a core α1,3-fucose residue is required for basipetal auxin transport and gravitropic response in rice (Oryza sativa).

    Science.gov (United States)

    Harmoko, Rikno; Yoo, Jae Yong; Ko, Ki Seong; Ramasamy, Nirmal Kumar; Hwang, Bo Young; Lee, Eun Ji; Kim, Ho Soo; Lee, Kyung Jin; Oh, Doo-Byoung; Kim, Dool-Yi; Lee, Sanghun; Li, Yang; Lee, Sang Yeol; Lee, Kyun Oh

    2016-10-01

    In plants, α1,3-fucosyltransferase (FucT) catalyzes the transfer of fucose from GDP-fucose to asparagine-linked GlcNAc of the N-glycan core in the medial Golgi. To explore the physiological significance of this processing, we isolated two Oryza sativa (rice) mutants (fuct-1 and fuct-2) with loss of FucT function. Biochemical analyses of the N-glycan structure confirmed that α1,3-fucose is missing from the N-glycans of allelic fuct-1 and fuct-2. Compared with the wild-type cv Kitaake, fuct-1 displayed a larger tiller angle, shorter internode and panicle lengths, and decreased grain filling as well as an increase in chalky grains with abnormal shape. The mutant allele fuct-2 gave rise to similar developmental abnormalities, although they were milder than those of fuct-1. Restoration of a normal tiller angle in fuct-1 by complementation demonstrated that the phenotype is caused by the loss of FucT function. Both fuct-1 and fuct-2 plants exhibited reduced gravitropic responses. Expression of the genes involved in tiller and leaf angle control was also affected in the mutants. We demonstrate that reduced basipetal auxin transport and low auxin accumulation at the base of the shoot in fuct-1 account for both the reduced gravitropic response and the increased tiller angle.

  19. Purification, characterization, and cloning of a Spodoptera frugiperda Sf9 beta-N-acetylhexosaminidase that hydrolyzes terminal N-acetylglucosamine on the N-glycan core.

    Science.gov (United States)

    Tomiya, Noboru; Narang, Someet; Park, Jung; Abdul-Rahman, Badarulhisam; Choi, One; Singh, Sundeep; Hiratake, Jun; Sakata, Kanzo; Betenbaugh, Michael J; Palter, Karen B; Lee, Yuan C

    2006-07-14

    Paucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a beta-N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been identified. We report the purification of a beta-N-acetylhexosaminidase from the culture medium of Spodoptera frugiperda Sf9 cells (Sfhex). The purified Sfhex protein showed 10 times higher activity for a terminal N-acetylglucosamine on the N-glycan core compared with tri-N-acetylchitotriose. Sfhex was found to be a homodimer of 110 kDa in solution, with a pH optimum of 5.5. With a biantennary N-glycan substrate, it exhibited a 5-fold preference for removal of the beta(1,2)-linked N-acetylglucosamine from the Man alpha(1,3) branch compared with the Man alpha(1,6) branch. We isolated two corresponding cDNA clones for Sfhex that encode proteins with >99% amino acid identity. A phylogenetic analysis suggested that Sfhex is an ortholog of mammalian lysosomal beta-N-acetylhexosaminidases. Recombinant Sfhex expressed in Sf9 cells exhibited the same substrate specificity and pH optimum as the purified enzyme. Although a larger amount of newly synthesized Sfhex was secreted into the culture medium by Sf9 cells, a significant amount of Sfhex was also found to be intracellular. Under a confocal microscope, cellular Sfhex exhibited punctate staining throughout the cytoplasm, but did not colocalize with a Golgi marker. Because secretory glycoproteins and Sfhex are cotransported through the same secretory pathway and because Sfhex is active at the pH of the secretory compartments, this study suggests that Sfhex may play a role as a processing beta-N-acetylhexosaminidase acting on N-glycans from Sf9 cells.

  20. Loss of α1,6-Fucosyltransferase Decreases Hippocampal Long Term Potentiation: IMPLICATIONS FOR CORE FUCOSYLATION IN THE REGULATION OF AMPA RECEPTOR HETEROMERIZATION AND CELLULAR SIGNALING.

    Science.gov (United States)

    Gu, Wei; Fukuda, Tomohiko; Isaji, Tomoya; Hang, Qinglei; Lee, Ho-hsun; Sakai, Seiichiro; Morise, Jyoji; Mitoma, Junya; Higashi, Hideyoshi; Taniguchi, Naoyuki; Yawo, Hiromu; Oka, Shogo; Gu, Jianguo

    2015-07-10

    Core fucosylation is catalyzed by α1,6-fucosyltransferase (FUT8), which transfers a fucose residue to the innermost GlcNAc residue via α1,6-linkage on N-glycans in mammals. We previously reported that Fut8-knock-out (Fut8(-/-)) mice showed a schizophrenia-like phenotype and a decrease in working memory. To understand the underlying molecular mechanism, we analyzed early form long term potentiation (E-LTP), which is closely related to learning and memory in the hippocampus. The scale of E-LTP induced by high frequency stimulation was significantly decreased in Fut8(-/-) mice. Tetraethylammonium-induced LTP showed no significant differences, suggesting that the decline in E-LTP was caused by postsynaptic events. Unexpectedly, the phosphorylation levels of calcium/calmodulin-dependent protein kinase II (CaMKII), an important mediator of learning and memory in postsynapses, were greatly increased in Fut8(-/-) mice. The expression levels of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) in the postsynaptic density were enhanced in Fut8(-/-) mice, although there were no significant differences in the total expression levels, implicating that AMPARs without core fucosylation might exist in an active state. The activation of AMPARs was further confirmed by Fura-2 calcium imaging using primary cultured neurons. Taken together, loss of core fucosylation on AMPARs enhanced their heteromerization, which increase sensitivity for postsynaptic depolarization and persistently activate N-methyl-d-aspartate receptors as well as Ca(2+) influx and CaMKII and then impair LTP. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Mass spectrometric profiling reveals association of N-glycan patterns with epithelial ovarian cancer progression.

    Science.gov (United States)

    Chen, Huanhuan; Deng, Zaian; Huang, Chuncui; Wu, Hongmei; Zhao, Xia; Li, Yan

    2017-07-01

    Aberrant changes of N-glycan modifications on proteins have been linked to various diseases including different cancers, suggesting possible avenue for exploring their etiologies based on N-glycomic analysis. Changes in N-glycan patterns during epithelial ovarian cancer development have so far been investigated mainly using serum, plasma, ascites, and cell lines. However, changes in patterns of N-glycans in tumor tissues during epithelial ovarian cancer progression have remained largely undefined. To investigate whether changes in N-glycan patterns correlate with oncogenesis and progression of epithelial ovarian cancer, we profiled N-glycans from formalin-fixed paraffin-embedded tissue slides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and quantitatively compared among different pathological grades of epithelial ovarian cancer and healthy controls. Our results show that among the 80 compositions of N-glycan detected, expression levels of high-mannose type were higher in epithelial ovarian cancer samples than that observed in healthy controls, accompanied by reduced levels of hybrid-type glycans. By applying receiver operating characteristic analysis, we show that a combined panel composed of four high-mannose and three fucosylated neutral complex N-glycans allows for good discrimination of epithelial ovarian cancer from healthy controls. Furthermore, using a statistical analysis of variance assay, we found that different N-glycan patterns, including 2 high-mannose-type, 2 fucosylated and sialylated complex structures, and 10 fucosylated neutral complex N-glycans, exhibited specific changes in N-glycan abundance across epithelial ovarian cancer grades. Together, our results provide strong evidence that N-glycomic changes are a strong indicator for epithelial ovarian cancer pathological grades and should provide avenues to identify novel biomarkers for epithelial ovarian cancer diagnosis and monitoring.

  2. Characterization of the N-glycans of female Angiostrongylus cantonensis worms.

    Science.gov (United States)

    Veríssimo, Carolina M; Morassutti, Alessandra L; von Itzstein, Mark; Sutov, Grigorij; Hartley-Tassell, Lauren; McAtamney, Sarah; Dell, Anne; Haslam, Stuart M; Graeff-Teixeira, Carlos

    2016-07-01

    Glycoconjugates play a crucial role in the host-parasite relationships of helminthic infections, including angiostrongyliasis. It has previously been shown that the antigenicity of proteins from female Angiostrongylus cantonensis worms may depend on their associated glycan moieties. Here, an N-glycan profile of A. cantonensis is reported. A total soluble extract (TE) was prepared from female A. cantonensis worms and was tested by western blot before and after glycan oxidation or N- and O-glycosidase treatment. The importance of N-glycans for the immunogenicity of A. cantonensis was demonstrated when deglycosylation of the TE with PNGase F completely abrogated IgG recognition. The TE was also fractionated using various lectin columns [Ulex europaeus (UEA), concanavalin A (Con A), Arachis hypogaea (PNA), Triticum vulgaris (WGA) and Lycopersicon esculentum (LEA)], and then each fraction was digested with PNGase F. Released N-glycans were analyzed with matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) and MALDI-TOF/TOF-MS/MS. Complex-type, high mannose, and truncated glycan structures were identified in all five fractions. Sequential MALDI-TOF-TOF analysis of the major MS peaks identified complex-type structures, with a α1-6 fucosylated core and truncated antennas. Glycoproteins in the TE were labeled with BodipyAF558-SE dye for a lectin microarray analysis. Fluorescent images were analyzed with ProScanArray imaging software followed by statistical analysis. A total of 29 lectins showed positive binding to the TE. Of these, Bandeiraea simplicifolia (BS-I), PNA, and Wisteria floribunda (WFA), which recognize galactose (Gal) and N-acetylgalactosamine (GalNAc), exhibited high affinity binding. Taken together, our findings demonstrate that female A. cantonensis worms have characteristic helminth N-glycans.

  3. Intrinsic hepatocyte dedifferentiation is accompanied by upregulation of mesenchymal markers, protein sialylation and core alpha 1,6 linked fucosylation

    Science.gov (United States)

    Mehta, Anand; Comunale, Mary Ann; Rawat, Siddhartha; Casciano, Jessica C.; Lamontagne, Jason; Herrera, Harmin; Ramanathan, Aarti; Betesh, Lucy; Wang, Mengjun; Norton, Pamela; Steel, Laura F.; Bouchard, Michael J.

    2016-01-01

    Alterations in N-linked glycosylation have long been associated with cancer but for the most part, the reasons why have remained poorly understood. Here we show that increased core fucosylation is associated with de-differentiation of primary hepatocytes and with the appearance of markers indicative of a transition of cells from an epithelial to a mesenchymal state. This increase in core fucosylation was associated with increased levels of two enzymes involved in α-1,6 linked fucosylation, GDP-mannose 4, 6-dehydratase (Gmds) and to a lesser extent fucosyltransferase 8 (Fut8). In addition, the activation of cancer-associated cellular signaling pathways in primary rat hepatocytes can increase core fucosylation and induce additional glycoform alterations on hepatocyte proteins. Specifically, we show that increased levels of protein sialylation and α-1,6-linked core fucosylation are observed following activation of the β-catenin pathway. Activation of the Akt signaling pathway or induction of hypoxia also results in increased levels of fucosylation and sialylation. We believe that this knowledge will help in the better understanding of the genetic factors associated with altered glycosylation and may allow for the development of more clinically relevant biomarkers. PMID:27328854

  4. N-glycan alterations are associated with drug resistance in human hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Nakagawa Takahito

    2007-05-01

    Full Text Available Abstract Background Correlations of disease phenotypes with glycosylation changes have been analysed intensively in the tumor biology field. Glycoforms potentially associated with carcinogenesis, tumor progression and cancer metastasis have been identified. In cancer therapy, drug resistance is a severe problem, reducing therapeutic effect of drugs and adding to patient suffering. Although multiple mechanisms likely underlie resistance of cancer cells to anticancer drugs, including overexpression of transporters, the relationship of glycans to drug resistance is not well understood. Results We established epirubicin (EPI – and mitoxantrone (MIT – resistant cell lines (HLE-EPI and HLE-MIT from the human hepatocellular carcinoma cell line (HLE. HLE-EPI and HLE-MIT overexpressed transporters MDR1/ABCB1 and BCRP/ABCG2, respectively. Here we compared the glycomics of HLE-EPI and HLE-MIT cells with the parental HLE line. Core fucosylated triantennary oligosaccharides were increased in the two resistant lines. We investigated mRNA levels of glycosyltransferases synthesizing this oligosaccharide, namely, N-acetylglucosaminyltransferase (GnT-IVa, GnT-IVb and α1,6-fucosyltransferase (α1,6-FucT, and found that α1,6-FucT was particularly overexpressed in HLE-MIT cells. In HLE-EPI cells, GnT-IVa expression was decreased, while GnT-IVb was increased. Both GnT-IVs were downregulated in HLE-MIT cells. HLE-MIT cells also showed decreases in fucosylated tetraantennary oligosaccharide, the product of GnT-V. GnT-V expression was decreased in both lines, but particularly so in HLE-MIT cells. Thus both N-glycan and glycosyltransferase expression was altered as cells acquired tolerance, suggesting novel mechanisms of drug resistance. Conclusion N-glycan and glycosyltransferase expression in HLE-EPI and HLE-MIT were analysed and presented that glycans altered according with acquired tolerance. These results suggested novel mechanisms of drug resistance.

  5. N-glycans of recombinant human acid alpha-glucosidase expressed in the milk of transgenic rabbits.

    Science.gov (United States)

    Jongen, Susanne P; Gerwig, Gerrit J; Leeflang, Bas R; Koles, Kate; Mannesse, Maurice L M; van Berkel, Patrick H C; Pieper, Frank R; Kroos, Marian A; Reuser, Arnold J J; Zhou, Qun; Jin, Xiaoying; Zhang, Kate; Edmunds, Tim; Kamerling, Johannis P

    2007-06-01

    Pompe disease is a lysosomal glycogen storage disorder characterized by acid alpha-glucosidase (GAA) deficiency. More than 110 different pathogenic mutations in the gene encoding GAA have been observed. Patients with this disease are being treated by intravenous injection of recombinant forms of the enzyme. Focusing on recombinant approaches to produce the enzyme means that specific attention has to be paid to the generated glycosylation patterns. Here, human GAA was expressed in the mammary gland of transgenic rabbits. The N-linked glycans of recombinant human GAA (rhAGLU), isolated from the rabbit milk, were released by peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The N-glycan pool was fractionated and purified into individual components by a combination of anion-exchange, normal-phase, and Sambucus nigra agglutinin-affinity chromatography. The structures of the components were analyzed by 500 MHz one-dimensional and 600 MHz cryo two-dimensional (total correlation spectroscopy [TOCSY] nuclear Overhauser enhancement spectroscopy) (1)H nuclear magnetic resonance spectroscopy, combined with two-dimensional (31)P-filtered (1)H-(1)H TOCSY spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and high-performance liquid chromatography (HPLC)-profiling of 2-aminobenzamide-labeled glycans combined with exoglycosidase digestions. The recombinant rabbit glycoprotein contained a broad array of different N-glycans, comprising oligomannose-, hybrid-, and complex-type structures. Part of the oligomannose-type glycans showed the presence of phospho-diester-bridged N-acetylglucosamine. For the complex-type glycans (partially) (alpha2-6)-sialylated (nearly only N-acetylneuraminic acid) diantennary structures were found; part of the structures were (alpha1-6)-core-fucosylated or (alpha1-3)-fucosylated in the upper antenna (Lewis x). Using HPLC-mass spectrometry of glycopeptides, information was generated with respect to the

  6. N-glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research.

    Science.gov (United States)

    Pedersen, Carina T; Loke, Ian; Lorentzen, Andrea; Wolf, Sara; Kamble, Manoj; Kristensen, Sebastian K; Munch, David; Radutoiu, Simona; Spillner, Edzard; Roepstorff, Peter; Thaysen-Andersen, Morten; Stougaard, Jens; Dam, Svend

    2017-08-01

    Studies of protein N-glycosylation are important for answering fundamental questions on the diverse functions of glycoproteins in plant growth and development. Here we generated and characterised a comprehensive collection of Lotus japonicusLORE1 insertion mutants, each lacking the activity of one of the 12 enzymes required for normal N-glycan maturation in the glycosylation machinery. The inactivation of the individual genes resulted in altered N-glycan patterns as documented using mass spectrometry and glycan-recognising antibodies, indicating successful identification of null mutations in the target glyco-genes. For example, both mass spectrometry and immunoblotting experiments suggest that proteins derived from the α1,3-fucosyltransferase (Lj3fuct) mutant completely lacked α1,3-core fucosylation. Mass spectrometry also suggested that the Lotus japonicus convicilin 2 was one of the main glycoproteins undergoing differential expression/N-glycosylation in the mutants. Demonstrating the functional importance of glycosylation, reduced growth and seed production phenotypes were observed for the mutant plants lacking functional mannosidase I, N-acetylglucosaminyltransferase I, and α1,3-fucosyltransferase, even though the relative protein composition and abundance appeared unaffected. The strength of our N-glycosylation mutant platform is the broad spectrum of resulting glycoprotein profiles and altered physiological phenotypes that can be produced from single, double, triple and quadruple mutants. This platform will serve as a valuable tool for elucidating the functional role of protein N-glycosylation in plants. Furthermore, this technology can be used to generate stable plant mutant lines for biopharmaceutical production of glycoproteins displaying relative homogeneous and mammalian-like N-glycosylation features. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  7. Correlative Fluorescence and Scanning Electron Microscopy of Labelled Core Fucosylated Glycans Using Cryosections Mounted on Carbon-Patterned Glass Slides.

    Science.gov (United States)

    Vancová, Marie; Nebesářová, Jana

    2015-01-01

    The aim of the study is co-localization of N-glycans with fucose attached to N-acetylglucosamine in α1,3 linkage, that belong to immunogenic carbohydrate epitopes in humans, and N-glycans with α1,6-core fucose typical for mammalian type of N-linked glycosylation. Both glycan epitopes were labelled in cryosections of salivary glands isolated from the tick Ixodes ricinus. Salivary glands secrete during feeding many bioactive molecules and influence both successful feeding and transmission of tick-borne pathogens. For accurate and reliable localization of labelled glycans in both fluorescence and scanning electron microscopes, we used carbon imprints of finder or indexed EM grids on glass slides. We discuss if the topographical images can provide information about labelled structures, the working setting of the field-emission scanning electron microscope and the influence of the detector selection (a below-the-lens Autrata improved YAG detector of back-scattered electrons; in-lens and conventional Everhart-Thornley detectors of secondary electrons) on the imaging of gold nanoparticles, quantum dots and osmium-stained membranes.

  8. Correlative Fluorescence and Scanning Electron Microscopy of Labelled Core Fucosylated Glycans Using Cryosections Mounted on Carbon-Patterned Glass Slides.

    Directory of Open Access Journals (Sweden)

    Marie Vancová

    Full Text Available The aim of the study is co-localization of N-glycans with fucose attached to N-acetylglucosamine in α1,3 linkage, that belong to immunogenic carbohydrate epitopes in humans, and N-glycans with α1,6-core fucose typical for mammalian type of N-linked glycosylation. Both glycan epitopes were labelled in cryosections of salivary glands isolated from the tick Ixodes ricinus. Salivary glands secrete during feeding many bioactive molecules and influence both successful feeding and transmission of tick-borne pathogens. For accurate and reliable localization of labelled glycans in both fluorescence and scanning electron microscopes, we used carbon imprints of finder or indexed EM grids on glass slides. We discuss if the topographical images can provide information about labelled structures, the working setting of the field-emission scanning electron microscope and the influence of the detector selection (a below-the-lens Autrata improved YAG detector of back-scattered electrons; in-lens and conventional Everhart-Thornley detectors of secondary electrons on the imaging of gold nanoparticles, quantum dots and osmium-stained membranes.

  9. Protein N-glycosylation and N-glycan trimming are required for postembryonic development of the pest beetle Tribolium castaneum

    Science.gov (United States)

    Walski, Tomasz; Van Damme, Els J. M.; Smargiasso, Nicolas; Christiaens, Olivier; De Pauw, Edwin; Smagghe, Guy

    2016-01-01

    In holometabolous insects the transition from larva to adult requires a complete body reorganization and relies on N-glycosylated proteins. N-glycosylation is an important posttranslational modification that influences protein activity but its impact on the metamorphosis has not been studied yet. Here we used the red flour beetle, Tribolium castaneum, to perform a first comprehensive study on the involvement of the protein N-glycosylation pathway in metamorphosis. The transcript levels for genes encoding N-glycan processing enzymes increased during later developmental stages and, in turn, transition from larva to adult coincided with an enrichment of more extensively modified paucimannose glycans, including fucosylated ones. Blockage of N-glycan attachment resulted in larval mortality, while RNAi of α-glucosidases involved in early N-glycan trimming and quality control disrupted the larva to pupa transition. Additionally, simultaneous knockdown of multiple genes responsible for N-glycan processing towards paucimannose structures revealed their novel roles in pupal appendage formation and adult eclosion. Our findings revealed that, next to hormonal control, insect post-embryonic development and metamorphosis depend on protein N-glycan attachment and efficient N-glycan processing. Consequently, disruption of these processes could be an effective new approach for insect control. PMID:27731363

  10. Biological significance of complex N-glycans in plants and their impact on plant physiology

    Directory of Open Access Journals (Sweden)

    Richard eStrasser

    2014-07-01

    Full Text Available Asparagine (N-linked protein glycosylation is a ubiquitous co- and post-translational modification which can alter the biological function of proteins and consequently affects the development, growth and physiology of organisms. Despite an increasing knowledge of N-glycan biosynthesis and processing, we still understand very little about the biological function of individual N-glycan structures in plants. In particular, the N-glycan processing steps mediated by Golgi-resident enzymes create a structurally diverse set of protein-linked carbohydrate structures. Some of these complex N-glycan modifications like the presence of beta1,2-xylose, core alpha1,3-fucose or the Lewis a-epitope are characteristic for plants and are evolutionary highly conserved. In mammals, complex N-glycans are involved in different cellular processes including molecular recognition and signalling events. By contrast, the complex N-glycan function is still largely unknown in plants. Here, in this short review I focus on important recent developments and discuss their implications for future research in plant glycobiology and plant biotechnology.

  11. Quantitative conformational analysis of the core region of N-glycans using residual dipolar couplings, aqueous molecular dynamics, and steric alignment

    Energy Technology Data Exchange (ETDEWEB)

    Almond, Andrew; Duus, Jens O. [Carlsberg Laboratory (Denmark)

    2001-08-15

    A method is described for quantitatively investigating the dynamic conformation of small oligosaccharides containing an {alpha}(1{sup {yields}}6) linkage. It was applied to the oligosaccharide Man-{alpha}(1{sup {yields}}3) {l_brace}Man-{alpha} (1{sup {yields}}6){r_brace}Man-{alpha}-O-Me, which is a core region frequently observed in N-linked glycans. The approach tests an aqueous molecular dynamics simulation, capable of predicting microscopic dynamics, against experimental residual dipolar couplings, by assuming that alignment is caused purely by steric hindrance. The experimental constraints were heteronuclear and homonuclear residual dipolar couplings, and in particular those within the {alpha}(1{sup {yields}}6) linkage itself. Powerful spin-state-selective pulse sequences and editing schemes were used to obtain the most relevant couplings for testing the model. Molecular dynamics simulations in water over a period of 50 ns were not able to predict the correct rotamer population at the {alpha}(1{sup {yields}}6) linkage to agree with the experimental data. However, this sampling problem could be corrected using a simple maximum likelihood optimisation, indicating that the simulation was modelling local dynamics correctly. The maximum likelihood prediction of the residual dipolar couplings was found to be an almost equal population of the gg and gt rotamer conformations at the {alpha}(1{sup {yields}}6) linkage, and the tg conformation was predicted to be unstable and unpopulated in aqueous solution. In this case all twelve measured residual dipolar couplings could be satisfied. This conformer population could also be used to make predictions of scalar couplings with the use of a previously derived empirical equation, and is qualitatively in agreement with previous predictions based on NMR, X-ray crystallography and optical data.

  12. Rapid high-resolution characterization of functionally important monoclonal antibody N-glycans by capillary electrophoresis.

    Science.gov (United States)

    Szabo, Zoltan; Guttman, András; Bones, Jonathan; Karger, Barry L

    2011-07-01

    Characterization of the N-glycosylation present in the Fc region of therapeutic monoclonal antibodies requires rapid, high-resolution separation methods to guarantee product safety and efficacy during all stages of process development. Determination of fucosylated oligosaccharides is particularly important during clone selection, product characterization, and lot release as fucose has been shown to adversely affect the ability of mAbs to induce antibody dependent cellular cytotoxicity (ADCC). Here, we apply a general capillary electrophoresis optimization strategy to separate functionally relevant fucosylated and afucosylated glycans on mononclonal antibody products in the presence of several high mannose oligosaccharides. The N-glycans chosen represent those most commonly reported on CHO cell derived therapeutic antibodies. A rapid (processing for automated 96 well plate-based glycosylation analyses of two nonproprietary therapeutic monoclonal antibodies, demonstrating ruggedness and suitability for high-throughput process and product monitoring applications.

  13. The presence of outer arm fucose residues on the N-glycans of tissue inhibitor of metalloproteinases-1 reduces its activity.

    Science.gov (United States)

    Kim, Han Ie; Saldova, Radka; Park, Jun Hyoung; Lee, Young Hun; Harvey, David J; Wormald, Mark R; Wynne, Kieran; Elia, Giuliano; Kim, Hwa-Jung; Rudd, Pauline M; Lee, Seung-Taek

    2013-08-02

    Tissue inhibitor of metalloproteinases-1 (TIMP-1) inhibits matrix metalloproteinases (MMPs) by binding at a 1:1 stoichiometry. Here we have shown the involvement of N-glycosylation in the MMP inhibitory ability of TIMP-1. TIMP-1, purified from HEK 293 cells overexpressing TIMP-1 (293 TIMP-1), showed less binding and inhibitory abilities to MMPs than TIMP-1 purified from fibroblasts or SF9 insect cells infected with TIMP-1 baculovirus. Following deglycosylation of TIMP-1, all forms of TIMP-1 showed similar levels of MMP binding and inhibition, suggesting that glycosylation is involved in the regulation of these TIMP-1 activities. Analysis of the N-glycan structures showed that SF9 TIMP-1 has the simplest N-glycan structures, followed by fibroblast TIMP-1 and 293 TIMP-1, in order of increasing complexity in their N-glycan structures. Further analyses showed that cleavage of outer arm fucose residues from the N-glycans of 293 TIMP-1 or knockdown of both FUT4 and FUT7 (which encode for fucosyltransferases that add outer arm fucose residues to N-glycans) enhanced the MMP-binding and catalytic abilities of 293 TIMP-1, bringing them up to the levels of the other TIMP-1. These results demonstrate that the ability of TIMP-1 to inhibit MMPs is at least in part regulated by outer arm fucosylation of its N-glycans.

  14. Posttranslational modification of E-cadherin by core fucosylation regulates Src activation and induces epithelial-mesenchymal transition-like process in lung cancer cells.

    Science.gov (United States)

    Shao, Kang; Chen, Zhong Yi; Gautam, Suraj; Deng, Nian Hui; Zhou, You; Wu, Xing Zhong

    2016-02-01

    E-cadherin is often dysregulated in aggressive lung cancer, the mechanism of which cannot always be explained at the level of transcription. In 66 patients with lung cancer, immunohistochemical staining demonstrated that co-localization of E-cadherin and core fucose by Lens culinaris agglutinin was significantly less extensive in tumor than in nontumor tissue. Through gain and loss of fucosylation experiments in the giant lung carcinoma cell lines 95C and 95D, our results revealed that E-cadherin core fucosylation in 95C cells overexpressing α-1, 6-fucosyltransferase (Fut8) inhibited Fut8-95C cell migration, whereas knockdown of Fut8 in 95D cells enhanced migration of short-interfering RNA-targeting Fut8 (siFut8)-95D cells. The level of active Src (phosphorylated Src [Y416]) was significantly reduced in Fut8-95C cells, but elevated in siFut8-95D cells. In protein complexes immunoprecipitated from Fut8-95C cell lysates with anti-E-cadherin, less phosphorylated Src (Y416) and more β-catenin were observed, but immunoprecipitates from siFut8-95D cells, containing less core fucosylated E-cadherin, contained an elevated level of phospho-Src Y416. In Fut8-95C cells, phosphorylation of Akt (Y315, Y326) and GSK-3β (S9) was significantly reduced, but β-catenin (S37) phosphorylation was enhanced. Expression of N-cadherin and Snail1 was also reduced in Fut8-95C cells, but significantly increased in siFut8-95D cells. Intriguingly, when Src kinase activity was inhibited by treatment of cells with PP2 and SU6656, regulation of N-cadherin, Snail1 and cell migration by E-cadherin core fucosylation was abrogated in both Fut8-95C and siFut8-95D cells. Therefore, posttranslational modification of E-cadherin by less core fucosylation recruited and activated Src, and induced an epithelial-mesenchymal transition-like process in lung cancer cells.

  15. High-mannose N-glycan-specific lectin from the red alga Kappaphycus striatum (Carrageenophyte).

    Science.gov (United States)

    Hung, Le Dinh; Sato, Yuichiro; Hori, Kanji

    2011-06-01

    From a fresh sample (1 kg) of cultivated red alga Kappaphycus striatum, three isolectins, KSA-1 (15.1 mg), KSA-2 (58.0 mg) and KSA-3 (6.9 mg), were isolated by a combination of extraction with aqueous ethanol, ethanol precipitation, and ion exchange chromatography. Isolated KSAs were monomeric proteins of about 28kDa having identical 20N-terminal amino acid sequences to each other. Their hemagglutination activities were not inhibited by monosaccharides, but inhibited by glycoproteins bearing high-mannose N-glycans. In a binding experiment with pyridylaminated oligosaccharides by centrifugal ultrafiltration-HPLC assay, the isolectin KSA-2 was exclusively bound to high-mannose type N-glycans, but not to other glycans. Including complex types and a pentasaccharide core of N-glycans, indicating that it recognized branched oligomannosides. The binding activity of KSA-2 was slightly different among high-mannose N-glycans examined, indicating that the lectin has a higher affinity for those having the exposed (α1-3) Man in the D2 arm. On the other hand, KSA-2 did not bind to a free oligomannose that is a constituent of the branched oligomannosides, implying that the portion of the core GlcNAc residue(s) of the N-glycans is also essential for binding. Thus, KSA-2 appears to recognize the extended carbohydrate structure with a minimal length of a tetrasaccharide, Man(α1-3)Man(α1-6)Man(β1-4)GlcNAc. This study indicates that K. striatum, which has extensively been cultivated as a source of carrageenan, is a good source of a valuable lectin(s) that is strictly specific for high-mannose N-glycans.

  16. LC-MS/MS analysis of permethylated free oligosaccharides and N-glycans derived from human, bovine, and goat milk samples.

    Science.gov (United States)

    Dong, Xue; Zhou, Shiyue; Mechref, Yehia

    2016-06-01

    Oligosaccharides in milk not only provide nutrition to the infants but also have significant immune biofunctions such as inhibition of pathogen binding to the host cell. The main component in milk oligosaccharides is free oligosaccharides. Since the proteins in milk are highly glycosylated, N-glycans in milk also play an import role. In this study, we investigated the permethylated free oligosaccharides and N-glycans extracted from bovine, goat, and human milks using LC-MS/MS. Quantitation profiles of free oligosaccharides and N-glycans were reported. The number of free oligosaccharides observed in bovine, goat, and human milk samples (without isomeric consideration) were 11, 8, and 11, respectively. Human milk had more complex free oligosaccharides structures than the other two milk samples. Totally 58, 21, and 43 N-glycan structures (without isomeric consideration) were associated with whey proteins extracted from bovine, goat, and human milk samples, respectively. Bovine milk free oligosaccharides and N-glycans from whey proteins were highly sialylated and to a lesser extend fucosylated. Goat and human milk free oligosaccharides and N-glycans from whey proteins were both highly fucosylated. Also, the isomeric glycans in milk samples were determined by porous graphitic carbon LC at elevated temperatures. For example, separation of human milk free oligosaccharide Gal-GlcNAc-(Fuc)-Gal-Glc and Gal-GlcNAc-Gal-Glc-Fuc isomers was achieved using porous graphitic carbon column. Permethylation of the glycan structures facilitated the interpretation of MS/MS. For example, internal cleavage and glycosidic bond cleavage are readily distinguished in the tandem mass spectra of permethylated glycans. This feature resulted in the identification of several isomers.

  17. Synthesis of core-shell hydrophilic polymer-silica hybrid material and its application in N-glycan enrichment%新型核壳型亲水聚合物-硅胶杂化填料的合成及其在蛋白质N-糖链富集中的应用

    Institute of Scientific and Technical Information of China (English)

    白海红; 范超; 沈丙权; 田芳; 邓玉林; 潘一廷; 秦伟捷; 钱小红

    2015-01-01

    蛋白质的 N-糖基化修饰与多种重要的生理、病理进程密切相关,是多种重大疾病诊断标志物研究的热点。由于糖蛋白本身多是低丰度表达的蛋白质,且糖链结构具有高度微不均一性,这使得蛋白质糖基化修饰的分析具有一定的挑战。本研究利用表面引发-原子转移自由基聚合( SI-ATRP )法,以带有双键的氨基葡萄糖为单体(GMAG),成功制备了新型核壳型亲水聚合物-硅胶杂化填料(pGMAG-SiO2)。由于在硅胶表面引入致密的亲水聚合物层,该填料不仅保持了硅胶良好的机械强度,而且显著提高了其亲水性,因此非常适合作为亲水填料用于蛋白质的N-糖链富集。以麦芽七糖和鸡卵清蛋白的N-糖链为研究对象,考察了该填料对N-糖链的富集效果,并将该杂化填料成功用于人血浆中糖蛋白 N-糖链的富集检测,共鉴定了47种糖型。以上结果表明,pGMAG-SiO2填料对N-糖链具有较高的亲和性,可以用于 N-糖链的高覆盖率鉴定。%Protein N-glycosylation is one of the most important post-translational modifications closely correlated with many important biological and pathological processes. The structural alterations of N-linked glycans in glycoproteins are always associated with many diseases,such as diabetes,heart failure and malignant tumors. Therefore,it is very important to establish sen-sitive methods for high-throughput N-glycan profiling. However,the low abundance of the N-glycoproteins and the heterogeneity of the N-glycans make it a challenge to analyse the pro-tein glycosylation sensitively. In this work,we had synthesized core-shell hydrophilic polymer-silica hybrid materials(pGMAG-SiO2)for the efficient enrichment of protein N-glycans. Firstly, pGMAG-SiO2 was prepared by in situ growth of glucose polymer on the surface of silica microp-articles using surface-initiated atom transfer radical polymerization( SI

  18. Tegument Glycoproteins and Cathepsins of Newly Excysted Juvenile Fasciola hepatica Carry Mannosidic and Paucimannosidic N-glycans.

    Science.gov (United States)

    Garcia-Campos, Andres; Ravidà, Alessandra; Nguyen, D Linh; Cwiklinski, Krystyna; Dalton, John P; Hokke, Cornelis H; O'Neill, Sandra; Mulcahy, Grace

    2016-05-01

    Recently, the prevalence of Fasciola hepatica in some areas has increased considerably and the availability of a vaccine to protect livestock from infection would represent a major advance in tools available for controlling this disease. To date, most vaccine-target discovery research on this parasite has concentrated on proteomic and transcriptomic approaches whereas little work has been carried out on glycosylation. As the F. hepatica tegument (Teg) may contain glycans potentially relevant to vaccine development and the Newly Excysted Juvenile (NEJ) is the first lifecycle stage in contact with the definitive host, our work has focused on assessing the glycosylation of the NEJTeg and identifying the NEJTeg glycoprotein repertoire. After in vitro excystation, NEJ were fixed and NEJTeg was extracted. Matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of released N-glycans revealed that oligomannose and core-fucosylated truncated N-glycans were the most dominant glycan types. By lectin binding studies these glycans were identified mainly on the NEJ surface, together with the oral and ventral suckers. NEJTeg glycoproteins were affinity purified after targeted biotinylation of the glycans and identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS). From the total set of proteins previously identified in NEJTeg, eighteen were also detected in the glycosylated fraction, including the F. hepatica Cathepsin B3 (FhCB3) and two of the Cathepsin L3 (FhCL3) proteins, among others. To confirm glycosylation of cathepsins, analysis at the glycopeptide level by LC-ESI-ion-trap-MS/MS with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) was carried out. We established that cathepsin B1 (FhCB1) on position N80, and FhCL3 (BN1106_s10139B000014, scaffold10139) on position N153, carry unusual paucimannosidic Man2GlcNAc2 glycans. To our knowledge, this is the first description of F

  19. Tegument Glycoproteins and Cathepsins of Newly Excysted Juvenile Fasciola hepatica Carry Mannosidic and Paucimannosidic N-glycans.

    Directory of Open Access Journals (Sweden)

    Andres Garcia-Campos

    2016-05-01

    Full Text Available Recently, the prevalence of Fasciola hepatica in some areas has increased considerably and the availability of a vaccine to protect livestock from infection would represent a major advance in tools available for controlling this disease. To date, most vaccine-target discovery research on this parasite has concentrated on proteomic and transcriptomic approaches whereas little work has been carried out on glycosylation. As the F. hepatica tegument (Teg may contain glycans potentially relevant to vaccine development and the Newly Excysted Juvenile (NEJ is the first lifecycle stage in contact with the definitive host, our work has focused on assessing the glycosylation of the NEJTeg and identifying the NEJTeg glycoprotein repertoire. After in vitro excystation, NEJ were fixed and NEJTeg was extracted. Matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS analysis of released N-glycans revealed that oligomannose and core-fucosylated truncated N-glycans were the most dominant glycan types. By lectin binding studies these glycans were identified mainly on the NEJ surface, together with the oral and ventral suckers. NEJTeg glycoproteins were affinity purified after targeted biotinylation of the glycans and identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS. From the total set of proteins previously identified in NEJTeg, eighteen were also detected in the glycosylated fraction, including the F. hepatica Cathepsin B3 (FhCB3 and two of the Cathepsin L3 (FhCL3 proteins, among others. To confirm glycosylation of cathepsins, analysis at the glycopeptide level by LC-ESI-ion-trap-MS/MS with collision-induced dissociation (CID and electron-transfer dissociation (ETD was carried out. We established that cathepsin B1 (FhCB1 on position N80, and FhCL3 (BN1106_s10139B000014, scaffold10139 on position N153, carry unusual paucimannosidic Man2GlcNAc2 glycans. To our knowledge, this is the first description

  20. Comparisons of Caenorhabditis Fucosyltransferase Mutants Reveal a Multiplicity of Isomeric N-Glycan Structures.

    Science.gov (United States)

    Yan, Shi; Jin, Chunsheng; Wilson, Iain B H; Paschinger, Katharina

    2015-12-01

    Recent studies have shown a remarkable degree of plasticity in the N-glycome of the model nematode Caenorhabditis elegans; ablation of glycosylation-relevant genes can result in radically altered N-glycan profiles despite only minor biological phenotypic effects. Up to four fucose residues and five different linkages of fucose are known on the N-glycans of C. elegans. Due to the complexity in the wild type, we established three mutant strains defective in two core fucosyltransferases each (fut-1;fut-6, fut-1;fut-8, and fut-6;fut-8). Enzymatically released N-glycans were subject to HPLC and MALDI-TOF MS/MS, in combination with various treatments, to verify structural details. The N-glycome of the fut-1;fut-6 mutant was the most complex of the three double-mutant strains due to the extension of the core α1,6-fucose as well as the presence of fucose on the bisecting galactose. In contrast, maximally two fucoses were found on N-glycans of the fut-1;fut-8 and fut-6;fut-8 strains. The different locations and capping of fucose meant that up to 13 isomeric structures, many highly galactosylated, were determined for some single masses. These data not only show the high variability of the N-glycomic capacity of a "simple" nematode but also exemplify the need for multiple approaches to reveal individual glycan structures within complex invertebrate glycomes.

  1. Predominant Expression of Hybrid N-Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N-Glycans

    Directory of Open Access Journals (Sweden)

    M. Kristen Hall

    2016-06-01

    Full Text Available Glycosylation modulates growth, maintenance, and stress signaling processes. Consequently, altered N-glycosylation is associated with reduced fitness and disease. Therefore, expanding our understanding of N-glycans in altering biological processes is of utmost interest. Herein, clustered regularly interspaced short palindromic repeats/caspase9 (CRISPR/Cas9 technology was employed to engineer a glycosylation mutant Chinese Hamster Ovary (CHO cell line, K16, which expresses predominantly hybrid type N-glycans. This newly engineered cell line enabled us to compare N-glycan effects on cellular properties of hybrid type N-glycans, to the well-established Pro−5 and Lec1 cell lines, which express complex and oligomannose types of N-glycans, respectively. Lectin binding studies revealed the predominant N-glycan expressed in K16 is hybrid type. Cell dissociation and migration assays demonstrated the greatest strength of cell–cell adhesion and fastest migratory rates for oligomannose N-glycans, and these properties decreased as oligomannose type were converted to hybrid type, and further decreased upon conversion to complex type. Next, we examined the roles of three general types of N-glycans on ectopic expression of E-cadherin, a cell–cell adhesion protein. Microscopy revealed more functional E-cadherin at the cell–cell border when N-glycans were oligomannose and these levels decreased as the oligomannose N-glycans were processed to hybrid and then to complex. Thus, we provide evidence that all three general types of N-glycans impact plasma membrane architecture and cellular properties.

  2. N-glycan abnormalities in children with galactosemia.

    Science.gov (United States)

    Coss, Karen P; Hawkes, Colin P; Adamczyk, Barbara; Stöckmann, Henning; Crushell, Ellen; Saldova, Radka; Knerr, Ina; Rubio-Gozalbo, Maria E; Monavari, Ardeshir A; Rudd, Pauline M; Treacy, Eileen P

    2014-02-07

    Galactose intoxication and over-restriction in galactosemia may affect glycosylation pathways and cause multisystem effects. In this study, we describe an applied hydrophilic interaction chromatography ultra-performance liquid chromatography high-throughput method to analyze whole serum and extracted IgG N-glycans with measurement of agalactosylated (G0), monogalactosylated (G1), and digalactosylated (G2) structures as a quantitative measure of galactose incorporation. This was applied to nine children with severe galactosemia (genotype Q188R/Q188R) and one child with a milder variant (genotype S135L/S135L). The profiles were also compared with those obtained from three age-matched children with PMM2-CDG (congenital disorder of glycosylation type Ia) and nine pediatric control samples. We have observed that severe N-glycan assembly defects correct in the neonate following dietary restriction of galactose. However, treated adult galactosemia patients continue to exhibit ongoing N-glycan processing defects. We have now applied informative galactose incorporation ratios as a method of studying the presence of N-glycan processing defects in children with galactosemia. We identified N-glycan processing defects present in galactosemia children from an early age. For G0/G1, G0/G2, and (G0/G1)/G2 ratios, the difference noted between galactosemia patients and controls was found to be statistically significant (p = 0.002, 0.01, and 0.006, respectively).

  3. A novel core fucose-specific lectin from the mushroom Pholiota squarrosa.

    Science.gov (United States)

    Kobayashi, Yuka; Tateno, Hiroaki; Dohra, Hideo; Moriwaki, Kenta; Miyoshi, Eiji; Hirabayashi, Jun; Kawagishi, Hirokazu

    2012-10-05

    Fucα1-6 oligosaccharide has a variety of biological functions and serves as a biomarker for hepatocellular carcinoma because of the elevated presence of fucosylated α-fetoprotein (AFP) in this type of cancer. In this study we purified a novel Fucα1-6-specific lectin from the mushroom Pholiota squarrosa by ion-exchange chromatography and affinity chromatography on thyroglobulin-agarose. The purified lectin was designated as PhoSL (P. squarrosa lectin). SDS-PAGE, MALDI-TOF mass spectrometry, and N-terminal amino acid sequencing indicate that PhoSL has a molecular mass of 4.5 kDa and consists of 40 amino acids (NH(2)-APVPVTKLVCDGDTYKCTAYLDFGDGRWVAQWDTNVFHTG-OH). Isoelectric focusing of the lectin showed bands near pI 4.0. The lectin activity was stable between pH 2.0 and 11.0 and at temperatures ranging from 0 to 100 °C for incubation times of 30 min. When PhoSL was investigated with frontal affinity chromatography using 132 pyridylaminated oligosaccharides, it was found that the lectin binds only to core α1-6-fucosylated N-glycans and not to other types of fucosylated oligosaccharides, such as α1-2-, α1-3-, and α1-4-fucosylated glycans. Furthermore, PhoSL bound to α1-6-fucosylated AFP but not to non-fucosylated AFP. In addition, PhoSL was able to demonstrate the differential expression of α1-6 fucosylation between primary and metastatic colon cancer tissues. Thus, PhoSL will be a promising tool for analyzing the biological functions of α1-6 fucosylation and evaluating Fucα1-6 oligosaccharides as cancer biomarkers.

  4. Strict specificity for high-mannose type N-glycans and primary structure of a red alga Eucheuma serra lectin.

    Science.gov (United States)

    Hori, Kanji; Sato, Yuichiro; Ito, Kaori; Fujiwara, Yoshifumi; Iwamoto, Yasumasa; Makino, Hiroyuki; Kawakubo, Akihiro

    2007-05-01

    We have elucidated the carbohydrate-binding profile of a non-monosaccharide-binding lectin named Eucheuma serra lectin (ESA)-2 from the red alga Eucheuma serra using a lectin-immobilized column and a centrifugal ultrafiltration-high performance liquid chromatography method with a variety of fluorescence-labeled oligosaccharides. In both methods, ESA-2 exclusively bound with high-mannose type (HM) N-glycans, but not with any of other N-glycans including complex type, hybrid type and core pentasaccharides, and oligosaccharides from glycolipids. These findings indicate that ESA-2 recognizes the branched oligomannosides of the N-glycans. However, ESA-2 did not bind with any of the free oligomannoses examined that are constituents of the branched oligomannosides implying that the portion of the core N-acetyl-D-glucosamine (GlcNAc) residue(s) of the N-glycans is also essential for binding. Thus, the algal lectin was strictly specific for HM N-glycans and recognized the extended carbohydrate structure with a minimum size of the pentasaccharide, Man(alpha1-3)Man(alpha1-6)Man(beta1-4)GlcNAc(beta1-4) GlcNAc. Kinetic analysis of binding with a HM heptasaccharide (M5) showed that ESA-2 has four carbohydrate-binding sites per polypeptide with a high association constant of 1.6x10(8) M-1. Sequence analysis, by a combination of Edman degradation and mass analyses of the intact protein and of peptides produced by its enzymic digestions, showed that ESA-2 is composed of 268 amino acids (molecular weight 27950) with four tandemly repeated domains of 67 amino acids. The number of repeats coincided with the number of carbohydrate-binding sites in the monomeric molecule. Surprisingly, the marine algal lectin was homologous to hemagglutinin from the soil bacterium Myxococcus xanthus.

  5. N-glycan transition of the early developmental stage in Oryza sativa.

    Science.gov (United States)

    Horiuchi, Risa; Hirotsu, Naoki; Miyanishi, Nobumitsu

    2016-08-26

    N-Glycosylation is one of the post-translational modifications. In animals, N-glycans linked to proteins function in cell-cell recognition, sorting, transport, and other biological phenomena. However, in plants, N-glycan-mediated biological functions remain obscure. In a previous study, we showed that the main type of N-glycan transition is from the paucimannosidic to complex type before and after germination in Oryza sativa, suggesting that transitions of N-glycan, including those of glycoproteins and glycosyltransferases, are closely associated with plant growth. To further elucidate the relationship between N-glycan structure and plant growth, we analyzed the structures of N-glycans expressed in O. sativa seedlings grown under light conditions and performed comparative analyses of the structures in the shoot and root. The analyses show that fundamental N-glycan structures are common to the shoot and root, whereas paucimannosidic-type N-glycans dramatically decreased in the root grown under light conditions. Further, to investigate the effects of light on N-glycan structures in O. sativa seedlings, we analyzed N-glycan structures in O. sativa seedlings grown in the dark. Understandably, N-glycan expression in the root was almost unaffected by light. However, despite a marked difference in phenotype, N-glycan expression in the shoot was also unaffected by light. This result suggests that the shoot and root of O. sativa have different glycoproteins and distinct N-glycan synthetic systems. Thus, we propose that the N-glycan synthetic system of the O. sativa shoot is almost unaffected by light conditions and that many photosynthesis-related proteins are not modified by N-glycans.

  6. Establishment and application of a method for the detection of the core fucosylated IgG level%IgG核心岩藻糖基化水平检测方法的建立及应用

    Institute of Scientific and Technical Information of China (English)

    衣常红; 王坤; 李慧; 高春芳

    2011-01-01

    To build up a method for the detection of the core fucosylated IgG level and to figure out the relationship between the core fucosylated level and aberrance of high IgG concentration in HBV-related liver cirrhosis, the core fucosylated serum protein was purified with specific core fucose binding lectin LCA agglutinin affinity column.and then the levels of the core fucosylated total protein (TP) and IgG of patients with HBV-related liver cirrhosis were calculated and analyzed.It was found that the level of LCA binding IgG was higher in patients with HBV-related liver cirrhosis in comparison with that of controls and chronic hepatitis B patients (P<0.05).This seemed to be a valuable alternative for the identification of HBV-related liver cirrhosis.The increased level of core fucosylated IgG may be attribute partially or totally to the attenuated function of IgG.This method of testing the core fucosylated IgG level is promising to be a noninvasive serum marker for the diagnosis and may be a potential therapeutic target for the immune regulation in liver cirrhosis.%为建立IgG核心岩藻糖基化水平检测方法,研究血清IgG核心岩藻糖基化水平与慢性乙肝(CHB)、肝硬化(LC)中IgG异常增高的关系.应用特异性结合核心岩藻糖的LCA凝集素亲和柱分离纯化血清中具有的核心岩藻糖基化蛋白,并对CHB、LC患者的血清总蛋白(TP)和免疫球蛋白G(IgG)的核心岩藻糖基化水平进行检测和对比分析.结果显示:LC患者IgG及其核心岩藻精基化水平较正常组和CHB组均升高且存在统计学差异(P<0.05),并对LC具有诊断提示作用.表明:IgG核心岩藻糖糖基化水平的增高可能是LC中IgG异常增高和IgG功能部分丧失的原因之一,有望成为一个新的无创性肝纤维化监测、诊断指标,也为可能的免疫调控干预提供了潜在的治疗靶点.

  7. Engineering the Campylobacter jejuni N-glycan to create an effective chicken vaccine

    Science.gov (United States)

    Nothaft, Harald; Davis, Brandi; Lock, Yee Ying; Perez-Munoz, Maria Elisa; Vinogradov, Evgeny; Walter, Jens; Coros, Colin; Szymanski, Christine M.

    2016-01-01

    Campylobacter jejuni is a predominant cause of human gastroenteritis worldwide. Source-attribution studies indicate that chickens are the main reservoir for infection, thus elimination of C. jejuni from poultry would significantly reduce the burden of human disease. We constructed glycoconjugate vaccines combining the conserved C. jejuni N-glycan with a protein carrier, GlycoTag, or fused to the Escherichia coli lipopolysaccharide-core. Vaccination of chickens with the protein-based or E. coli-displayed glycoconjugate showed up to 10-log reduction in C. jejuni colonization and induced N-glycan-specific IgY responses. Moreover, the live E. coli vaccine was cleared prior to C. jejuni challenge and no selection for resistant campylobacter variants was observed. Analyses of the chicken gut communities revealed that the live vaccine did not alter the composition or complexity of the microbiome, thus representing an effective and low-cost strategy to reduce C. jejuni in chickens and its subsequent entry into the food chain. PMID:27221144

  8. Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Zhengliang L., E-mail: Leon.wu@bio-techne.com [Bio-Techne Inc., 614 McKinley Place NE, Minneapolis, MN 55413 (United States); Zhou, Hui [Gregg Hall, UNH Glycomics Center, University of New Hampshire (United States); Ethen, Cheryl M. [Bio-Techne Inc., 614 McKinley Place NE, Minneapolis, MN 55413 (United States); Reinhold, Vernon N., E-mail: Vernon.Reinhold@unh.edu [Gregg Hall, UNH Glycomics Center, University of New Hampshire (United States)

    2016-04-29

    The 1918 H1N1 influenza virus was responsible for one of the most deadly pandemics in human history. Yet to date, the structure component responsible for its virulence is still a mystery. In order to search for such a component, the neuraminidase (NA) antigen of the virus was expressed, which led to the discovery of an active form (tetramer) and an inactive form (dimer and monomer) of the protein due to different glycosylation. In this report, the N-glycans from both forms were released and characterized by mass spectrometry. It was found that the glycans from the active form had 26% core-6 fucosylated, while the glycans from the inactive form had 82% core-6 fucosylated. Even more surprisingly, the stalk region of the active form was almost completely devoid of core-6-linked fucose. These findings were further supported by the results obtained from in vitro incorporation of azido fucose and {sup 3}H-labeled fucose using core-6 fucosyltransferase, FUT8. In addition, the incorporation of fucose did not change the enzymatic activity of the active form, implying that core-6 fucose is not directly involved in the enzymatic activity. It is postulated that core-6 fucose prohibits the oligomerization and subsequent activation of the enzyme. - Graphical abstract: Proposed mechanism for how core-fucose prohibits the tetramerization of the 1918 pandemic viral neuraminidase. Only the cross section of the stalk region with two N-linked glycans are depicted for clarity. (A) Carbohydrate–carbohydrate interaction on non-fucosylated monomer allows tetramerization. (B) Core-fucosylation disrupts the interaction and prevents the tetramerization. - Highlights: • Expressed 1918 pandemic influenza viral neuraminidase has inactive and active forms. • The inactive form contains high level of core-6 fucose, while the active form lacks such modification. • Core fucose could interfere the oligomerization of the neuraminidase and thus its activation. • This discovery may explain

  9. Piperidine-based glycodendrons as protein N-glycan prosthetics.

    Science.gov (United States)

    Hudak, Jason E; Belardi, Brian; Appel, Mason J; Solania, Angelo; Robinson, Peter V; Bertozzi, Carolyn R

    2016-10-15

    The generation of homogeneously glycosylated proteins is essential for defining glycoform-specific activity and improving protein-based therapeutics. We present a novel glycodendron prosthetic which can be site-selectively appended to recombinant proteins to create 'N-glycosylated' glycoprotein mimics. Using computational modeling, we designed the dendrimer scaffold and protein attachment point to resemble the native N-glycan architecture. Three piperidine-melamine glycodendrimers were synthesized via a chemoenzymatic route and attached to human growth hormone and the Fc region of human IgG. These products represent a new class of engineered biosimilars bearing novel glycodendrimer structures.

  10. Intramolecular N-glycan/polypeptide interactions observed at multiple N-glycan remodeling steps through [(13)C,(15)N]-N-acetylglucosamine labeling of immunoglobulin G1.

    Science.gov (United States)

    Barb, Adam W

    2015-01-20

    Asparagine-linked (N) glycosylation is a common eukaryotic protein modification that affects protein folding, function, and stability through intramolecular interactions between N-glycan and polypeptide residues. Attempts to characterize the structure-activity relationship of each N-glycan are hindered by inherent properties of the glycoprotein, including glycan conformational and compositional heterogeneity. These limitations can be addressed by using a combination of nuclear magnetic resonance techniques following enzymatic glycan remodeling to simultaneously generate homogeneous glycoforms. However, widely applicable methods do not yet exist. To address this technological gap, immature glycoforms of the immunoglobulin G1 fragment crystallizable (Fc) were isolated in a homogeneous state and enzymatically remodeled with [(13)C,(15)N]-N-acetylglucosamine (GlcNAc). UDP-[(13)C,(15)N]GlcNAc was synthesized enzymatically in a one-pot reaction from [(13)C]glucose and [(15)N-amido]glutamine. Modifying Fc with recombinantly expressed glycosyltransferases (Gnt1 and Gnt2) and UDP-[(13)C,(15)N]GlcNAc resulted in complete glycoform conversion as judged by mass spectrometry. Two-dimensional heteronuclear single-quantum coherence spectra of the Gnt1 product, containing a single [(13)C,(15)N]GlcNAc residue on each N-glycan, showed that the N-glycan is stabilized through interactions with polypeptide residues. Similar spectra of homogeneous glycoforms, halted at different points along the N-glycan remodeling pathway, revealed the presence of an increased level of interaction between the N-glycan and polypeptide at each step, including mannose trimming, as the N-glycan was converted to a complex-type, biantennary form. Thus, conformational restriction increases as Fc N-glycan maturation proceeds. Gnt1 and Gnt2 catalyze fundamental reactions in the synthesis of every glycoprotein with a complex-type N-glycan; thus, the strategies presented herein can be applied to a broad range of

  11. Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [13C,15N]-N-Acetylglucosamine Labeling of Immunoglobulin G1

    Science.gov (United States)

    2014-01-01

    Asparagine-linked (N) glycosylation is a common eukaryotic protein modification that affects protein folding, function, and stability through intramolecular interactions between N-glycan and polypeptide residues. Attempts to characterize the structure–activity relationship of each N-glycan are hindered by inherent properties of the glycoprotein, including glycan conformational and compositional heterogeneity. These limitations can be addressed by using a combination of nuclear magnetic resonance techniques following enzymatic glycan remodeling to simultaneously generate homogeneous glycoforms. However, widely applicable methods do not yet exist. To address this technological gap, immature glycoforms of the immunoglobulin G1 fragment crystallizable (Fc) were isolated in a homogeneous state and enzymatically remodeled with [13C,15N]-N-acetylglucosamine (GlcNAc). UDP-[13C,15N]GlcNAc was synthesized enzymatically in a one-pot reaction from [13C]glucose and [15N-amido]glutamine. Modifying Fc with recombinantly expressed glycosyltransferases (Gnt1 and Gnt2) and UDP-[13C,15N]GlcNAc resulted in complete glycoform conversion as judged by mass spectrometry. Two-dimensional heteronuclear single-quantum coherence spectra of the Gnt1 product, containing a single [13C,15N]GlcNAc residue on each N-glycan, showed that the N-glycan is stabilized through interactions with polypeptide residues. Similar spectra of homogeneous glycoforms, halted at different points along the N-glycan remodeling pathway, revealed the presence of an increased level of interaction between the N-glycan and polypeptide at each step, including mannose trimming, as the N-glycan was converted to a complex-type, biantennary form. Thus, conformational restriction increases as Fc N-glycan maturation proceeds. Gnt1 and Gnt2 catalyze fundamental reactions in the synthesis of every glycoprotein with a complex-type N-glycan; thus, the strategies presented herein can be applied to a broad range of glycoprotein

  12. Two pathways for importing GDP-fucose into the endoplasmic reticulum lumen function redundantly in the O-fucosylation of Notch in Drosophila.

    Science.gov (United States)

    Ishikawa, Hiroyuki O; Ayukawa, Tomonori; Nakayama, Minoru; Higashi, Shunsuke; Kamiyama, Shin; Nishihara, Shoko; Aoki, Kazuhisa; Ishida, Nobuhiro; Sanai, Yutaka; Matsuno, Kenji

    2010-02-05

    Notch is a transmembrane receptor that shares homology with proteins containing epidermal growth factor-like repeats and mediates the cell-cell interactions necessary for many cell fate decisions. In Drosophila, O-fucosyltransferase 1 catalyzes the O-fucosylation of these epidermal growth factor-like repeats. This O-fucose elongates, resulting in an O-linked tetrasaccharide that regulates the signaling activities of Notch. Fucosyltransferases utilize GDP-fucose, which is synthesized in the cytosol, but fucosylation occurs in the lumen of the endoplasmic reticulum (ER) and Golgi. Therefore, GDP-fucose uptake into the ER and Golgi is essential for fucosylation. However, although GDP-fucose biosynthesis is well understood, the mechanisms and intracellular routes of GDP-fucose transportation remain unclear. Our previous study on the Drosophila Golgi GDP-fucose transporter (Gfr), which specifically localizes to the Golgi, suggested that another GDP-fucose transporter(s) exists in Drosophila. Here, we identified Efr (ER GDP-fucose transporter), a GDP-fucose transporter that localizes specifically to the ER. Efr is a multifunctional nucleotide sugar transporter involved in the biosynthesis of heparan sulfate-glycosaminoglycan chains and the O-fucosylation of Notch. Comparison of the fucosylation defects in the N-glycans in Gfr and Efr mutants revealed that Gfr and Efr made distinct contributions to this modification; Gfr but not Efr was crucial for the fucosylation of N-glycans. We also found that Gfr and Efr function redundantly in the O-fucosylation of Notch, although they had different localizations and nucleotide sugar transportation specificities. These results indicate that two pathways for the nucleotide sugar supply, involving two nucleotide sugar transporters with distinct characteristics and distributions, contribute to the O-fucosylation of Notch.

  13. Systems analysis of N-glycan processing in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Patrick Hossler

    Full Text Available N-glycosylation plays a key role in the quality of many therapeutic glycoprotein biologics. The biosynthesis reactions of these oligosaccharides are a type of network in which a relatively small number of enzymes give rise to a large number of N-glycans as the reaction intermediates and terminal products. Multiple glycans appear on the glycoprotein molecules and give rise to a heterogeneous product. Controlling the glycan distribution is critical to the quality control of the product. Understanding N-glycan biosynthesis and the etiology of microheterogeneity would provide physiological insights, and facilitate cellular engineering to enhance glycoprotein quality. We developed a mathematical model of glycan biosynthesis in the Golgi and analyzed the various reaction variables on the resulting glycan distribution. The Golgi model was modeled as four compartments in series. The mechanism of protein transport across the Golgi is still controversial. From the viewpoint of their holding time distribution characteristics, the two main hypothesized mechanisms, vesicular transport and Golgi maturation models, resemble four continuous mixing-tanks (4CSTR and four plug-flow reactors (4PFR in series, respectively. The two hypotheses were modeled accordingly and compared. The intrinsic reaction kinetics were first evaluated using a batch (or single PFR reactor. A sufficient holding time is needed to produce terminally-processed glycans. Altering enzyme concentrations has a complex effect on the final glycan distribution, as the changes often affect many reaction steps in the network. Comparison of the glycan profiles predicted by the 4CSTR and 4PFR models points to the 4PFR system as more likely to be the true mechanism. To assess whether glycan heterogeneity can be eliminated in the biosynthesis of biotherapeutics the 4PFR model was further used to assess whether a homogeneous glycan profile can be created through metabolic engineering. We demonstrate by

  14. Mass + retention time = structure: a strategy for the analysis of N-glycans by carbon LC-ESI-MS and its application to fibrin N-glycans.

    Science.gov (United States)

    Pabst, Martin; Bondili, Jayakumar Singh; Stadlmann, Johannes; Mach, Lukas; Altmann, Friedrich

    2007-07-01

    Analysis of the numerous possible, often isobaric structures of protein-bound oligosaccharides calls for a high-performance two-dimensional method that combines liquid chromatography's ability to separate isomers and mass spectrometry's ability to determine glycan composition. Here we investigate the usefulness of porous graphitic carbon columns coupled to ESI-MS for the separation of N-glycans with two or more sialic acids. Internal standards helped to rectify retention time fluctuations and thus allowed elution times to play an essential role in the structural assignment of peaks. For generation of a retention time library, standards representing the possible isomers of diantennary non-, mono-, and disialylated N-glycans, differing in the linkage of galactose and sialic acids as well as isobaric hybrid-type N-glycans, were produced using recombinant glycosyltransferases. Once the retention times library was established, isomers could be identified by LC-ESI-MS in the positive mode without additional MS/MS experiments. The method was applied for the detailed structural analysis of fibrin(ogen) N-glycans from various species (human, cow, pig, mouse, rat, cat, dog, Chinese hamster, horse, sheep, and chicken). All fibrins contained diantennary N-glycans. They differed in the occurrence of beta1,3-linked galactose, alpha2,3-linked sialic acids, and N-glycolylneuraminic acid, in the mono/diantennary glycan ratio, and in the O-acetylation of neuraminic acids. The separation system's potential for analyzing tri- and tetrasialylated N-glycans was demonstrated.

  15. Improved sample preparation for CE-LIF analysis of plant N-glycans.

    Science.gov (United States)

    Nagels, Bieke; Santens, Francis; Weterings, Koen; Van Damme, Els J M; Callewaert, Nico

    2011-12-01

    In view of glycomics studies in plants, it is important to have sensitive tools that allow one to analyze and characterize the N-glycans present on plant proteins in different species. Earlier methods combined plant-based sample preparations with CE-LIF N-glycan analysis but suffered from background contaminations, often resulting in non-reproducible results. This publication describes a reproducible and sensitive protocol for the preparation and analysis of plant N-glycans, based on a combination of the 'in-gel release method' and N-glycan analysis on a multicapillary DNA sequencer. Our protocol makes it possible to analyze plant N-glycans starting from low amounts of plant material with highly reproducible results. The developed protocol was validated for different plant species and plant cells.

  16. Clinical implications of serum N-glycan profiling as a diagnostic and prognostic biomarker in germ-cell tumors.

    Science.gov (United States)

    Narita, Takuma; Hatakeyama, Shingo; Yoneyama, Tohru; Narita, Shintaro; Yamashita, Shinichi; Mitsuzuka, Koji; Sakurai, Toshihiko; Kawamura, Sadafumi; Tochigi, Tatsuo; Takahashi, Ippei; Nakaji, Shigeyuki; Tobisawa, Yuki; Yamamoto, Hayato; Koie, Takuya; Tsuchiya, Norihiko; Habuchi, Tomonori; Arai, Yoichi; Ohyama, Chikara

    2017-03-20

    Serum biomarker monitoring is essential for management of germ-cell tumors (GCT). However, not all GCT are positive for conventional tumor markers. We examined whether serum N-glycan-based biomarkers can be applied for detection and prognosis in patients with GCT. We performed a comprehensive N-glycan structural analysis of sera from 54 untreated GCT patients and 103 age-adjusted healthy volunteers using glycoblotting methods and mass spectrometry. Candidate N-glycans were selected from those with the highest association; cutoff concentration values were established, and an N-glycan score was created based on the number of positive N-glycans present. The validity of this score for diagnosis and prognosis was analyzed using a receiver operating characteristic (ROC) curve. We identified five candidate N-glycans significantly associated with GCT patients. The accuracy of the N-glycan score for GCT was significant with an area-under-the-curve (AUC) value of 0.87. Diagnostically, the N-glycan score detected 10 of 12 (83%) patients with negative conventional tumor markers. Prognostically, the N-glycan score comprised four candidate N-glycans. The predictive value of the prognostic N-glycan score was significant, with an AUC value of 0.89. A high value prognostic N-glycan score was significantly associated with poor prognosis. Finally, to identify a potential carrier protein, immunoglobulin (Ig) fractions of sera were subjected to N-glycan analysis and compared to whole sera. Candidate N-glycans in Ig-fractions were significantly decreased; therefore, the carrier protein for candidate N-glycans is likely not an immunoglobulin. In summary, our newly developed N-glycan score seems to be a practical diagnostic and prognostic method for GCT.

  17. Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk.

    Science.gov (United States)

    Gil, Geun-Cheol; Velander, William H; Van Cott, Kevin E

    2008-07-01

    Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) produced in the transgenic pig mammary gland were determined. The majority of the N-glycans of transgenic pig-derived Factor IX (tg-FIX) are complex, bi-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan structures of tg-FIX produced in the porcine mammary epithelial cells differed with respect to N-glycans from glycoproteins produced in other porcine tissues. tg-FIX contains no detectable Neu5Gc, the sialic acid commonly found in porcine glycoproteins produced in other tissues. Additionally, we were unable to detect glycans in tg-FIX that have a terminal Galalpha(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. The N-glycan structures of tg-FIX are also compared to the published N-glycan structures of recombinant human glycoproteins produced in other transgenic animal species. While tg-FIX contains only complex structures, antithrombin III (goat), C1 inhibitor (rabbit), and lactoferrin (cow) have both high mannose and complex structures. Collectively, these data represent a beginning point for the future investigation of species-specific and tissue/cell-specific differences in N-glycan structures among animals used for transgenic animal bioreactors.

  18. Analysis of site-specific N-glycan remodeling in the endoplasmic reticulum and the Golgi

    Science.gov (United States)

    Hang, Ivan; Lin, Chia-wei; Grant, Oliver C; Fleurkens, Susanna; Villiger, Thomas K; Soos, Miroslav; Morbidelli, Massimo; Woods, Robert J; Gauss, Robert; Aebi, Markus

    2015-01-01

    The hallmark of N-linked protein glycosylation is the generation of diverse glycan structures in the secretory pathway. Dynamic, non-template-driven processes of N-glycan remodeling in the endoplasmic reticulum and the Golgi provide the cellular setting for structural diversity. We applied newly developed mass spectrometry-based analytics to quantify site-specific N-glycan remodeling of the model protein Pdi1p expressed in insect cells. Molecular dynamics simulation, mutational analysis, kinetic studies of in vitro processing events and glycan flux analysis supported the defining role of the protein in N-glycan processing. PMID:26240167

  19. Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk

    OpenAIRE

    Gil, Geun-Cheol; Velander, William H.; Van Cott, Kevin E

    2008-01-01

    Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) produced in the transgenic pig mammary gland were determined. The majority of the N-glycans of transgenic pig-derived Factor IX (tg-FIX) are complex, bi-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan stru...

  20. Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase.

    Science.gov (United States)

    Wu, Zhengliang L; Zhou, Hui; Ethen, Cheryl M; N Reinhold, Vernon

    2016-04-29

    The 1918 H1N1 influenza virus was responsible for one of the most deadly pandemics in human history. Yet to date, the structure component responsible for its virulence is still a mystery. In order to search for such a component, the neuraminidase (NA) antigen of the virus was expressed, which led to the discovery of an active form (tetramer) and an inactive form (dimer and monomer) of the protein due to different glycosylation. In this report, the N-glycans from both forms were released and characterized by mass spectrometry. It was found that the glycans from the active form had 26% core-6 fucosylated, while the glycans from the inactive form had 82% core-6 fucosylated. Even more surprisingly, the stalk region of the active form was almost completely devoid of core-6-linked fucose. These findings were further supported by the results obtained from in vitro incorporation of azido fucose and (3)H-labeled fucose using core-6 fucosyltransferase, FUT8. In addition, the incorporation of fucose did not change the enzymatic activity of the active form, implying that core-6 fucose is not directly involved in the enzymatic activity. It is postulated that core-6 fucose prohibits the oligomerization and subsequent activation of the enzyme.

  1. Enzymes for N-Glycan Branching and Their Genetic and Nongenetic Regulation in Cancer

    Directory of Open Access Journals (Sweden)

    Yasuhiko Kizuka

    2016-04-01

    Full Text Available N-glycan, a fundamental and versatile protein modification in mammals, plays critical roles in various physiological and pathological events including cancer progression. The formation of N-glycan branches catalyzed by specific N-acetylglucosaminyltransferases [GnT-III, GnT-IVs, GnT-V, GnT-IX (Vb] and a fucosyltransferase, Fut8, provides functionally diverse N-glycosylated proteins. Aberrations of these branches are often found in cancer cells and are profoundly involved in cancer growth, invasion and metastasis. In this review, we focus on the GlcNAc and fucose branches of N-glycans and describe how their expression is dysregulated in cancer by genetic and nongenetic mechanisms including epigenetics and nucleotide sugar metabolisms. We also survey the roles that these N-glycans play in cancer progression and therapeutics. Finally, we discuss possible applications of our knowledge on basic glycobiology to the development of medicine and biomarkers for cancer therapy.

  2. The Mechanism by which 146-N-Glycan Affects the Active Site of Neuraminidase.

    Directory of Open Access Journals (Sweden)

    Pi Liu

    Full Text Available One of the most conserved glycosylation sites of neuraminidase (NA is 146-N-glycan. This site is adjacent to the 150-cavity of NA, which is found within the active site and thought to be a target for rational drug development against the antiviral resistance of influenza. Here, through a total of 2.4 μs molecular dynamics (MD simulations, we demonstrated that 146-N-glycan can stabilize the conformation of the 150-loop that controls the volume of the 150-cavity. Moreover, with 146-N-glycan, our simulation result was more consistent with crystal structures of NAs than simulations conducted without glycans. Cluster analysis of the MD trajectories showed that 146-N-glycan adopted three distinct conformations: monomer-bridged, dimer-bridged and standing. Of these conformations, the dimer-bridged 146-N-glycan was the most stable one and contributed to stabilization of the 150-loop conformation. Furthermore, our simulation revealed that various standing conformations of 146-N-glycan could block the entrance of the binding pocket. This result was consistent with experimental data and explained the relatively low activity of inhibitors with flexible substituents toward the 150-cavity. Together, our results lead us to hypothesize that rigid and hydrophobic substituents could serve as better inhibitors targeting the 150-cavity.

  3. N-glycans and metastasis in galectin-3 transgenic mice.

    Science.gov (United States)

    More, Shyam K; Srinivasan, Nithya; Budnar, Srikanth; Bane, Sanjay M; Upadhya, Archana; Thorat, Rahul A; Ingle, Arvind D; Chiplunkar, Shubhada V; Kalraiya, Rajiv D

    2015-05-01

    Poly-N-acetyl-lactosamine (polyLacNAc) on N-glycans facilitate lung specific metastasis of melanoma cells by serving as high affinity ligands for galectin-3, expressed in highest amounts in the lungs, on almost all its tissue compartments including on the surface of vascular endothelium. PolyLacNAc not only aids in initial arrest on the organ endothelium but in all the events of extravasation. Inhibition of polyLacNAc synthesis, or competitive inhibition of its interaction with galectin-3 all inhibited these processes and experimental metastasis. Transgenic galectin-3 mice, viz., gal-3(+/+) (wild type), gal-3(+/-) (hemizygous) and gal-3(-/-) (null) have been used to prove that galectin-3/polyLacNAc interactions are indeed critical for lung specific metastasis. Gal-3(+/-) mice which showed metastasis. However, surprisingly, the number and size of metastatic colonies in gal-3(-/-) mice was very similar as that seen in gal-3(+/+) mice. The levels of lactose binding lectins on the lungs and the transcripts of other galectins (galectin-1, -8 and -9) which are expressed on lungs and have similar sugar binding specificities as galectins-3, remain unchanged in gal-3(+/+) and gal-3(-/-) mice. Further, inhibition of N-glycosylation with Swainsonine (SW) which drastically reduces metastasis of B16F10 cells in gal-3(+/+) mice, did not affect lung metastasis when assessed in gal-3(-/-) mice. Together, these results rule out the possibility of some other galectin taking over the function of galectin-3 in gal-3(-/-) mice. Chimeric mice generated to assess if absence of any effect on metastasis is due to compromised tumor immunity by replacing bone marrow of gal-3(-/-) mice with that from gal-3(+/+) mice, also failed to impact melanoma metastasis. As galectin-3 regulates several immune functions including maturation of different immune cells, compromised tumor immunity could be the major determinant of melanoma metastasis in gal-3(-/-) mice and warrants thorough investigation.

  4. Construction of N-glycan microarrays by using modular synthesis and on-chip nanoscale enzymatic glycosylation.

    Science.gov (United States)

    Serna, Sonia; Etxebarria, Juan; Ruiz, Nerea; Martin-Lomas, Manuel; Reichardt, Niels-Christian

    2010-11-22

    An effective chemoenzymatic strategy is reported that has allowed the construction, for the first time, of a focused microarray of synthetic N-glycans. Based on modular approaches, a variety of N-glycan core structures have been chemically synthesized and covalently immobilized on a glass surface. The printed structures were then enzymatically diversified by the action of three different glycosyltransferases in nanodroplets placed on top of individual spots of the microarray by a printing robot. Conversion was followed by lectin binding specific for the terminal sugars. This enzymatic extension of surface-bound ligands in nanodroplets reduces the amount of precious glycosyltransferases needed by seven orders of magnitude relative to reactions carried out in the solution phase. Moreover, only those ligands that have been shown to be substrates to a specific glycosyltransferase can be individually chosen for elongation on the array. The methodology described here, combining focused modular synthesis and nanoscale on-chip enzymatic elongation, could open the way for the much needed rapid construction of large synthetic glycan arrays.

  5. Serum N-glycans outperform CA19-9 in diagnosis of extrahepatic cholangiocarcinoma.

    Science.gov (United States)

    Wang, Mengmeng; Fang, Meng; Zhu, Jianhui; Feng, Huijuan; Warner, Elisa; Yi, Changhong; Ji, Jun; Gu, Xing; Gao, Chunfang

    2017-07-28

    Extensive efforts have been devoted to improve the diagnosis of extrahepatic cholangiocarcinoma (ECCA) due to its silent clinical character and lack of effective diagnostic biomarkers. Specific alterations in N-glycosylation of glycoproteins are considered a key component in cancer progression, which can serve as a distinct molecular signature for cancer detection. This study aims to find potential serum N-glycan markers for ECCA. In total, 255 serum samples from patients with ECCA (n = 106), benign bile tract disease (BBD, n = 60) and healthy controls (HC, n = 89) were recruited. Only 2 μL of serum from individual patients was used in this assay where the N-glycome of serum glycoproteins was profiled by DNA sequencer-assisted fluorophore-assisted capillary electrophoresis (DSA-FACE) technology. Multi-parameter models were constructed by combining the N-glycans and carbohydrate antigen 19-9 (CA19-9) which is currently used clinically. Quantitative analyses showed that among 13 N-glycan structures, the bifucosylated triantennary N-glycan (peak10, NA3F2) presented the best diagnostic performance for distinguishing ECCA from BBD and HC. Two diagnostic models (Glycotest1 and Glycotest2) performed better than single N-glycan or CA19-9. Additionally, two N-glycan structures (peak9, NA3Fb; peak12, NA4Fb) were tightly related to lymph node metastasis in ECCA patients. In conclusion, sera of ECCA showed relatively specific N-glycome profiling patterns. Serum N-glycan markers and models are novel, valuable and noninvasive alternatives in ECCA diagnosis and progression monitoring. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. IgG N-glycans as potential biomarkers for determining galactose tolerance in Classical Galactosaemia.

    LENUS (Irish Health Repository)

    Coss, K P

    2012-02-01

    N-glycan processing and assembly defects have been demonstrated in untreated and partially treated patients with Classical Galactosaemia. These defects may contribute to the ongoing pathophysiology of this disease. The aim of this study was to develop an informative method of studying differential galactose tolerance levels and diet control in individuals with Galactosaemia, compared to the standard biochemical markers. Ten Galactosaemia adults with normal intellectual outcomes were analyzed in the study. Five subjects followed galactose liberalization, increments of 300 mg to 4000 mg\\/day over 16 weeks, and were compared to five adult Galactosaemia controls on a galactose restricted diet. All study subjects underwent clinical and biochemical monitoring of red blood cell galactose-1-phosphate (RBC Gal-1-P) and urinary galactitol levels. Serum N-glycans were isolated and analyzed by normal phase high-performance liquid chromatography (NP-HPLC) with galactosylation of IgG used as a specific biomarker of galactose tolerance. IgG N-glycan profiles showed consistent individual alterations in response to diet liberalization. The individual profiles were improved for all, but one study subject, at a galactose intake of 1000 mg\\/day, with decreases in agalactosylated (G0) and increases in digalactosylated (G2) N-glycans. We conclude that IgG N-glycan profiling is an improved method of monitoring variable galactosylation and determining individual galactose tolerance in Galactosaemia compared to the standard methods.

  7. A comparison of anti-HER2 IgA and IgG1 in vivo efficacy is facilitated by high N-glycan sialylation of the IgA.

    Science.gov (United States)

    Rouwendal, Gerard Ja; van der Lee, Miranda M; Meyer, Saskia; Reiding, Karli R; Schouten, Jan; de Roo, Guy; Egging, David F; Leusen, Jeanette Hw; Boross, Peter; Wuhrer, Manfred; Verheijden, Gijs F; Dokter, Wim H; Timmers, Marco; Ubink, Ruud

    2016-01-01

    Monomeric IgA has been proposed as an alternative antibody format for cancer therapy. Here, we present our studies on the production, purification and functional evaluation of anti-HER2 IgA antibodies as anti-cancer agents in comparison to the anti-HER2 IgG1 trastuzumab. MALDI-TOF MS analysis showed profound differences in glycosylation traits across the IgA isotypes and cell lines used for production, including sialylation and linkage thereof, fucosylation (both core and antennary) and the abundance of high-mannose type species. Increases in sialylation proved to positively correlate with in vivo plasma half-lives. The polymerization propensity of anti-HER2 IgA2m2 could be suppressed by an 18-aa deletion of the heavy chain tailpiece - coinciding with the loss of high-mannose type N-glycan species - as well as by 2 cysteine to serine mutations at positions 320 and 480. The HER2 F(ab')2-mediated anti-proliferative effect of the IgA2m1 and IgA2m2 subtypes was similar to IgG1, whereas the IgA1 isotype displayed considerably lower potency and efficacy. The Fc-mediated induction of antibody-dependent cell-mediated cytotoxicity (ADCC) using human whole blood ADCC assays did not demonstrate such clear differences between the IgA isotypes. However, the potency of the anti-HER2 IgA antibodies in these ADCC assays was found to be significantly lower than that of trastuzumab. In vivo anti-tumor activity of the anti-HER2 IgA antibodies was compared to that of trastuzumab in a BT-474 breast cancer xenograft model. Multiple dosing and sialylation of the IgA antibodies compensated for the short in vivo half-life of native IgA antibodies in mice compared to a single dose of IgG1. In the case of the IgA2m2 antibody, the resulting high plasma exposure levels were sufficient to cause clear tumor stasis comparable to that observed for trastuzumab at much lower plasma exposure levels.

  8. Novel Kv3 glycoforms differentially expressed in adult mammalian brain contain sialylated N-glycans.

    Science.gov (United States)

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

    2008-02-01

    The N-glycan pool of mammalian brain contains remarkably high levels of sialylated N-glycans. This study provides the first evidence that voltage-gated K+ channels Kv3.1, Kv3.3, and Kv3.4, possess distinct sialylated N-glycan structures throughout the central nervous system of the adult rat. Electrophoretic migration patterns of Kv3.1, Kv3.3, and Kv3.4 glycoproteins from spinal cord, hypothalamus, thalamus, cerebral cortex, hippocampus, and cerebellum membranes digested with glycosidases were used to identify the various glycoforms. Differences in the migration of Kv3 proteins were attributed to the desialylated N-glycans. Expression levels of the Kv3 proteins were highest in cerebellum, whereas those of Kv3.1 and Kv3.3 were much lower in the other 5 regions. The lowest level of Kv3.1 was expressed in the hypothalamus, whereas the lowest levels of Kv3.3 were expressed in both thalamus and hypothalamus. The other regions expressed intermediate levels of Kv3.3, with spinal cord expressing the highest. The expression level of Kv3.4 in the hippocampus was slightly lower than that in cerebellum, and was closely followed by the other 4 regions, with spinal cord expressing the lowest level. We suggest that novel Kv3 glycoforms may endow differences in channel function and expression among regions throughout the central nervous system.

  9. Sulfated di-, tri- and tetraantennary N-glycans in human Tamm-Horsfall glycoprotein

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Rooijen, J.J.M. van; Kamerling, J.P.

    1998-01-01

    The primary structures of 32 sulfated di-, tri- and tetraantennary N-glycans of human Tamm-Horsfall glycoprotein (THP) have been determined. THP was isolated from the urine of one healthy male donor. The intact carbohydrate chains were released by PNGase-F and fractionated via FPLC on Resource Q, HP

  10. A novel lectin (Morniga M) from mulberry (Morus nigra) bark recognizes oligomannosyl residues in N-glycans.

    Science.gov (United States)

    Wu, Albert M; Wu, June H; Singh, Tanuja; Chu, Kang-Chuang; Peumans, Willy J; Rougé, Pierre; Van Damme, Els J M

    2004-01-01

    Morniga M is a jacalin-related and mannose-specific lectin isolated from the bark of the mulberry (Morus nigra). In order to understand the function and application of this novel lectin, the binding property of Morniga M was studied in detail using an enzyme-linked lectinosorbent assay and lectin-glycan inhibition assay with extended glycan/ligand collection. From the results, it was found that the di-, tri-, and oligomannosyl structural units of N-glycans such as those of the bovine alpha1-acid glycoprotein (gp) and lactoferrin were the most active gps, but not the O-glycans or polysaccharides including mannan from yeast. The binding affinity of Morniga M for ligands can be ranked in decreasing order as follows: gps carrying multiple N-glycans with oligomannosyl residues > N-glycopeptide with a single trimannosyl core > Tri-Man oligomer [Man alpha1-->6(Man alpha1-->3) Man], Penta-Man oligomer [Man alpha1-->6(Man alpha1-->3)Man alpha1-->6(Man alpha1-->3) Man] > or = Man alpha1-->2, 3 or 6 Man > Man > GlcNAc, Glc > L-Fuc, Gal, GalNAc (inactive), demonstrating the unique specificity of this lectin that may not only assist in our understanding of cell surface carbohydrate ligand-lectin recognition, but also provide informative guidelines for the application of this structural probe in biotechnological and clinical regimens, especially in the detection and purification of N-linked glycans. 2004 National Science Council, ROC and S. Karger AG, Basel

  11. Structural features of N-glycans linked to glycoproteins expressed in three kinds of water plants: Predominant occurrence of the plant complex type N-glycans bearing Lewis a epitope.

    Science.gov (United States)

    Maeda, Megumi; Tani, Misato; Yoshiie, Takeo; Vavricka, Christopher J; Kimura, Yoshinobu

    2016-11-29

    The Japanese cedar pollen allergen (Cry j1) and the mountain cedar pollen allergen (Jun a1) are glycosylated with plant complex type N-glycans bearing Lewis a epitope(s) (Galβ1-3[Fucα1-4]GlcNAc-). The biological significance of Lewis a type plant N-glycans and their effects on the human immune system remain to be elucidated. Since a substantial amount of such plant specific N-glycans are required to evaluate immunological activity, we have searched for good plant-glycan sources to characterize Lewis a epitope-containing plant N-glycans. In this study, we have found that three water plants, Elodea nuttallii, Egeria densa, and Ceratophyllum demersum, produce glycoproteins bearing Lewis a units. Structural analysis of the N-glycans revealed that almost all glycoproteins expressed in these three water plants predominantly carry plant complex type N-glycans including the Lewis a type, suggesting that these water plants are good sources for preparation of Lewis a type plant N-glycans in substantial amounts.

  12. [Lactobacillus plantarum and phosprenyl competitively distinguish ovalbumin oligomannoside n-glycan].

    Science.gov (United States)

    Sobolev, S M; Nikolaeva, T N; Grigor'eva, E A; Pronin, A V

    2011-01-01

    Demonstration of the ability of native products of lactobacteria to bind mannose containing N-glycans as a display of one of the mechanisms of probiotic behavior of these symbiont microorganism in gastrointestinal tract (GIT). Lactobacillus plantarum 30 grown on selective medium (MRS-agar) and their ultrasound lysates were used in the study. Standard technique of delayed type hypersensitivity (DTH) reaction was used with inactivated Listeria monocytogenes culture as a priming agent. DTH reaction in vivo has demonstrated that oligomannoside N-glycan of egg albumin is a general acceptor for adhesins that are present in the native preparations of certain L. plantarum strains, as well as for phosprenyl (PHP) immunomodulator--an inhibitor of cell IL-2 reception. The data obtained give evidence that mannose specific adhesins that are conserved in lactobacteria preparations have the same binding sites in egg albumin as PHP and, therefore, IL-2.

  13. Serum protein N-glycans profiling for the discovery of potential biomarkers for nonalcoholic steatohepatitis.

    Science.gov (United States)

    Chen, Cuiying; Schmilovitz-Weiss, Hemda; Liu, Xue-en; Pappo, Orit; Halpern, Marisa; Sulkes, Jaqueline; Braun, Marius; Cohen, Maya; Barak, Nir; Tur-Kaspa, Ran; Vanhooren, Valerie; Van Vlierberghe, Hans; Libert, Claude; Contreras, Roland; Ben-Ari, Ziv

    2009-02-01

    The hepatic histology in nonalcoholic fatty liver disease can vary from isolated hepatic steatosis to steatohepatitis can progress to cirrhosis and liver-related death. The aim was to evaluate the use of blood serum N-glycan fingerprinting as a tool for differential diagnosis of nonalcoholic steatohepatitis from steatosis. A group of 47 patients with NAFLD was diagnosed by clinical laboratory analysis and ultrasonography, and was studied histologically using the Brunt's scoring system. The control group included 13 healthy individuals. N-glycan profiles of serum proteins were determined by DNA sequencer-based carbohydrate analytical profiling. We have found that the concentrations of two glycans (NGA2F and NA2) and their logarithm ratio of NGA2F versus NA2 (named GlycoNashTest) were associated with the degree of NASH-related fibrosis, but had no correlation with the grade of inflammation nor steatosis severity. When used to screen NAFLD patients, GlycoNashTest could identify advanced NASH-related fibrosis (F3-F4) with the diagnosis sensitivity of 89.5% and specificity of 71.4%. The serum N-glycan profile is a promising noninvasive method for detecting NASH or NASH-related fibrosis in NAFLD patients, which could be a valuable supplement to other markers currently used in diagnosis of NASH.

  14. Modifying an Insect Cell N-Glycan Processing Pathway Using CRISPR-Cas Technology.

    Science.gov (United States)

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

    2015-10-16

    Fused lobes (FDL) is an enzyme that simultaneously catalyzes a key trimming reaction and antagonizes elongation reactions in the insect N-glycan processing pathway. Accordingly, FDL function accounts, at least in part, for major differences in the N-glycosylation patterns of glycoproteins produced by insect and mammalian cells. In this study, we used the CRISPR-Cas9 system to edit the fdl gene in Drosophila melanogaster S2 cells. CRISPR-Cas9 editing produced a high frequency of site-specific nucleotide insertions and deletions, reduced the production of insect-type, paucimannosidic products (Man3GlcNAc2), and led to the production of partially elongated, mammalian-type complex N-glycans (GlcNAc2Man3GlcNAc2) in S2 cells. As CRISPR-Cas9 has not been widely used to analyze or modify protein glycosylation pathways or edit insect cell genes, these results underscore its broad utility as a tool for these purposes. Our results also confirm the key role of FDL at the major branch point distinguishing insect and mammalian N-glycan processing pathways. Finally, the new FDL-deficient S2 cell derivative produced in this study will enable future bottom-up glycoengineering efforts designed to isolate insect cell lines that can efficiently produce recombinant glycoproteins with chemically predefined oligosaccharide side-chain structures.

  15. Isolation and quantification of N-glycans from immunoglobulin G antibodies for quantitative glycosylation analysis

    Directory of Open Access Journals (Sweden)

    Venkata S. Tayi

    2015-06-01

    Full Text Available N-glycosylation is one of the critical quality attributes for the therapeutic monoclonal antibodies. Characterization of N-glycans of monoclonal antibodies provides valuable information about its therapeutic efficacy. We present a non-invasive method of isolating N-glycans from Immunoglobulin G based antibodies for glycosylation analysis. The method consists of purification of antibodies from biological solution and release of N-glycans with peptide-N-glycosidase F in a single consolidated process using a mini affinity ligand column (e.g. protein-A column. The method is highly reproducible with average coefficient of variation of 0.012 in the glycoform percentage distributions between the replicates. The method provides quantification of the molar yield of glycans as a function of molar concentration of antibody in a single analysis. To our knowledge, this is the first time this approach was used to detect and quantify any macro-heterogeneity of N-glycosylation in monoclonal antibody samples. This fairly rapid and very cost-efficient method would be of great interest for academic labs and biopharmaceutical industries.

  16. Alteration of a recombinant protein N-glycan structure in silkworms by partial suppression of N-acetylglucosaminidase gene expression.

    Science.gov (United States)

    Kato, Tatsuya; Kikuta, Kotaro; Kanematsu, Ayumi; Kondo, Sachiko; Yagi, Hirokazu; Kato, Koichi; Park, Enoch Y

    2017-09-01

    To synthesize complex type N-glycans in silkworms, shRNAs against the fused lobe from Bombyx mori (BmFDL), which codes N-acetylglucosaminidase (GlcNAcase) in the Golgi, was expressed by recombinant B. mori nucleopolyhedrovirus (BmNPV) in silkworm larvae. Expression was under the control of the actin promoter of B. mori or the U6-2 and i.e.-2 promoters from Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). The reduction of specific GlcNAcase activity was observed in Bm5 cells and silkworm larvae using the U6-2 promoter. In silkworm larvae, the partial suppression of BmFDL gene expression was observed. When shRNA against BmFDL was expressed under the control of U6-2 promoter, the Man3GlcNAc(Fuc)GlcNAc structure appeared in a main N-glycans of recombinant human IgG. These results suggested that the control of BmFDL expression by its shRNA in silkworms caused the modification of its N-glycan synthetic pathway, which may lead to the alteration of N-glycans in the expressed recombinant proteins. Suppression of BmFDL gene expression by shRNA is not sufficient to synthesize complex N-glycans in silkworm larvae but can modify the N-glycan synthetic pathway.

  17. Deletion of fucose residues in plant N-glycans by repression of the GDP-mannose 4,6-dehydratase gene using virus-induced gene silencing and RNA interference.

    Science.gov (United States)

    Matsuo, Kouki; Matsumura, Takeshi

    2011-02-01

    Production of pharmaceutical glycoproteins in plants has many advantages in terms of safety and reduced costs. However, plant-produced glycoproteins have N-glycans with plant-specific sugar residues (core β-1,2-xylose and α-1,3-fucose) and a Lewis a (Le(a) ) epitope, i.e., Galβ(1-3)[Fucα(1-4)]GlcNAc. Because these sugar residues and glycan structures seemed to be immunogenic, several attempts have been made to delete them by repressing their respective glycosyltransferase genes. However, until date, such deletions have not been successful in completely eliminating the fucose residues. In this study, we simultaneously reduced the plant-specific core α-1,3-fucose and α-1,4-fucose residues in the Le(a) epitopes by repressing the Guanosine 5'-diphosphate (GDP)-D-mannose 4,6-dehydratase (GMD) gene, which is associated with GDP-L-fucose biosynthesis, in Nicotiana benthamiana plants. Repression of GMD was achieved using virus-induced gene silencing (VIGS) and RNA interference (RNAi). The proportion of fucose-free N-glycans found in total soluble protein from GMD gene-repressed plants increased by 80% and 95% following VIGS and RNAi, respectively, compared to wild-type plants. A small amount of putative galactose substitution in N-glycans from the NbGMD gene-repressed plants was observed, similar to what has been previously reported GMD-knockout Arabidopsis mutant. On the other hand, the recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) with fucose-deleted N-glycans was successfully produced in NbGMD-RNAi transgenic N. benthamiana plants. Thus, repression of the GMD gene is thus very useful for deleting immunogenic total fucose residues and facilitating the production of pharmaceutical glycoproteins in plants.

  18. An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides

    Science.gov (United States)

    Chen, Minyong; Shi, Xiaofeng; Duke, Rebecca M.; Ruse, Cristian I.; Dai, Nan; Taron, Christopher H.; Samuelson, James C.

    2017-01-01

    A method for selective and comprehensive enrichment of N-linked glycopeptides was developed to facilitate detection of micro-heterogeneity of N-glycosylation. The method takes advantage of the inherent properties of Fbs1, which functions within the ubiquitin-mediated degradation system to recognize the common core pentasaccharide motif (Man3GlcNAc2) of N-linked glycoproteins. We show that Fbs1 is able to bind diverse types of N-linked glycomolecules; however, wild-type Fbs1 preferentially binds high-mannose-containing glycans. We identified Fbs1 variants through mutagenesis and plasmid display selection, which possess higher affinity and improved recovery of complex N-glycomolecules. In particular, we demonstrate that the Fbs1 GYR variant may be employed for substantially unbiased enrichment of N-linked glycopeptides from human serum. Most importantly, this highly efficient N-glycopeptide enrichment method enables the simultaneous determination of N-glycan composition and N-glycosites with a deeper coverage (compared to lectin enrichment) and improves large-scale N-glycoproteomics studies due to greatly reduced sample complexity. PMID:28534482

  19. An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides.

    Science.gov (United States)

    Chen, Minyong; Shi, Xiaofeng; Duke, Rebecca M; Ruse, Cristian I; Dai, Nan; Taron, Christopher H; Samuelson, James C

    2017-05-23

    A method for selective and comprehensive enrichment of N-linked glycopeptides was developed to facilitate detection of micro-heterogeneity of N-glycosylation. The method takes advantage of the inherent properties of Fbs1, which functions within the ubiquitin-mediated degradation system to recognize the common core pentasaccharide motif (Man3GlcNAc2) of N-linked glycoproteins. We show that Fbs1 is able to bind diverse types of N-linked glycomolecules; however, wild-type Fbs1 preferentially binds high-mannose-containing glycans. We identified Fbs1 variants through mutagenesis and plasmid display selection, which possess higher affinity and improved recovery of complex N-glycomolecules. In particular, we demonstrate that the Fbs1 GYR variant may be employed for substantially unbiased enrichment of N-linked glycopeptides from human serum. Most importantly, this highly efficient N-glycopeptide enrichment method enables the simultaneous determination of N-glycan composition and N-glycosites with a deeper coverage (compared to lectin enrichment) and improves large-scale N-glycoproteomics studies due to greatly reduced sample complexity.

  20. Characterization of N-Glycans from Arabidopsis. Application to a Fucose-Deficient Mutant1

    Science.gov (United States)

    Rayon, Catherine; Cabanes-Macheteau, Marion; Loutelier-Bourhis, Corinne; Salliot-Maire, Isabelle; Lemoine, Jérome; Reiter, Wolf-Dieter; Lerouge, Patrice; Faye, Loïc

    1999-01-01

    The structures of glycans N-linked to Arabidopsis proteins have been fully identified. From immuno- and affinodetections on blots, chromatography, nuclear magnetic resonance, and glycosidase sequencing data, we show that Arabidopsis proteins are N-glycosylated by high-mannose-type N-glycans from Man5GlcNAc2 to Man9GlcNAc2, and by xylose- and fucose (Fuc)-containing oligosaccharides. However, complex biantenary structures containing the terminal Lewis a epitope recently reported in the literature (A.-C. Fitchette-Lainé, V. Gomord, M. Cabanes, J.-C. Michalski, M. Saint Macary, B. Foucher, B. Cavalier, C. Hawes, P. Lerouge, and L. Faye [1997] Plant J 12: 1411–1417) were not detected. A similar study was done on the Arabidopsis mur1 mutant, which is affected in the biosynthesis of l-Fuc. In this mutant, one-third of the Fuc residues of the xyloglucan has been reported to be replaced by l-galactose (Gal) (E. Zablackis, W.S. York, M. Pauly, S. Hantus, W.D. Reiter, C.C.S. Chapple, P. Albersheim, and A. Darvill [1996] Science 272: 1808–1810). N-linked glycans from the mutant were identified and their structures were compared with those isolated from the wild-type plants. In about 95% of all N-linked glycans from the mur1 plant, l-Fuc residues were absent and were not replaced by another monosaccharide. However, in the remaining 5%, l-Fuc was found to be replaced by a hexose residue. From nuclear magnetic resonance and mass spectrometry data of the mur1 N-glycans, and by analogy with data reported on mur1 xyloglucan, this subpopulation of N-linked glycans was proposed to be l-Gal-containing N-glycans resulting from the replacement of l-Fuc by l-Gal. PMID:9952469

  1. Deglycosylating enzymes acting on N-glycans in fungi: Insights from a genome survey.

    Science.gov (United States)

    Tzelepis, Georgios; Karlsson, Magnus; Suzuki, Tadashi

    2017-11-01

    N-Glycosylation, one of the most prominent post-translational modifications of proteins, is found in all domains of life, i.e. eukaryotes, bacteria and archaea, and has been shown to play a crucial role in modulating the physicochemical/physiological properties of carrier proteins. Deglycosylating enzymes that act on N-glycans are widely used in analyzing the structures/functions of N-glycans. Fungi are known to produce various deglycosylating enzymes, some of which are fungi-specific. While such enzymes likely are biologically relevant in fungal biology, their properties as well as their functions have not been explored in detail. In this review, we summarize the current knowledge of fungal deglycosylating enzymes and discuss their biological significance. As of this writing, five types of deglycosylating enzymes that act on N-glycans are known to occur in fungi; (1) the cytosolic peptide: N-glycanase (PNGase), (2) the acidic PNGase, (3) the glycoside hydrolase family (GH) 85 endo-β-N-acetylglucosaminidase (ENGase), (4) the GH18 cytosolic ENGase, and (5) the GH18 secreted ENGase. Interestingly, genome surveys indicate that the loss of cytosolic PNGase activity in certain fungi coincide with the occurrence of GH18 cytosolic ENGase, implying that the GH18 ENGase serves to replace the deglycosylation function of the cytosolic PNGase in those filamentous ascomycete fungi. Our review concludes that fungi promise to be valuable organisms for developing an understanding of the biological functions of PNGases/ENGases. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. A Lectin from Platypodium elegans with Unusual Specificity and Affinity for Asymmetric Complex N-Glycans*

    Science.gov (United States)

    Benevides, Raquel Guimarães; Ganne, Géraldine; Simões, Rafael da Conceição; Schubert, Volker; Niemietz, Mathäus; Unverzagt, Carlo; Chazalet, Valérie; Breton, Christelle; Varrot, Annabelle; Cavada, Benildo Sousa; Imberty, Anne

    2012-01-01

    Lectin activity with specificity for mannose and glucose has been detected in the seed of Platypodium elegans, a legume plant from the Dalbergieae tribe. The gene of Platypodium elegans lectin A has been cloned, and the resulting 261-amino acid protein belongs to the legume lectin family with similarity with Pterocarpus angolensis agglutinin from the same tribe. The recombinant lectin has been expressed in Escherichia coli and refolded from inclusion bodies. Analysis of specificity by glycan array evidenced a very unusual preference for complex type N-glycans with asymmetrical branches. A short branch consisting of one mannose residue is preferred on the 6-arm of the N-glycan, whereas extensions by GlcNAc, Gal, and NeuAc are favorable on the 3-arm. Affinities have been obtained by microcalorimetry using symmetrical and asymmetrical Asn-linked heptasaccharides prepared by the semi-synthetic method. Strong affinity with Kd of 4.5 μm was obtained for both ligands. Crystal structures of Platypodium elegans lectin A complexed with branched trimannose and symmetrical complex-type Asn-linked heptasaccharide have been solved at 2.1 and 1.65 Å resolution, respectively. The lectin adopts the canonical dimeric organization of legume lectins. The trimannose bridges the binding sites of two neighboring dimers, resulting in the formation of infinite chains in the crystal. The Asn-linked heptasaccharide binds with the 6-arm in the primary binding site with extensive additional contacts on both arms. The GlcNAc on the 6-arm is bound in a constrained conformation that may rationalize the higher affinity observed on the glycan array for N-glycans with only a mannose on the 6-arm. PMID:22692206

  3. Characterization of N-Glycan Structures on the Surface of Mature Dengue 2 Virus Derived from Insect Cells.

    Directory of Open Access Journals (Sweden)

    Y Lei

    Full Text Available DENV envelope glycoprotein (E is responsible for interacting with host cell receptors and is the main target for the development of a dengue vaccine based on an induction of neutralizing antibodies. It is well known that DENV E glycoprotein has two potential N-linked glycosylation sites at Asn67 and Asn153. The N-glycans of E glycoprotein have been shown to influence the proper folding of the protein, its cellular localization, its interactions with receptors and its immunogenicity. However, the precise structures of the N-glycans that are attached to E glycoprotein remain elusive, although the crystal structure of DENV E has been determined. This study characterized the structures of envelope protein N-linked glycans on mature DENV-2 particles derived from insect cells via an integrated method that used both lectin microarray and MALDI-TOF-MS. By combining these methods, a high heterogeneity of DENV N-glycans was found. Five types of N-glycan were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid; high mannose-type N-linked oligosaccharides and the galactosylation of N-glycans were the major structures that were found. Furthermore, a complex between a glycan on DENV and the carbohydrate recognition domain (CRD of DC-SIGN was mimicked with computational docking experiments. For the first time, this study provides a comprehensive understanding of the N-linked glycan profile of whole DENV-2 particles derived from insect cells.

  4. Serum Aberrant N-Glycan Profile as a Marker Associated with Early Antibody-Mediated Rejection in Patients Receiving a Living Donor Kidney Transplant.

    Science.gov (United States)

    Noro, Daisuke; Yoneyama, Tohru; Hatakeyama, Shingo; Tobisawa, Yuki; Mori, Kazuyuki; Hashimoto, Yasuhiro; Koie, Takuya; Tanaka, Masakazu; Nishimura, Shin-Ichiro; Sasaki, Hideo; Saito, Mitsuru; Harada, Hiroshi; Chikaraishi, Tatsuya; Ishida, Hideki; Tanabe, Kazunari; Satoh, Shigeru; Ohyama, Chikara

    2017-08-08

    We determined if the serum N-glycan profile can be used as a diagnostic marker of antibody-mediated rejection (ABMR) in living donor kidney transplant (LKTx) recipients. Glycoblotting, combined with mass spectrometry, was used to retrospectively examine N-glycan levels in the postoperative sera of 197 LKTx recipients of whom 16 recipients had ABMR with or without T-cell-mediated rejection (TCMR), 40 recipients had TCMR, and 141 recipients had no adverse events. Multivariate discriminant analysis for prediction of ABMR was performed by inputting an ABMR event as an explanatory variable and sex, age, and serum N-glycan level as objective variables. The N-glycan score was calculated by multiplying the level of candidate objective variables by objective function values. The ABMR predictive performance of the N-glycan score was assessed by receiver operator characteristic curve and Kaplan-Meier curve analyses. The N-glycan score discriminated ABMR with 81.25% sensitivity, 87.85% specificity, and an area under the curve (AUC) of 0.892 that was far superior to that of preformed donor-specific antibody status (AUC, 0.761). Recipients with N-glycan-positive scores >0.8770 had significantly shorter ABMR survival than that of recipients with N-glycan-negative scores. Although the limitations of our study includ its small sample size and retrospective nature, the serum N-glycan score may contribute to prediction of ABMR.

  5. Enhanced detection of in-gel released N-glycans by MALDI-TOF-MS.

    Science.gov (United States)

    Weiz, Stefan; Kamalakumar, Aryaline; Biskup, Karina; Blanchard, Véronique

    2015-05-01

    Many biologically relevant glycoproteins need to be separated on 1D- or 2D-gels prior to analysis and are available in picomole amounts. Therefore, it is important to have optimized methods to unravel the glycome that combine in-gel digestions with MALDI-TOF-MS. In this technical report, we investigated how the detection of in-gel released N-glycans could be improved by MALDI-TOF-MS. First, an AnchorChip target was tested and compared to ground steel target using several reference oligosaccharides. The highest signals were obtained with an AnchorChip target and D-arabinosazone as the matrix; a LOD of 1.3 to 10 fmol was attained. Then, the effect of octyl-β-glucopyranoside, a nonionic detergent, was studied during in-gel peptide-N(4) -(acetyl-ß-glucosaminyl) asparagine amidase F digestion of standard glycoproteins and during glycan extraction. Octyl-β-glucopyranoside increased the intensity and the amount of detected neutral as well as acidic N-glycans. A LOD of under 7 pmol glycoprotein could be achieved.

  6. Benchmark study of automatic annotation of MALDI-TOF N-glycan profiles.

    Science.gov (United States)

    Brito, Alejandro E; Kletter, Doron; Singhal, Mudita; Bern, Marshall

    2015-11-01

    Human experts can annotate peaks in MALDI-TOF profiles of detached N-glycans with some degree of accuracy. Even though MALDI-TOF profiles give only intact masses without any fragmentation information, expert knowledge of the most common glycans and biosynthetic pathways in the biological system can point to a small set of most likely glycan structures at the "cartoon" level of detail. Cartoonist is a recently developed, fully automatic annotation tool for MALDI-TOF glycan profiles. Here we benchmark Cartoonist's automatic annotations against human expert annotations on human and mouse N-glycan data from the Consortium for Functional Glycomics. We find that Cartoonist and expert annotations largely agree, but the expert tends to annotate more specifically, meaning fewer suggested structures per peak, and Cartoonist more comprehensively, meaning more annotated peaks. On peaks for which both Cartoonist and the expert give unique cartoons, the two cartoons agree in over 90% of all cases. This article is part of a Special Issue entitled: Computational Proteomics.

  7. Multi-Site N-glycan mapping study 1: Capillary electrophoresis - laser induced fluorescence.

    Science.gov (United States)

    Szekrényes, Ákos; Park, SungAe Suhr; Santos, Marcia; Lew, Clarence; Jones, Aled; Haxo, Ted; Kimzey, Michael; Pourkaveh, Shiva; Szabó, Zoltán; Sosic, Zoran; Feng, Peng; Váradi, Csaba; de l'Escaille, François; Falmagne, Jean-Bernard; Sejwal, Preeti; Niedringhaus, Thomas; Michels, David; Freckleton, Gordon; Hamm, Melissa; Manuilov, Anastasiya; Schwartz, Melissa; Luo, Jiann-Kae; van Dyck, Jonathan; Leung, Pui-King; Olajos, Marcell; Gu, Yingmei; Gao, Kai; Wang, Wenbo; Wegstein, Jo; Tep, Samnang; Guttman, András

    2016-01-01

    An international team that included 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia was formed to evaluate the reproducibility of sample preparation and analysis of N-glycans using capillary electrophoresis of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans with laser induced fluorescence (CE-LIF) detection (16 sites) and ultra high-performance liquid chromatography (UHPLC, 12 sites; results to be reported in a subsequent publication). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. Migration time, peak area and peak area percent values were determined for all peaks with >0.1% peak area. Our results demonstrated low variability and high reproducibility, both, within any given site as well across all sites, which indicates that a standard N-glycan analysis platform appropriate for general use (clone selection, process development, lot release, etc.) within the industry can be established.

  8. Multi-Site N-glycan mapping study 1: Capillary electrophoresis – laser induced fluorescence

    Science.gov (United States)

    Szekrényes, Ákos; Park, SungAe Suhr; Santos, Marcia; Lew, Clarence; Jones, Aled; Haxo, Ted; Kimzey, Michael; Pourkaveh, Shiva; Szabó, Zoltán; Sosic, Zoran; Feng, Peng; Váradi, Csaba; de l'Escaille, François; Falmagne, Jean-Bernard; Sejwal, Preeti; Niedringhaus, Thomas; Michels, David; Freckleton, Gordon; Hamm, Melissa; Manuilov, Anastasiya; Schwartz, Melissa; Luo, Jiann-Kae; van Dyck, Jonathan; Leung, Pui-King; Olajos, Marcell; Gu, Yingmei; Gao, Kai; Wang, Wenbo; Wegstein, Jo; Tep, Samnang; Guttman, András

    2016-01-01

    An international team that included 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia was formed to evaluate the reproducibility of sample preparation and analysis of N-glycans using capillary electrophoresis of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans with laser induced fluorescence (CE-LIF) detection (16 sites) and ultra high-performance liquid chromatography (UHPLC, 12 sites; results to be reported in a subsequent publication). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. Migration time, peak area and peak area percent values were determined for all peaks with >0.1% peak area. Our results demonstrated low variability and high reproducibility, both, within any given site as well across all sites, which indicates that a standard N-glycan analysis platform appropriate for general use (clone selection, process development, lot release, etc.) within the industry can be established. PMID:26466659

  9. Sialylated β1, 6 branched N-glycans modulate the adhesion, invasion and metastasis of hepatocarcinoma cells.

    Science.gov (United States)

    Yu, Xiao; Zhao, Yujie; Wang, Liping; Chen, Xixi; Su, Zhen; Zhang, Han; Yuan, Qingmin; Wang, Shujing

    2016-12-01

    The mouse hepatocarcinoma cell lines Hca-F and Hca-P have been derived from hepatocarcinoma in mice and metastasize only to the lymph node. Hca-F cells displayed greater lymphatic metastasis ability than Hca-P cells. When the two cell lines were compared for cell surface sialylated β1,6 branched N-glycans by flow cytometry using L-PHA and SNA, Hca-F cells were found to express significantly higher levels. To explore the effect of increased sialylated β1,6 branched N-glycans on hepatocarcinoma progression, we inhibit their expression in Hca-F cells by using swainsonine treatment and RNA interference. We found that swainsonine treatment or GnT-V-shRNA transfection significantly inhibited the formation of β1,6 branched N-glycans, and partially inhibited the expression of α2,6 sialic acids. Knockdown of sialylated β1,6 branched N-glycans significantly attenuated the invasive and metastatic capability both in vitro and in vivo. Blockade of α2,6 sialic acid expression on Hca-F cell surface by the treatment with neuraminidase caused reduction in cellular adherence to lymph node. In addition, knockdown of sialylated β1,6 branched N-glycans could decrease the expression of Notch1, NICD1, NICD2 and HES1 in Hca-F cells. Collectively, these findings suggest that increased sialylated β1,6 branched N-glycans may contribute to hepatocarcinoma progression by altering the adhesive, invasive and metastatic ability to lymph node via Notch signaling pathway.

  10. Complementary LC-MS/MS-Based N-Glycan, N-Glycopeptide, and Intact N-Glycoprotein Profiling Reveals Unconventional Asn71-Glycosylation of Human Neutrophil Cathepsin G

    Directory of Open Access Journals (Sweden)

    Ian Loke

    2015-08-01

    Full Text Available Neutrophil cathepsin G (nCG is a central serine protease in the human innate immune system, but the importance of its N-glycosylation remains largely undescribed. To facilitate such investigations, we here use complementary LC-MS/MS-based N-glycan, N-glycopeptide, and intact glycoprotein profiling to accurately establish the micro- and macro-heterogeneity of nCG from healthy individuals. The fully occupied Asn71 carried unconventional N-glycosylation consisting of truncated chitobiose core (GlcNAcβ: 55.2%; Fucα1,6GlcNAcβ: 22.7%, paucimannosidic N-glycans (Manβ1,4GlcNAcβ1,4GlcNAcβ: 10.6%; Manβ1,4GlcNAcβ1,4(Fucα1,6GlcNAcβ: 7.9%; Manα1,6Manβ1,4GlcNAcβ1,4GlcNAcβ: 3.7%, trace level of Manα1,6Manβ1,4GlcNAcβ1,4(Fucα1,6GlcNAcβ, and trace levels of monoantennary α2,6- and α2,3-sialylated complex N-glycans. High-resolution/mass accuracy LC-MS profiling of intact nCG confirmed the Asn71-glycoprofile and identified two C-terminal truncation variants at Arg243 (57.8% and Ser244 (42.2%, both displaying oxidation of solvent-accessible Met152. Asn71 appeared proximal (~19 Å to the active site of nCG, but due to the truncated nature of Asn71-glycans (~5–17 Å we questioned their direct modulation of the proteolytic activity of the protein. This work highlights the continued requirement of using complementary technologies to accurately profile even relatively simple glycoproteins and illustrates important challenges associated with the analysis of unconventional protein N-glycosylation. Importantly, this study now facilitates investigation of the functional role of nCG Asn71-glycosylation.

  11. Structural Analysis of Free N-Glycans in α-Glucosidase Mutants of Saccharomyces cerevisiae: Lack of the Evidence for the Occurrence of Catabolic α-Glucosidase Acting on the N-Glycans.

    Science.gov (United States)

    Hossain, Tanim Jabid; Harada, Yoichiro; Hirayama, Hiroto; Tomotake, Haruna; Seko, Akira; Suzuki, Tadashi

    2016-01-01

    Saccharomyces cerevisiae produces two different α-glucosidases, Glucosidase 1 (Gls1) and Glucosidase 2 (Gls2), which are responsible for the removal of the glucose molecules from N-glycans (Glc3Man9GlcNAc2) of glycoproteins in the endoplasmic reticulum. Whether any additional α-glucosidases playing a role in catabolizing the glucosylated N-glycans are produced by this yeast, however, remains unknown. We report herein on a search for additional α-glucosidases in S. cerevisiae. To this end, the precise structures of cytosolic free N-glycans (FNGs), mainly derived from the peptide:N-glycanase (Png1) mediated deglycosylation of N-glycoproteins were analyzed in the endoplasmic reticulum α-glucosidase-deficient mutants. 12 new glucosylated FNG structures were successfully identified through 2-dimentional HPLC analysis. On the other hand, non-glucosylated FNGs were not detected at all under any culture conditions. It can therefore be safely concluded that no catabolic α-glucosidases acting on N-glycans are produced by this yeast.

  12. Structural characterization of the N-glycans of gp273, the ligand for sperm-egg interaction in the mollusc bivalve Unio elongatulus

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Di Patrizi, L.; Capone, A.; Focarelli, R.; Rosati, F.; Gutiérrez Gallego, R.; Gerwig, G.J.

    2001-01-01

    Gp273, a glycoprotein of the egg extracellular coats of the mollusc bivalve Unio elongatulus, is the ligand molecule for sperm-egg interaction during fertilization. In this study we have analyzed the N-glycans from gp273. N-glycans were enzymatically released by PNGase F digestion and their structur

  13. Decreased fucosylated PSA as a urinary marker for high Gleason score prostate cancer

    Science.gov (United States)

    Fujita, Kazutoshi; Hayashi, Takuji; Matsuzaki, Kyosuke; Nakata, Wataru; Masuda, Mika; Kawashima, Atsunari; Ujike, Takeshi; Nagahara, Akira; Tsuchiya, Mutsumi; Kobayashi, Yuka; Nojima, Satoshi; Uemura, Motohide; Morii, Eiichi; Miyoshi, Eiji; Nonomura, Norio

    2016-01-01

    Fucosylation is an important oligosaccharide modification associated with cancer and inflammation. We investigated whether urinary fucosylated PSA (Fuc-PSA) levels could be used for the detection of high Gleason score prostate cancer. Urine samples were collected from men with abnormal digital rectal examination findings or elevated serum PSA levels, before prostate biopsy. Lectin-antibody ELISA was used to quantify the Lewis-type or core-type fucosylated PSA (PSA-AAL) and core-type fucosylated PSA (PSA-PhoSL) in the urine samples. Both types of urinary Fuc-PSA were significantly decreased in the men with prostate cancer compared with the men whose biopsies were negative for cancer (P = 0.026 and P biopsy specimens (P = 0.001 and P < 0.001, respectively). Multivariate analysis showed that PSA density, urinary PSA-AAL, and urinary PSA-PhoSL were independent predictors of high Gleason score prostate cancer. The area under the receiver-operator characteristic curve (AUC) value for the prediction of cancers of Gleason score ≥ 7 was 0.69 for urinary PSA-AAL and 0.72 for urinary PSA-PhoSL. In contrast, the AUC value was 0.59 for serum PSA, 0.63 for PSA density, and 0.58 for urinary PSA. In conclusion, a decreased urinary Fuc-PSA level is a potential marker for the detection of high Gleason score prostate cancer. PMID:27494861

  14. Presence of beta-linked GalNAc residues on N-glycans of human thyroglobulin.

    Science.gov (United States)

    Takeya, Akira; Hosomi, Osamu; Nishijima, Hironori; Ohe, Yoshihide; Sugahara, Kunio; Sagi, Morihisa; Yamazaki, Kentaro; Hayakawa, Hideyuki; Takeshita, Hiroshi; Sasaki, Chizuko; Kogure, Tadahisa; Mukai, Toshiji

    2007-01-16

    Hepatic asialoglycoprotein receptor, which may mediate the clearance of circulating thyroglobulin, is known to have a high affinity for GalNAc. Recently, the receptor has been reported to be present also in the thyroid, implicating interaction with thyroglobulin. Here, mammalian thyroglobulins were analyzed for GalNAc termini by Western blotting with GalNAc-recognizing lectins labeled with peroxidase or (125)I. Wistaria floribunda lectin was found to bind human thyroglobulin and, to some extent, bovine, but not porcine thyroglobulin. After desialylation, the lectin bound all of the thyroglobulins tested. The binding was inhibited by competitive inhibitor GalNAc. Peptide N-glycanase treatment of human desialylated thyroglobulin resulted in the complete loss of reactivity with W. floribunda lectin, indicating that the binding sites are exclusively on N-glycans. The binding sites on human desialylated thyroglobulin were partly sensitive to beta-galactosidase, and the remainder was essentially sensitive to beta-N-acetylhexosaminidase. On the other hand, the binding sites of bovine and porcine desialylated thyroglobulins were totally sensitive to beta-galactosidase. Thus the lectin binds beta-Gal termini, as well as beta-GalNAc. GalNAc-specific Dolichos biflorus lectin also bound human thyroglobulin weakly. In contrast to W. floribunda lectin, desialylation diminished binding, suggesting that these two lectins recognize different GalNAc-terminated structures. Again, the binding was inhibited by GalNAc and by treatment with peptide N-glycanase. These results strongly indicate the presence of distinct GalNAc termini of N-glycans on human thyroglobulin.

  15. The recognition of N-glycans by the lectin ArtinM mediates cell death of a human myeloid leukemia cell line.

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    Fernanda Caroline Carvalho

    Full Text Available ArtinM, a D-mannose-binding lectin from Artocarpus heterophyllus (jackfruit, interacts with N-glycosylated receptors on the surface of several cells of hematopoietic origin, triggering cell migration, degranulation, and cytokine release. Because malignant transformation is often associated with altered expression of cell surface glycans, we evaluated the interaction of ArtinM with human myelocytic leukemia cells and investigated cellular responses to lectin binding. The intensity of ArtinM binding varied across 3 leukemia cell lines: NB4>K562>U937. The binding, which was directly related to cell growth suppression, was inhibited in the presence of Manα1-3(Manα1-6Manβ1, and was reverted in underglycosylated NB4 cells. ArtinM interaction with NB4 cells induced cell death (IC(50 = 10 µg/mL, as indicated by cell surface exposure of phosphatidylserine and disruption of mitochondrial membrane potential unassociated with caspase activation or DNA fragmentation. Moreover, ArtinM treatment of NB4 cells strongly induced reactive oxygen species generation and autophagy, as indicated by the detection of acidic vesicular organelles in the treated cells. NB4 cell death was attributed to ArtinM recognition of the trimannosyl core of N-glycans containing a ß1,6-GlcNAc branch linked to α1,6-mannose. This modification correlated with higher levels of N-acetylglucosaminyltransferase V transcripts in NB4 cells than in K562 or U937 cells. Our results provide new insights into the potential of N-glycans containing a β1,6-GlcNAc branch linked to α1,6-mannose as a novel target for anti-leukemia treatment.

  16. Deletion of plant-specific sugar residues in plant N-glycans by repression of GDP-D-mannose 4,6-dehydratase and β-1,2-xylosyltransferase genes.

    Science.gov (United States)

    Matsuo, Kouki; Kagaya, Uiko; Itchoda, Noriko; Tabayashi, Noriko; Matsumura, Takeshi

    2014-10-01

    Production of pharmaceutical glycoproteins, such as therapeutic antibodies and cytokines, in plants has many advantages in safety and reduced costs. However, plant-made glycoproteins have N-glycans with plant-specific sugar residues (core β-1,2-xylose and α-1,3-fucose) and a Lewis a (Le(a)) epitope, Galβ(1-3)[Fucα(1-4)]GlcNAc. Because it is likely that these sugar residues and glycan structures are immunogenic, many attempts have been made to delete them. Previously, we reported the simultaneous deletion of the plant-specific core α-1,3-fucose and α-1,4-fucose residues in Le(a) epitopes by repressing the GDP-D-mannose 4,6-dehydratase (GMD) gene, which is associated with GDP-L-fucose biosynthesis, in Nicotiana benthamiana plants (rGMD plants, renamed to ΔGMD plants) (Matsuo and Matsumura, Plant Biotechnol. J., 9, 264-281, 2011). In the present study, we generated a core β-1,2-xylose residue-repressed transgenic N. benthamiana plant by co-suppression of β-1,2-xylosyltransferase (ΔXylT plant). By crossing ΔGMD and ΔXylT plants, we successfully generated plants in which plant-specific sugar residues were repressed (ΔGMDΔXylT plants). The proportion of N-glycans with deleted plant-specific sugar residues found in total soluble protein from ΔGMDΔXylT plants increased by 82.41%. Recombinant mouse granulocyte/macrophage-colony stimulating factor (mGM-CSF) and human monoclonal immunoglobulin G (hIgG) harboring N-glycans with deleted plant-specific sugar residues were successfully produced in ΔGMDΔXylT plants. Simultaneous repression of the GMD and XylT genes in N. benthamiana is thus very useful for deleting plant-specific sugar residues.

  17. Discovery of specific metastasis-related N-glycan alterations in epithelial ovarian cancer based on quantitative glycomics.

    Directory of Open Access Journals (Sweden)

    Xingwang Zhang

    Full Text Available Generally, most of ovarian cancer cannot be detected until large scale and remote metastasis occurs, which is the major cause of high mortality in ovarian cancer. Therefore, it is urgent to discover metastasis-related biomarkers for the detection of ovarian cancer in its occult metastasis stage. Altered glycosylation is a universal feature of malignancy and certain types of glycan structures are well-known markers for tumor progressions. Thus, this study aimed to reveal specific changes of N-glycans in the secretome of the metastatic ovarian cancer. We employed a quantitative glycomics approach based on metabolic stable isotope labeling to compare the differential N-glycosylation of secretome between an ovarian cancer cell line SKOV3 and its high metastatic derivative SKOV3-ip. Intriguingly, among total 17 N-glycans identified, the N-glycans with bisecting GlcNAc were all significantly decreased in SKOV3-ip in comparison to SKOV3. This alteration in bisecting GlcNAc glycoforms as well as its corresponding association with ovarian cancer metastatic behavior was further validated at the glycotransferase level with multiple techniques including real-time PCR, western blotting, transwell assay, lectin blotting and immunohistochemistry analysis. This study illustrated metastasis-related N-glycan alterations in ovarian cancer secretome in vitro for the first time, which is a valuable source for biomarker discovery as well. Moreover, N-glycans with bisecting GlcNAc shed light on the detection of ovarian cancer in early peritoneal metastasis stage which may accordingly improve the prognosis of ovarian cancer patients.

  18. Quantitative analysis of N-glycans from human alfa-acid-glycoprotein using stable isotope labeling and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry as tool for pancreatic disease diagnosis

    Energy Technology Data Exchange (ETDEWEB)

    Giménez, Estela, E-mail: estelagimenez@ub.edu [Department of Analytical Chemistry, University of Barcelona, Diagonal 647, E-08028 Barcelona (Spain); Balmaña, Meritxell [Biochemistry and Molecular Biology Unit, Department of Biology, University of Girona, Campus Montilivi s/n, 17071 Girona (Spain); Figueras, Joan [Department of Surgery, Dr. Josep Trueta University Hospital, IdlBGi, 17007 Girona (Spain); Fort, Esther [Digestive Unit, Dr. Josep Trueta University Hospital, 17007 Girona (Spain); Bolós, Carme de [Gastroesophagic Cancer Research Group, Research Programme in Cancer, Hospital del Mar Medical Research Institute (IMIM), Dr. Aiguader, 88, 08003 Barcelona (Spain); Sanz-Nebot, Victòria [Department of Analytical Chemistry, University of Barcelona, Diagonal 647, E-08028 Barcelona (Spain); Peracaula, Rosa [Biochemistry and Molecular Biology Unit, Department of Biology, University of Girona, Campus Montilivi s/n, 17071 Girona (Spain); Rizzi, Andreas [Institute of Analytical Chemistry, University of Vienna, Währinger Straße 38, A-1090 Vienna (Austria)

    2015-03-25

    AGP as a candidate structure worth to be corroborated by an extended study including more clinical cases; especially those with chronic pancreatitis and initial stages of pancreatic cancer. Importantly, the results demonstrate that the presented methodology combining an enrichment of a target protein by IAC with isotope coded relative quantitation of N-glycans can be successfully used for targeted glycomics studies. The methodology is assumed being suitable as well for other such studies aimed at finding novel cancer associated glycoprotein biomarkers.

  19. Aided Diagnosis of Hepatocellular Carcinoma Using Serum Fucosylated Haptoglobin Ratios

    Science.gov (United States)

    Shang, Shuxin; Li, Wei; Qin, Xue; Zhang, Shu; Liu, Yinkun

    2017-01-01

    Aberrant fucosylation plays a functional role in regulating ontogeny and celluar differentiation and are differentially regulated in cancerous condition, which could provide hallmarks for cancer diagnostics and surveillance. We previously developed a magnetic beads-based lectin ELISA system to measure fucosylated haptoglobin (Hp), which has been reported to be a cancer biomarker through a series of glycoproteomic analysis. In this study, serum fucosylated Hp ratios were measured using our ELISA kit in a separate cohort of 260 patients independently, including 130 healthy controls and 130 patients with hepatocellular carcinoma (HCC). Fucosylated Hp /Hp ratio (levels of fucosylated Hp /levels of protein Hp) and ELISA Index (OD value of fucosylated Hp /OD value of protein Hp) were calculated respectively to reflect Hp fucosylation level on its protein level. Our data showed that fucosylated Hp /Hp ratio (AUC=0.8449) and ELISA Index (AUC=0.8581) had better performance in distinguishing HCC from controls, which indicated that fucosylated Hp ratios could improve the diagnosis and prediction of HCC even with a low level of alpha-fetoprotein (AFP). Additionally, the combination analysis of AFP and fucosylated Hp ratios increased the AUC value for HCC diagnosis. PMID:28382152

  20. Abolishment of N-glycan mannosylphosphorylation in glyco-engineered Saccharomyces cerevisiae by double disruption of MNN4 and MNN14 genes.

    Science.gov (United States)

    Kim, Yeong Hun; Kang, Ji-Yeon; Gil, Jin Young; Kim, Sang-Yoon; Shin, Keun Koo; Kang, Hyun Ah; Kim, Jeong-Yoon; Kwon, Ohsuk; Oh, Doo-Byoung

    2017-04-01

    Mannosylphosphorylated glycans are found only in fungi, including yeast, and the elimination of mannosylphosphates from glycans is a prerequisite for yeast glyco-engineering to produce human-compatible glycoproteins. In Saccharomyces cerevisiae, MNN4 and MNN6 genes are known to play roles in mannosylphosphorylation, but disruption of these genes does not completely remove the mannosylphosphates in N-glycans. This study was performed to find unknown key gene(s) involved in N-glycan mannosylphosphorylation in S. cerevisiae. For this purpose, each of one MNN4 and five MNN6 homologous genes were deleted from the och1Δmnn1Δmnn4Δmnn6Δ strain, which lacks yeast-specific hyper-mannosylation and the immunogenic α(1,3)-mannose structure. N-glycan profile analysis of cell wall mannoproteins and a secretory recombinant protein produced in mutants showed that the MNN14 gene, an MNN4 paralog with unknown function, is essential for N-glycan mannosylphosphorylation. Double disruption of MNN4 and MNN14 genes was enough to eliminate N-glycan mannosylphosphorylation. Our results suggest that the S. cerevisiae och1Δmnn1Δmnn4Δmnn14Δ strain, in which all yeast-specific N-glycan structures including mannosylphosphorylation are abolished, may have promise as a useful platform for glyco-engineering to produce therapeutic glycoproteins with human-compatible N-glycans.

  1. Structural and immunological characterization of the N-glycans from the major yellow jacket allergen Ves v 2: The N-glycan structures are needed for the human antibody recognition

    DEFF Research Database (Denmark)

    Seppälä, Ulla; Selby, David; Monsalve, Rafael

    2009-01-01

    .01, and residues 66 and 81 of Ves v 2.02. Structural analysis of the glycopeptides showed that the majority of the N-glycans contained at least one alpha1,3-fucose and/or alpha1,6-fucose residues in a structure. Interestingly, serum IgE antibodies from vespid allergic patients recognized nVes v 2 but not rVes v 2...

  2. Expression of LacdiNAc Groups on N-Glycans among Human Tumors Is Complex

    Directory of Open Access Journals (Sweden)

    Kiyoko Hirano

    2014-01-01

    Full Text Available Aberrant glycosylation of proteins and lipids is one of the characteristic features of malignantly transformed cells. The GalNAcβ1 → 4GlcNAc (LacdiNAc or LDN group at the nonreducing termini of both N- and O-glycans is not generally found in mammalian cells. We previously showed that the expression level of the LacdiNAc group in N-glycans decreases dramatically during the progression of human breast cancer. In contrast, the enhanced expression of the LacdiNAc group has been shown to be associated with the progression of human prostate, ovarian, and pancreatic cancers. Therefore, the expression of the disaccharide group appears to be dependent on types of tumors. The mechanism of formation of the LacdiNAc group in human tumors and cancer cells has been studied, and two β4-N-acetylgalacto-saminyltransferases (β4GalNAcTs, β4GalNAcT3 and β4GalNAcT4, have been shown to be involved in the biosynthesis of this disaccharide group in a tissue-dependent manner. Transfection of the β4GalNAcT3 gene brought about significant changes in the malignant phenotypes of human neuroblastoma, indicating that this disaccharide group is important for suppressing the tumor growth.

  3. Pancreatic α-Amylase Controls Glucose Assimilation by Duodenal Retrieval through N-Glycan-specific Binding, Endocytosis, and Degradation.

    Science.gov (United States)

    Date, Kimie; Satoh, Ayano; Iida, Kaoruko; Ogawa, Haruko

    2015-07-10

    α-Amylase, a major pancreatic protein and starch hydrolase, is essential for energy acquisition. Mammalian pancreatic α-amylase binds specifically to glycoprotein N-glycans in the brush-border membrane to activate starch digestion, whereas it significantly inhibits glucose uptake by Na(+)/glucose cotransporter 1 (SGLT1) at high concentrations (Asanuma-Date, K., Hirano, Y., Le, N., Sano, K., Kawasaki, N., Hashii, N., Hiruta, Y., Nakayama, K., Umemura, M., Ishikawa, K., Sakagami, H., and Ogawa, H. (2012) Functional regulation of sugar assimilation by N-glycan-specific interaction of pancreatic α-amylase with glycoproteins of duodenal brush border membrane. J. Biol. Chem. 287, 23104-23118). However, how the inhibition is stopped was unknown. Here, we show a new mechanism for the regulation of intestinal glucose absorption. Immunohistochemistry revealed that α-amylase in the duodena of non-fasted, but not fasted, pigs was internalized from the pancreatic fluid and immunostained. We demonstrated that after N-glycan binding, pancreatic α-amylase underwent internalization into lysosomes in a process that was inhibited by α-mannoside. The internalized α-amylase was degraded, showing low enzymatic activity and molecular weight at the basolateral membrane. In a human intestinal Caco-2 cell line, Alexa Fluor 488-labeled pancreatic α-amylase bound to the cytomembrane was transported to lysosomes through the endocytic pathway and then disappeared, suggesting degradation. Our findings indicate that N-glycan recognition by α-amylase protects enterocytes against a sudden increase in glucose concentration and restores glucose uptake by gradual internalization, which homeostatically controls the postprandial blood glucose level. The internalization of α-amylase may also enhance the supply of amino acids required for the high turnover of small intestine epithelial cells. This study provides novel and significant insights into the control of blood sugar during the absorption

  4. Pancreatic α-Amylase Controls Glucose Assimilation by Duodenal Retrieval through N-Glycan-specific Binding, Endocytosis, and Degradation*

    Science.gov (United States)

    Date, Kimie; Satoh, Ayano; Iida, Kaoruko; Ogawa, Haruko

    2015-01-01

    α-Amylase, a major pancreatic protein and starch hydrolase, is essential for energy acquisition. Mammalian pancreatic α-amylase binds specifically to glycoprotein N-glycans in the brush-border membrane to activate starch digestion, whereas it significantly inhibits glucose uptake by Na+/glucose cotransporter 1 (SGLT1) at high concentrations (Asanuma-Date, K., Hirano, Y., Le, N., Sano, K., Kawasaki, N., Hashii, N., Hiruta, Y., Nakayama, K., Umemura, M., Ishikawa, K., Sakagami, H., and Ogawa, H. (2012) Functional regulation of sugar assimilation by N-glycan-specific interaction of pancreatic α-amylase with glycoproteins of duodenal brush border membrane. J. Biol. Chem. 287, 23104–23118). However, how the inhibition is stopped was unknown. Here, we show a new mechanism for the regulation of intestinal glucose absorption. Immunohistochemistry revealed that α-amylase in the duodena of non-fasted, but not fasted, pigs was internalized from the pancreatic fluid and immunostained. We demonstrated that after N-glycan binding, pancreatic α-amylase underwent internalization into lysosomes in a process that was inhibited by α-mannoside. The internalized α-amylase was degraded, showing low enzymatic activity and molecular weight at the basolateral membrane. In a human intestinal Caco-2 cell line, Alexa Fluor 488-labeled pancreatic α-amylase bound to the cytomembrane was transported to lysosomes through the endocytic pathway and then disappeared, suggesting degradation. Our findings indicate that N-glycan recognition by α-amylase protects enterocytes against a sudden increase in glucose concentration and restores glucose uptake by gradual internalization, which homeostatically controls the postprandial blood glucose level. The internalization of α-amylase may also enhance the supply of amino acids required for the high turnover of small intestine epithelial cells. This study provides novel and significant insights into the control of blood sugar during the absorption

  5. Regulation of Mac-2BP secretion is mediated by its N-glycan binding to ERGIC-53.

    Science.gov (United States)

    Chen, Yang; Hojo, Sanae; Matsumoto, Naoki; Yamamoto, Kazuo

    2013-07-01

    The leguminous-type (L-type) lectin ER-Golgi intermediate compartment (ERGIC)-53, a homo-oligomeric endoplasmic reticulum (ER)-Golgi recycling protein, functions as a transport receptor for newly synthesized glycoproteins in the early secretory pathway. Although a limited subset of cargo glycoproteins transported by ERGIC-53, such as the coagulation factors V and VIII, cathepsin C and Z and α1-antitrypsin, has been identified, the exact role of the N-glycan binding of ERGIC-53 in the transport of secretory glycoproteins for ER exit has yet to be clarified. By screening a cDNA library isolated from HepG2 cells via a green fluorescent protein fragment complementation assay, we assessed several candidate luminal ERGIC-53-interacting partners and identified Mac-2 binding protein (Mac-2BP) as a novel ERGIC-53-transported cargo glycoprotein. Using an N-glycan-binding-deficient mutant of ERGIC-53 (N156A) or treatment with N-glycosylation processing inhibitors, as well as the introduction of the ER-mis-targeting mutant (KKAA), we demonstrated that the high-mannose-type N-glycan binding of ERGIC-53 contributes to its interaction with Mac-2BP, which is essential for the ERGIC-53-mediated ER-Golgi transport of nascent proteins during early secretion. Furthermore, we also provide evidence that MCFD2 is involved in the secretion of Mac-2BP. These observations reveal a distinct role for the N-glycan binding of ERGIC-53 in the receptor-mediated ER exit of newly synthesized Mac-2BP in the early secretion pathway.

  6. LC-MS/MS Peptide Mapping with Automated Data Processing for Routine Profiling of N-Glycans in Immunoglobulins

    Science.gov (United States)

    Shah, Bhavana; Jiang, Xinzhao Grace; Chen, Louise; Zhang, Zhongqi

    2014-06-01

    Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.

  7. alpha-mannosidase involved in turnover of plant complex type N-glycans in tomato (Lycopersicum esculentum) fruits.

    Science.gov (United States)

    Hossain, Md Anowar; Nakamura, Kosuke; Kimura, Yoshinobu

    2009-01-01

    In this study, we purified and characterized an alpha-mannosidase to homogeneity from mature red tomato fruits. Purified alpha-mannosidase (alpha-Man LE-1) gave two separate bands, of molecular masses of 70 kDa (L-subunit) and 47 kDa (S-subunit), on SDS-PAGE under non-reducing and reducing conditions. On the other hand, the molecular weight was estimated to be 230 kDa by gel filtration, indicating that alpha-Man LE-1 functions in a tetrameric structure in plant cells. The N-terminal sequence of the L-subunit and the S-subunit were determined to be L-Y-M-V-Y-M-T-K-Q-G- and X-X-L-E-Q/K-S-F-S-Y-Y respectively. When pyridylaminated N-glycans were used as substrates, alpha-Man LE-1 showed optimum activity at about pH 6 and at 40 degrees C, and the activity was completely inhibited by both swainsonine and 1-deoxy-mannojirimycin. alpha-Man LE-1 hydrolyzed the alpha-mannosidic linkages from both high-mannose type and plant complex type N-glycan, but preferred a truncated plant complex type structure to high-mannose type N-glycans bearing alpha1-2 mannosyl residues.

  8. Agl16, a thermophilic glycosyltransferase mediating the last step of N-Glycan biosynthesis in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Meyer, Benjamin H; Peyfoon, Elham; Dietrich, Carsten; Hitchen, Paul; Panico, Maria; Morris, Howard R; Dell, Anne; Albers, Sonja-Verena

    2013-05-01

    Recently, the S-layer protein of Sulfolobus acidocaldarius was shown to be N-linked with a tribranched hexasaccharide, composed of Man2Glc1GlcNAc2 and a sulfated sugar called sulfoquinovose. To identify genes involved in the biosynthesis and attachment of this glycan, markerless in-frame deletions of genes coding for predicted glycosyltransferases were created. The successful deletion of agl16, coding for a glycosyltransferase, resulted in the S-layer protein and archaellins having reduced molecular weights, as visualized by Coomassie staining or immunoblotting. This analysis indicated a change in the N-glycan composition. Nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses confirmed that the glycan of the S-layer protein from the agl16 deletion mutant was a pentasaccharide, which was missing a terminal hexose residue. High-performance liquid chromatography (HPLC) analyses of the hydrolyzed N-glycan indicated that the missing hexose is a glucose residue. A physiological characterization of the agl16 deletion mutant revealed a significant effect on the growth at elevated salt concentrations. At 300 mM NaCl, the doubling time of the Δagl16 mutant was increased 2-fold compared to that of the background strain. Furthermore, the incomplete glycan structure of the Δagl16 deletion strain affected the assembly and function of the archaellum, as exemplified by semisolid Gelrite plate analysis, in which the motility is decreased according to the N-glycan size.

  9. Electrostatics and N-glycan-mediated membrane tethering of SCUBE1 is critical for promoting bone morphogenetic protein signalling.

    Science.gov (United States)

    Liao, Wei-Ju; Tsao, Ku-Chi; Yang, Ruey-Bing

    2016-03-01

    SCUBE1 (S1), a secreted and membrane-bound glycoprotein, has a modular protein structure composed of an N-terminal signal peptide sequence followed by nine epidermal growth factor (EGF)-like repeats, a spacer region and three cysteine-rich (CR) motifs with multiple potential N-linked glycosylation sites, and one CUB domain at the C-terminus. Soluble S1 is a biomarker of platelet activation but an active participant of thrombosis via its adhesive EGF-like repeats, whereas its membrane-associated form acts as a bone morphogenetic protein (BMP) co-receptor in promoting BMP signal activity. However, the mechanism responsible for the membrane tethering and the biological importance of N-glycosylation of S1 remain largely unknown. In the present study, molecular mapping analysis identified a polycationic segment (amino acids 501-550) in the spacer region required for its membrane tethering via electrostatic interactions possibly with the anionic heparan sulfate proteoglycans. Furthermore, deglycosylation by peptide N-glycosidase F treatment revealed that N-glycans within the CR motif are essential for membrane recruitment through lectin-mediated surface retention. Injection of mRNA encoding zebrafish wild-type but not N-glycan-deficient scube1 restores the expression of haematopoietic and erythroid markers (scl and gata1) in scube1-knockdown embryos. We describe novel mechanisms in targeting S1 to the plasma membrane and demonstrate that N-glycans are required for S1 functions during primitive haematopoiesis in zebrafish.

  10. Carboxylated N-glycans on RAGE promote S100A12 binding and signaling

    Science.gov (United States)

    Srikrishna, Geetha; Nayak, Jonamani; Weigle, Bernd; Temme, Achim; Foell, Dirk; Hazelwood, Larnele; Olsson, Anna; Volkmann, Niels; Hanein, Dorit; Freeze, Hudson H.

    2010-01-01

    RAGE, the Receptor for Advanced Glycation End Products, is a signaling receptor protein of the immunoglobulin superfamily implicated in multiple pathologies. It binds a diverse repertoire of ligands, but the structural basis for the interaction of different ligands is not well understood. We earlier showed that carboxylated glycans on the V-domain of RAGE promote the binding of HMGB1 and S100A8/A9. Here we study the role of these glycans on the binding and intracellular signaling mediated by another RAGE ligand, S100A12. S100A12 binds carboxylated glycans, and a subpopulation of RAGE enriched for carboxylated glycans shows more than ten fold higher binding potential for S100A12 than total RAGE. When expressed in mammalian cells, RAGE is modified by complex glycans predominantly at the first glycosylation site (N25IT) that retains S100A12 binding. Glycosylation of RAGE and maximum binding sites for S100A12 on RAGE are also cell type dependent. Carboxylated glycan-enriched population of RAGE forms higher order multimeric complexes with S100A12, and this ability to multimerize is reduced upon deglycosylation or by using non-glycosylated sRAGE expressed in E.coli. mAbGB3.1, an antibody against carboxylated glycans, blocks S100A12 mediated NF-κB signaling in HeLa cells expressing full length RAGE. These results demonstrate that carboxylated N-glycans on RAGE enhance binding potential and promote receptor clustering and subsequent signaling events following oligomeric S100A12 binding. PMID:20512925

  11. Fucosylation Is a Promising Target for Cancer Diagnosis and Therapy

    Directory of Open Access Journals (Sweden)

    Shinichiro Shinzaki

    2012-01-01

    Full Text Available Oligosaccharides, sequences of carbohydrates conjugated to proteins and lipids, are arguably the most abundant and structurally diverse class of molecules. Fucosylation is one of the most important oligosaccharide modifications involved in cancer and inflammation. Recent advances in glycomics have identified several types of glyco-biomarkers containing fucosylation that are linked to certain types of cancer. Fucosylated alpha-fetoprotein (AFP is widely used in the diagnosis of hepatocellular carcinoma because it is more specific than alpha-fetoprotein. High levels of fucosylated haptoglobin have also been found in sera of patients with various carcinomas. We have recently established a simple lectin-antibody ELISA to measure fucosylated haptoglobin and to investigate its clinical use. Cellular fucosylation is dependent upon fucosyltransferase activity and the level of its donor substrate, guanosine diphosphate (GDP-fucose. GDP-mannose-4,6-dehydratase (GMDS is a key enzyme involved in the synthesis of GDP-fucose. Mutations of GMDS found in colon cancer cells induced a malignant phenotype, leading to rapid growth in athymic mice resistant to natural killer cells. This review describes the role of fucosylated haptoglobin as a cancer biomarker, and discusses the possible biological role of fucosylation in cancer development.

  12. A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in pregnancy.

    Science.gov (United States)

    Kapur, Rick; Kustiawan, Iwan; Vestrheim, Anne; Koeleman, Carolien A M; Visser, Remco; Einarsdottir, Helga K; Porcelijn, Leendert; Jackson, Dave; Kumpel, Belinda; Deelder, André M; Blank, Dennis; Skogen, Björn; Killie, Mette Kjaer; Michaelsen, Terje E; de Haas, Masja; Rispens, Theo; van der Schoot, C Ellen; Wuhrer, Manfred; Vidarsson, Gestur

    2014-01-23

    Immunoglobulin G (IgG) formed during pregnancy against human platelet antigens (HPAs) of the fetus mediates fetal or neonatal alloimmune thrombocytopenia (FNAIT). Because antibody titer or isotype does not strictly correlate with disease severity, we investigated by mass spectrometry variations in the glycosylation at Asn297 in the IgG Fc because the composition of this glycan can be highly variable, affecting binding to phagocyte IgG-Fc receptors (FcγR). We found markedly decreased levels of core fucosylation of anti-HPA-1a-specific IgG1 from FNAIT patients (n = 48), but not in total serum IgG1. Antibodies with a low amount of fucose displayed higher binding affinity to FcγRIIIa and FcγRIIIb, but not to FcγRIIa, compared with antibodies with a high amount of Fc fucose. Consequently, these antibodies with a low amount of Fc fucose showed enhanced phagocytosis of platelets using FcγRIIIb(+) polymorphonuclear cells or FcγRIIIa(+) monocytes as effector cells, but not with FcγRIIIa(-) monocytes. In addition, the degree of anti-HPA-1a fucosylation correlated positively with the neonatal platelet counts in FNAIT, and negatively to the clinical disease severity. In contrast to the FNAIT patients, no changes in core fucosylation were observed for anti-HLA antibodies in refractory thrombocytopenia (post platelet transfusion), indicating that the level of fucosylation may be antigen dependent and/or related to the immune milieu defined by pregnancy.

  13. The S-layer glycoprotein of the crenarchaeote Sulfolobus acidocaldarius is glycosylated at multiple sites with chitobiose-linked N-glycans.

    Science.gov (United States)

    Peyfoon, Elham; Meyer, Benjamin; Hitchen, Paul G; Panico, Maria; Morris, Howard R; Haslam, Stuart M; Albers, Sonja-Verena; Dell, Anne

    2010-09-29

    Glycosylation of the S-layer of the crenarchaea Sulfolobus acidocaldarius has been investigated using glycoproteomic methodologies. The mature protein is predicted to contain 31 N-glycosylation consensus sites with approximately one third being found in the C-terminal domain spanning residues L(1004)-Q(1395). Since this domain is rich in Lys and Arg and therefore relatively tractable to glycoproteomic analysis, this study has focused on mapping its N-glycosylation. Our analysis identified nine of the 11 consensus sequence sites, and all were found to be glycosylated. This constitutes a remarkably high glycosylation density in the C-terminal domain averaging one site for each stretch of 30-40 residues. Each of the glycosylation sites observed was shown to be modified with a heterogeneous family of glycans, with the largest having a composition Glc(1)Man(2)GlcNAc(2) plus 6-sulfoquinovose (QuiS), consistent with the tribranched hexasaccharide previously reported in the cytochrome b(558/566) of S. acidocaldarius. S. acidocaldarius is the only archaeal species whose N-glycans are known to be linked via the chitobiose core disaccharide that characterises the N-linked glycans of Eukarya.

  14. The S-Layer Glycoprotein of the Crenarchaeote Sulfolobus acidocaldarius Is Glycosylated at Multiple Sites with Chitobiose-Linked N-Glycans

    Directory of Open Access Journals (Sweden)

    Elham Peyfoon

    2010-01-01

    Full Text Available Glycosylation of the S-layer of the crenarchaea Sulfolobus acidocaldarius has been investigated using glycoproteomic methodologies. The mature protein is predicted to contain 31 N-glycosylation consensus sites with approximately one third being found in the C-terminal domain spanning residues L1004-Q1395. Since this domain is rich in Lys and Arg and therefore relatively tractable to glycoproteomic analysis, this study has focused on mapping its N-glycosylation. Our analysis identified nine of the 11 consensus sequence sites, and all were found to be glycosylated. This constitutes a remarkably high glycosylation density in the C-terminal domain averaging one site for each stretch of 30–40 residues. Each of the glycosylation sites observed was shown to be modified with a heterogeneous family of glycans, with the largest having a composition Glc1Man2GlcNAc2 plus 6-sulfoquinovose (QuiS, consistent with the tribranched hexasaccharide previously reported in the cytochrome b558/566 of S. acidocaldarius. S. acidocaldarius is the only archaeal species whose N-glycans are known to be linked via the chitobiose core disaccharide that characterises the N-linked glycans of Eukarya.

  15. Roles of N-glycans in the polymerization-dependent aggregation of mutant Ig-μ chains in the early secretory pathway.

    Science.gov (United States)

    Giannone, Chiara; Fagioli, Claudio; Valetti, Caterina; Sitia, Roberto; Anelli, Tiziana

    2017-02-03

    The polymeric structure of secretory IgM allows efficient antigen binding and complement fixation. The available structural models place the N-glycans bound to asparagines 402 and 563 of Ig-μ chains within a densely packed core of native IgM. These glycans are found in the high mannose state also in secreted IgM, suggesting that polymerization hinders them to Golgi processing enzymes. Their absence alters polymerization. Here we investigate their role following the fate of aggregation-prone mutant μ chains lacking the Cμ1 domain (μ∆). Our data reveal that μ∆ lacking 563 glycans (μ∆5) form larger intracellular aggregates than μ∆ and are not secreted. Like μ∆, they sequester ERGIC-53, a lectin previously shown to promote polymerization. In contrast, μ∆ lacking 402 glycans (μ∆4) remain detergent soluble and accumulate in the ER, as does a double mutant devoid of both (μ∆4-5). These results suggest that the two C-terminal Ig-μ glycans shape the polymerization-dependent aggregation by engaging lectins and acting as spacers in the alignment of individual IgM subunits in native polymers.

  16. The role of N-glycans and the C-terminal loop of the subunit rBAT in the biogenesis of the cystinuria-associated transporter.

    Science.gov (United States)

    Rius, Mònica; Sala, Laura; Chillarón, Josep

    2016-02-01

    The transport system b(0,+) mediates reabsorption of dibasic amino acids and cystine in the kidney. It is made up of two disulfide-linked membrane subunits: the carrier, b(0,+)AT and the helper, rBAT (related to b(0,+) amino acid transporter). rBAT mutations that impair biogenesis of the transporter cause type I cystinuria. It has been shown that upon assembly, b(0,+)AT prevents degradation and promotes folding of rBAT; then, rBAT traffics b(0,+)AT from the endoplasmic reticulum (ER) to the plasma membrane. The role of the N-glycans of rBAT and of its C-terminal loop, which has no homology to any other sequence, in biogenesis of system b(0,+) is unknown. In the present study, we studied these points. We first identified the five N-glycans of rBAT. Elimination of the N-glycan Asn(575), but not of the others, delayed transporter maturation, as measured by pulse chase experiments and endoglycosidase H assays. Moreover, a transporter with only the N-glycan Asn(575) displayed similar maturation compared with wild-type, suggesting that this N-glycan was necessary and sufficient to achieve the maximum rate of transporter maturation. Deletion of the rBAT C-terminal disulfide loop (residues 673-685) prevented maturation and prompted degradation of the transporter. Alanine-scanning mutagenesis uncovered loop residues important for stability and/or maturation of system b(0,+). Further, double-mutant cycle analysis showed partial additivity of the effects of the Asn(679) loop residue and the N-glycan Asn(575) on transporter maturation, indicating that they may interact during system b(0,+) biogenesis. These data highlight the important role of the N-glycan Asn(575) and the C-terminal disulfide loop of rBAT in biogenesis of the rBAT-b(0,+)AT heterodimer.

  17. Specific N-glycan alterations are coupled in epithelial-mesenchymal transition induced by EGF in GE11 epithelial cells.

    Science.gov (United States)

    Xu, Qingsong; Qu, Chen; Wang, Wenjing; Gu, Jianguo; Du, Yuguang; Song, Linsheng

    2017-02-01

    Epithelial-mesenchymal transition (EMT) is a phenomenon in cancer progression during which cancer cells undergo remarkable alteration acquiring highly invasive property. The aim of this study was to evaluate specific N-glycan alterations during EMT induced by epidermal growth factor (EGF) in GE11 epithelial cells. Herein, we demonstrated that EGF activated epidermal growth factor receptor (EGFR)/Akt/extracellular signal-regulated kinase (ERK) phosphorylation and promoted GE11 cell proliferation. Meanwhile, EGF stimulated the epithelial cells to undergo morphological alteration, destroying cell-cell inter-contact and exhibiting mesenchymal cells higher metastatic potential. A wound-healing assay showed the migratory ability increased 1.5-fold after EGF treatment. Moreover, the relative intensity of N-cadherin versus E-cadherin increased 2.6-fold, and the E-cadherin distribution in cell-cell junctions became jagged and faint after EGF incubation for 72 h. Interestingly, the amounts of bisecting GlcNAc structure were dramatically declined, by contrast, the formation of β1,6 GlcNAc branches on cell surface was upregulated during EMT induced by EGF. To understand the roles of N-glycans in EGF-induced EMT, the cells were stably transfected with N-acetylglucosaminyltransferase III (GnT-III), which catalyzes the bisecting GlcNAc structure formation. As the markers for EMT, EGF-induced E-cadherin decrease and fibronectin increase were delayed in GnT-III-overexpressing cells. Taken together, these results demonstrated that specific N-glycan alterations were coupled in EMT induced by EGF, which might be contributed to diagnosis and therapy of tumor metastasis.

  18. Reversal effect of GnT-V on the radioresistance of human nasopharyngeal carcinoma cells by alteration β1, 6-GlcNAc branched N-glycans.

    Science.gov (United States)

    Wu, Jun-Bo; Shen, Li; Qiu, Li; Duan, Qi-Wen; Luo, Zhi-Guo; Dong, Xiao-Xia

    2015-01-01

    Radiotherapy is the primary treatment for human nasopharyngeal carcinoma (NPC), yet radioresistance remains a major obstacle to successful treatment in many cases. N-acetylglucosaminyltransferase V (GnT-V), which synthesizes β1, 6-GlcNAc branched N-glycans, is closely related to the radiosensitivity of NPC cells. However, a better understanding of the functional role of GnT-V in NPC radioresistance and the related mechanisms is urgently needed. In the present study, a radioresistant NPC cell line, CNE-2R, was established by repeated γ-irradiation. We found that GnT-V levels, as well as β1, 6-GlcNAc branched N-glycans were significantly increased in the CNE-2R cells as compared with that in the parental cells. Meanwhile, knockdown of GnT-V in the CNE-2R cells enhanced cell radiosensitivity and inhibited the formation of β1, 6-branched N-glycans. In addition, the regulated expression of GnT-V in the CNE-2R cells converted the heterogeneous N-glycosylated forms of CD147. Furthermore, swainsonine, an inhibitor of N-glycan biosynthesis, was also able to reverse the radioresistance of the CNE-2R cells. Taken together, the present study revealed a novel mechanism of GnT-V as a regulator of radioresistance in NPC cells, which may be useful for fully understanding the biological role of N-glycans in NPC radioresistance.

  19. Emerging Structural Insights into Glycoprotein Quality Control Coupled with N-Glycan Processing in the Endoplasmic Reticulum

    Directory of Open Access Journals (Sweden)

    Tadashi Satoh

    2015-01-01

    Full Text Available In the endoplasmic reticulum (ER, the sugar chain is initially introduced onto newly synthesized proteins as a triantennary tetradecasaccharide (Glc3Man9GlcNAc2. The attached oligosaccharide chain is subjected to stepwise trimming by the actions of specific glucosidases and mannosidases. In these processes, the transiently expressed N-glycans, as processing intermediates, function as signals for the determination of glycoprotein fates, i.e., folding, transport, or degradation through interactions of a series of intracellular lectins. The monoglucosylated glycoforms are hallmarks of incompletely folded states of glycoproteins in this system, whereas the outer mannose trimming leads to ER-associated glycoprotein degradation. This review outlines the recently emerging evidence regarding the molecular and structural basis of this glycoprotein quality control system, which is regulated through dynamic interplay among intracellular lectins, glycosidases, and glycosyltransferase. Structural snapshots of carbohydrate-lectin interactions have been provided at the atomic level using X-ray crystallographic analyses. Conformational ensembles of uncomplexed triantennary high-mannose-type oligosaccharides have been characterized in a quantitative manner using molecular dynamics simulation in conjunction with nuclear magnetic resonance spectroscopy. These complementary views provide new insights into glycoprotein recognition in quality control coupled with N-glycan processing.

  20. 精神分裂症患者血清免疫球蛋白核心岩藻糖基化水平与体液免疫指标的相关性研究%Study of correlation between the levels of serum immunoglobulin core fucosylation and humoral immune parameters in patients with schizophrenia

    Institute of Scientific and Technical Information of China (English)

    郭静; 邱锦云; 冯方波

    2014-01-01

    目的:研究精神分裂症患者血清免疫球蛋白核心岩藻糖基化水平与体液免疫各项指标之间的关系,探讨其免疫学意义。方法采用直线相关分析法,研究46例精神分裂症组和47例对照组血清小扁豆凝集素(LCA )-IgM、LCA-IgG、LCA-IgA水平,与血清IgM、IgG、IgA、循环免疫复合物(CIC)水平的相关性。结果对照组血清LCA-IgA 水平与 IgG水平呈正相关(r=0.3117,P<0.05)。精神分裂症组血清LCA-IgG水平与IgA水平呈负相关(r=-0.3236,P<0.05),精神分裂症组血清LCA-IgM、LCA-IgG、LCA-IgA水平均与CIC水平呈正相关(r=0.3548,r=0.1898,r=0.4797,P<0.05)。结论精神分裂症患者血清免疫球蛋白发生了过度岩藻糖基化,并参与患者的中枢神经系统免疫损伤的病理过程。%Objective To investigate the correlation between the levels of serum immunoglobulin core fucosylation and humoral immune parameters in patients with schizophrenia and its immunological significance .Methods The levels of serum lens culinaris agglutinin(LCA)-IgM ,LCA-IgG ,LCA-IgA ,IgM ,IgG ,IgA ,circular immune complex(CIC) in patients with schizophrenia(n= 46) and health people(n=47) were determined .Their correlations were investigated by linear regression analysis .Results There was a positive correlation between the LCA-IgA and IgG in healthy people(r=0 .311 7 ,P<0 .05) .However ,there was a negative correla-tion between LCA-IgG and IgA in patients with schizophrenia(r=0 .323 6 ,P<0 .05) .In addition ,there were positive correlations between LCA-IgM ,LCA-IgG ,LCA-IgA and CIC(r=0 .354 8 ,r=0 .189 8 ,r=0 .479 7 ,P<0 .05) .Conclusion Increased fucosyla-tion levels of immunoglobulin were detected in patients with schizophrenia ,which may play important roles in immunological inju-ries of central nervous system in schizophrenia .

  1. Identification of physiologically relevant substrates for cloned Gal: 3-O-sulfotransferases (Gal3STs): distinct high affinity of Gal3ST-2 and LS180 sulfotransferase for the globo H backbone, Gal3ST-3 for N-glycan multiterminal Galbeta1, 4GlcNAcbeta units and 6-sulfoGalbeta1, 4GlcNAcbeta, and Gal3ST-4 for the mucin core-2 trisaccharide.

    Science.gov (United States)

    Chandrasekaran, E V; Lakhaman, Sukhwinder S; Chawda, Ram; Piskorz, Conrad F; Neelamegham, Sriram; Matta, Khushi L

    2004-03-12

    Sulfated glycoconjugates regulate biological processes such as cell adhesion and cancer metastasis. We examined the acceptor specificities and kinetic properties of three cloned Gal:3-O-sulfotransferases (Gal3STs) ST-2, ST-3, and ST-4 along with a purified Gal3ST from colon carcinoma LS180 cells. Gal3ST-2 was the dominant Gal3ST in LS180. While the mucin core-2 structure Galbeta1,4GlcNAcbeta1,6(3-O-MeGalbeta1,3)GalNAcalpha-O-Bn (where Bn is benzyl) and the disaccharide Galbeta1,4GlcNAc served as high affinity acceptors for Gal3ST-2 and Gal3ST-3, 3-O-MeGalbeta1,4GlcNAcbeta1,-6(Galbeta1,3)GalNAcalpha-O-Bn and Galbeta1,3GalNAcalpha-O-Al (where Al is allyl) were efficient acceptors for Gal3ST-4. The activities of Gal3ST-2 and Gal3ST-3 could be distinguished with the Globo H precursor (Galbeta1,3GalNAcbeta1,3Galalpha-O-Me) and fetuin triantennary asialoglycopeptide. Gal3ST-2 acted efficiently on the former, while Gal3ST-3 showed preference for the latter. Gal3ST-4 also acted on the Globo H precursor but not the glycopeptide. In support of the specificity, Gal3ST-2 activity toward the Galbeta1,4GlcNAcbeta unit on mucin core-2 as well as the Globo H precursor could be inhibited competitively by Galbeta1,4GlcNAcbeta1,6(3-O-sulfoGalbeta1,3)GalNAcalpha-O-Bn but not 3-O-sulfoGalbeta1,-4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-O-Bn. Remarkably these sulfotransferases were uniquely specific for sulfated substrates: Gal3ST-3 utilized Galbeta1,4(6-O-sulfo)-GlcNAcbeta-O-Al as acceptor, Gal3ST-2 acted efficiently on Galbeta1,3(6-O-sulfo)GlcNAcbeta-O-Al, and Gal3ST-4 acted efficiently on Galbeta1,3(6-O-sulfo)GalNAcalpha-O-Al. Mg(2+), Mn(2+), and Ca(2+) stimulated the activities of Gal3ST-2, whereas only Mg(2+) augmented Gal3ST-3 activity. Divalent cations did not stimulate Gal3ST-4, although inhibition was noted at high Mn(2+) concentrations. The fine substrate specificities of Gal3STs indicate a distinct physiological role for each enzyme.

  2. Preparative scale purification of fucosyl-N-acetylglucosamine disaccharides and their evaluation as potential prebiotics and antiadhesins.

    Science.gov (United States)

    Becerra, Jimmy E; Coll-Marqués, José M; Rodríguez-Díaz, Jesús; Monedero, Vicente; Yebra, María J

    2015-09-01

    Fucosyl-N-acetylglucosamine disaccharides are important core structures that form part of human mucosal and milk glyco-complexes. We have previously shown that AlfB and AlfC α-L-fucosidases from Lactobacillus casei are able to synthesize fucosyl-α-1,3--N-acetylglucosamine (Fuc-α1,3-GlcNAc) and fucosyl-α-1,6-N-acetylglucosamine (Fuc-α1,6-GlcNAc), respectively, in transglycosylation reactions. Here, these reactions were performed in a semipreparative scale, and the produced disaccharides were purified. The maximum yields obtained of Fuc-α1,3-GlcNAc and Fuc-α1,6-GlcNAc were 4.2 and 9.3 g/l, respectively. The purified fucosyl-disaccharides were then analyzed for their prebiotic effect in vitro using strains from the Lactobacillus casei/paracasei/rhamnosus group and from Bifidobacterium species. The results revealed that 6 out of 11 L. casei strains and 2 out of 6 L. rhamnosus strains tested were able to ferment Fuc-α1,3-GlcNAc, and L. casei BL87 and L. rhamnosus BL327 strains were also able to ferment Fuc-α1,6-GlcNAc. DNA hybridization experiments suggested that the metabolism of Fuc-α1,3-GlcNAc in those strains relies in an α-L-fucosidase homologous to AlfB. Bifidobacterium breve and Bibidobacterium pseudocatenolatum species also metabolized Fuc-α1,3-GlcNAc. Notably, L-fucose was excreted from all the Lactobacillus and Bifidobacterium strains fermenting fucosyl-disaccharides, except from strains L. rhamnosus BL358 and BL377, indicating that in these latest strains, L-fucose was catabolized. The fucosyl-disaccharides were also tested for their inhibitory potential of pathogen adhesion to human colon adenocarcinoma epithelial (HT29) cell line. Enteropathogenic Escherichia coli (EPEC) strains isolated from infantile gastroenteritis were used, and the results showed that both fucosyl-disaccharides inhibited adhesion to different extents of certain EPEC strains to HT29 cells in tissue culture.

  3. The Analysis of Sialylation, N-Glycan Branching, and Expression of O-Glycans in Seminal Plasma of Infertile Men

    Directory of Open Access Journals (Sweden)

    Ewa M. Kratz

    2015-01-01

    Full Text Available Carbohydrates are known to mediate some events involved in successful fertilization. Although some studies on the glycosylation of seminal plasma proteins are available, the total glycan profile was rarely analyzed as a feature influencing fertilization potential. In this work we aimed to compare some glycosylation traits in seminal plasma glycoproteins of fertile and infertile men. The following findings emerge from our studies: (1 in human seminal plasma the presence and alterations of O-linked glycans were observed; (2 the expression of SNA-reactive sialic acid significantly differs between asthenozoospermia and both normozoospermic (fertile and infertile groups; (3 the expression of PHA-L-reactive highly branched N-glycans was significantly lower in oligozoospermic patients than in both normozoospermic groups. Indication of the appropriate lectins that would enable the possibly precise determination of the glycan profile seems to be a good supplement to mass spectrum analysis. Extension of the lectin panel is useful for the further research.

  4. N-glycan analysis of recombinant L-Selectin reveals sulfated GalNAc and GalNAc-GalNAc motifs.

    Science.gov (United States)

    Wedepohl, Stefanie; Kaup, Matthias; Riese, Sebastian B; Berger, Markus; Dernedde, Jens; Tauber, Rudolf; Blanchard, Véronique

    2010-07-01

    The leukocytic adhesion receptor L-selectin plays a crucial role in the first step of the adhesion cascade, enabling leukocytes to migrate into surrounding tissues during inflammation and immune surveillance. We analyzed the site-specific N-glycosylation of the lectin and EGF-like domain of L-selectin using recombinant variants ("LEHis"). The three glycosylation sites of LEHis were mutated to obtain singly glycosylated variants that were expressed in HEK293F cells. alpha1-Acid glycoprotein (AGP), expressed in the same system, was used to distinguish between cell type- and protein-specific glycosylation. Using mass spectrometry and exoglycosidase digestions, we established that LEHis was mostly bearing multifucosylated diantennary N-glycans with a major fraction terminating with GalNAc residues replacing the more common Gal. We could also show that parts of the GalNAc residues were sulfated. Furthermore, we identified novel diantennary glycan structures terminating with the motif GalNAc-GalNAc or SO(4)-GalNAc-GalNAc, which have not been described for N-glycans yet. Interestingly, none of these specific features were found in the N-glycan profile of AGP. This indicates that protein intrinsic information of L-selectin leads to decoration with specific N-glycans, which in turn may be related to L-selectin function.

  5. N-glycan structures of human transferrin produced by Lymantria dispar (gypsy moth)cells using the LdMNPV expression system

    Science.gov (United States)

    One Choi; Noboru Tomiya; Jung H. Kim; James M. Slavicek; Michael J. Betenbaugh; Yuan C. Lee

    2003-01-01

    N-glycan structures of recombinant human serum transferrin (hTf) expressed by Lymantria dispar (gypsy moth) 652Y cells were determined. The gene encoding hTf was incorporated into a Lymantria dispar nucleopolyhedrovirus (LdMNPV) under the control of the polyhedrin promoter. This virus was then...

  6. Highly specific purification of N-glycans using phosphate-based derivatization as an affinity tag in combination with Ti(4+)-SPE enrichment for mass spectrometric analysis.

    Science.gov (United States)

    Zhang, Ying; Peng, Ye; Bin, Zhichao; Wang, Huijie; Lu, Haojie

    2016-08-31

    N-linked protein glycosylation is involved in regulation of a wide variety of cellular processes and associated with numerous diseases. Highly specific identification of N-glycome remains a challenge while its biological significance is acknowledged. The relatively low abundance of glycan in complex biological mixtures, lack of basic sites for protonation, and suppression by other highly abundant proteins/peptides lead to the particularly poor detection sensitivity of N-glycans in the MS analysis. Therefore, the highly specific purification procedure becomes a crucial step prior to MS analysis of the N-glycome. Herein, a novel N-glycans enrichment approach based on phosphate derivatization combined with Ti(4+)-SPE (solid phase extraction) was developed. Briefly, in this strategy, N-glycans were chemically labeled with a phospho-group at their reducing ends, such that the Ti(4+)-SPE microspheres were able to capture the phospho-containing glycans. The enrichment method was developed and optimized using model oligosaccharides (maltoheptaose DP7 and sialylated glycan A1) and also glycans from a standard glycoprotein (asialofetuin, ASF). This method experimentally showed high derivatization efficiency (almost 100%), excellent selectivity (analyzing DP7 in the digests of bovine serum albumin at a mass ratio of 1:100), high enriching recovery (90%), good reproducibility (CVN-glycome in human serum, in which a total of 31 N-glycan masses were identified.

  7. Specifically Binding of L-ficolin to N-glycans of HCV Envelope Glycoproteins E1 and E2 Leads to Complement Activation

    Institute of Scientific and Technical Information of China (English)

    Jun Liu; Mohammed A.M.Ali; Yinghua Shi; Yinglan Zhao; Fenglin Luo; Jin Yu; Tian Xiang; Jie Tang; Dongqing Li; Quan Hu; Wenzhe Ho; Xiaolian Zhang

    2009-01-01

    L-ficolin and HCV E1 and E2 glycoproteins was attributed to the N-glycans of E1 and E2.These findings provide new insights into the biological functions of L-ficolin in clinically important hepatic viral diseases.

  8. Targeted N-glycan deletion at the receptor-binding site retains HIV Env NFL trimer integrity and accelerates the elicited antibody response.

    Science.gov (United States)

    Dubrovskaya, Viktoriya; Guenaga, Javier; de Val, Natalia; Wilson, Richard; Feng, Yu; Movsesyan, Arlette; Karlsson Hedestam, Gunilla B; Ward, Andrew B; Wyatt, Richard T

    2017-09-13

    Extensive shielding by N-glycans on the surface of the HIV envelope glycoproteins (Env) restricts B cell recognition of conserved neutralizing determinants. Elicitation of broadly neutralizing antibodies (bNAbs) in selected HIV-infected individuals reveals that Abs capable of penetrating the glycan shield can be generated by the B cell repertoire. Accordingly, we sought to determine if targeted N-glycan deletion might alter antibody responses to Env. We focused on the conserved CD4 binding site (CD4bs) since this is a known neutralizing determinant that is devoid of glycosylation to allow CD4 receptor engagement, but is ringed by surrounding N-glycans. We selectively deleted potential N-glycan sites (PNGS) proximal to the CD4bs on well-ordered clade C 16055 native flexibly linked (NFL) trimers to potentially increase recognition by naïve B cells in vivo. We generated glycan-deleted trimer variants that maintained native-like conformation and stability. Using a panel of CD4bs-directed bNAbs, we demonstrated improved accessibility of the CD4bs on the N-glycan-deleted trimer variants. We showed that pseudoviruses lacking these Env PNGSs were more sensitive to neutralization by CD4bs-specific bNAbs but remained resistant to non-neutralizing mAbs. We performed rabbit immunogenicity experiments using two approaches comparing glycan-deleted to fully glycosylated NFL trimers. The first was to delete 4 PNGS sites and then boost with fully glycosylated Env; the second was to delete 4 sites and gradually re-introduce these N-glycans in subsequent boosts. We demonstrated that the 16055 PNGS-deleted trimers more rapidly elicited serum antibodies that more potently neutralized the CD4bs-proximal-PNGS-deleted viruses in a statistically significant manner and strongly trended towards increased neutralization of fully glycosylated autologous virus. This approach elicited serum IgG capable of cross-neutralizing selected tier 2 viruses lacking N-glycans at residue N276 (natural or

  9. Glycoprotein fucosylation is increased in seminal plasma of subfertile men

    Directory of Open Access Journals (Sweden)

    Beata Olejnik

    2015-04-01

    Full Text Available Fucose, the monosaccharide frequent in N- and O-glycans, is a part of Lewis-type antigens that are known to mediate direct sperm binding to the zona pellucida. Such interaction was found to be inhibited in vitroby fucose-containing oligo- and polysaccharides, as well as neoglycoproteins. The objective of this study was to screen seminal plasma proteins of infertile/subfertile men for the content and density of fucosylated glycoepitopes, and compare them to samples of fertile normozoospermic subjects. Seminal proteins were separated in polyacrylamide gel electrophoresis and blotted onto nitrocellulose membrane and probed with fucose-specific Aleuria aurantia lectin (AAL. Twelve electrophoretic bands were selected for quantitative densitometric analysis. It was found that the content, and especially the density of fucosylated glycans, were higher in glycoproteins present in seminal plasma of subfertile men. No profound differences in fucosylation density were found among the groups of normozoospermic, oligozoospermic, asthenozoospermic, and oligoasthenozoospermic subfertile men. According to the antibody probing, AAL-reactive bands can be attributed to male reproductive tract glycoproteins, including prostate-specific antigen, prostatic acid phosphatase, glycodelin and chorionic gonadotropin. Fibronectin, α1 -acid glycoprotein, α1 -antitrypsin, immunoglobulin G and antithrombin III may also contribute to this high fucosylation. It is suggested that the abundant fucosylated glycans in the sperm environment could interfere with the sperm surface and disturb the normal course of the fertilization cascade.

  10. Unambiguous characterization of N-glycans of monoclonal antibody cetuximab by integration of LC-MS/MS and ¹H NMR spectroscopy.

    Science.gov (United States)

    Wiegandt, Alena; Meyer, Bernd

    2014-05-20

    Monoclonal antibodies are most rapidly emerging as therapeutic drugs for the treatment of cancer and of various other diseases such as autoimmunity or inflammation. Recently, it was found that nonhuman glycosylation of recombinant antibodies can cause tremendous problems for some patients. Therefore, unambiguous assignment of the glycosylation pattern of therapeutic antibodies is of high importance for assessment of human compatibility. Here we present results from a broad and detailed N-glycan analysis of the therapeutic antibody cetuximab by LC-MS/MS analyses tightly integrated with (1)H NMR to obtain unambiguous structures. Thirty-seven N-glycan compositions were identified by LC-MS(/MS). Subsequently, ten abundant structures were structurally characterized by applying the recently introduced method called three-dimensional cross correlation (3DCC). It was possible to extract NMR spectra of pure N-glycans that were heavily overlapping in a chromatographic separation by mathematically dissecting the NMR spectra obtained from chromatographic fractions. Even mass isobaric structures that differ only in the branching position of one monosaccharide unit were distinguished and characterized. We also developed an improvement of the 3DCC method by introducing singular value decomposition (SVD) for processing of the data. The smallest amount of the N-glycan characterized by 3DCC was approximately 400 pmol (836 ng). Among the ten unambiguously identified glycans, six N-glycans, representing 24% of all detected glycans, possess the immunogenic α-1,3-Gal epitope and/or N-glycolylneuraminic acid. These results illustrate the importance of integrated use of LC-MS(/MS) and (1)H NMR for the glycome analysis of biopharmaceuticals in research, development, and quality control.

  11. Mutations in four glycosyl hydrolases reveal a highly coordinated pathway for rhodopsin biosynthesis and N-glycan trimming in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Erica E Rosenbaum

    2014-05-01

    Full Text Available As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II, α-mannosidase-IIb (α-Man-IIb, a β-N-acetylglucosaminidase called fused lobes (Fdl, and hexosaminidase 1 (Hexo1. We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights

  12. Capillary electrophoresis-electrospray ionization mass spectrometry for rapid and sensitive N-glycan analysis of glycoproteins as 9-fluorenylmethyl derivatives.

    Science.gov (United States)

    Nakano, Miyako; Higo, Daisuke; Arai, Etsuo; Nakagawa, Takatoshi; Kakehi, Kazuaki; Taniguchi, Naoyuki; Kondo, Akihiro

    2009-02-01

    It is well known that most protein therapeutics such as monoclonal antibody pharmaceuticals and other biopharmaceuticals including cancer biomarkers are glycoproteins, and thus the development of high-throughput and sensitive analytical methods for glycans is essential in terms of their determination and quality control. We previously reported a novel alternative labeling method for glycans involving 9-fluorenylmethyl chloroformate (Fmoc-Cl) instead of the conventional reductive amination procedure. The derivatives were analyzed by high-performance liquid chromatography (HPLC) (Kamoda S, Nakano M, Ishikawa R, Suzuki S, Kakehi K. 2005. Rapid and sensitive screening of N-glycans as 9-fluorenylmethyl derivatives by high-performance liquid chromatography: A method which can recover free oligosaccharides after analysis. J Proteome Res. 4:146-152). This method was rapid and simple; however, it was time-consuming in terms of analysis by HPLC and did not provide so much information such as the detailed structures and mass numbers of glycans. Here we have developed a high-throughput and highly sensitive method. It comprises three steps, i.e., release of glycans, derivatization with Fmoc, and capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI MS) analysis. We analyzed several glycoproteins such as fetuin, alpha1 acid glycoprotein, IgG, and transferrin in order to validate this method. We were able to analyze the above glycoproteins with the three-step procedure within only 5 h, which provided detailed N-glycan patterns. Moreover, the MS/MS analysis allowed identification of the N-glycan structures. As novel applications, the method was employed for the analysis of N-glycans derived from monoclonal antibody pharmaceuticals and also from alpha-fetoprotein; the latter is known as one of the tumor markers of hepatocellular carcinomas. We were able to easily and rapidly determine the detailed structures of the N-glycans. The present method is very useful

  13. Mass spectrometric detection of multiple extended series of neutral highly fucosylated N-acetyllactosamine oligosaccharides in human milk

    Science.gov (United States)

    Pfenninger, Anja; Chan, Shiu-Yung; Karas, Michael; Finke, Berndt; Stahl, Bernd; Costello, Catherine E.

    2008-12-01

    Complex mixtures of high-molecular weight fractions of pooled neutral human milk oligosaccharides (obtained via gel permeation chromatography) have been investigated. The subfractions were each permethylated and analyzed by high-resolution mass spectrometry, using matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, in order to investigate their oligosaccharide compositions. The obtained spectra reveal that human milk contains more complex neutral oligosaccharides than have been described previously; the data show that these oligosaccharides can be highly fucosylated, and that their poly-N-acetyllactosamine cores are substituted with up to 10 fucose residues on an oligosaccharide that has 7-N-acetyllactosamine units. This is the first report of the existence in human milk of this large range of highly fucosylated oligosaccharides which possess novel, potentially immunologically active structures.

  14. Development of a hydrophilic interaction liquid chromatography coupled with matrix-assisted laser desorption/ionization-mass spectrometric imaging platform for N-glycan relative quantitation using stable-isotope labeled hydrazide reagents.

    Science.gov (United States)

    Chen, Zhengwei; Zhong, Xuefei; Tie, Cai; Chen, Bingming; Zhang, Xinxiang; Li, Lingjun

    2017-07-01

    In this work, the capability of newly developed hydrophilic interaction liquid chromatography (HILIC) coupled with matrix-assisted laser desorption/ionization-mass spectrometric imaging (MALDI-MSI) platform for quantitative analysis of N-glycans has been demonstrated. As a proof-of-principle experiment, heavy and light stable-isotope labeled hydrazide reagents labeled maltodextrin ladder were used to demonstrate the feasibility of the HILIC-MALDI-MSI platform for reliable quantitative analysis of N-glycans. MALDI-MSI analysis by an Orbitrap mass spectrometer enabled high-resolution and high-sensitivity detection of N-glycans eluted from HILIC column, allowing the re-construction of LC chromatograms as well as accurate mass measurements for structural inference. MALDI-MSI analysis of the collected LC traces showed that the chromatographic resolution was preserved. The N-glycans released from human serum was used to demonstrate the utility of this novel platform in quantitative analysis of N-glycans from a complex sample. Benefiting from the minimized ion suppression provided by HILIC separation, comparison between MALDI-MS and the newly developed platform HILIC-MALDI-MSI revealed that HILIC-MALDI-MSI provided higher N-glycan coverage as well as better quantitation accuracy in the quantitative analysis of N-glycans released from human serum. Graphical abstract Reconstructed chromatograms based on HILIC-MALDI-MSI results of heavy and light labeled maltodextrin enabling quantitative glycan analysis.

  15. Genomics meets glycomics-the first GWAS study of human N-Glycome identifies HNF1α as a master regulator of plasma protein fucosylation.

    Directory of Open Access Journals (Sweden)

    Gordan Lauc

    2010-12-01

    Full Text Available Over half of all proteins are glycosylated, and alterations in glycosylation have been observed in numerous physiological and pathological processes. Attached glycans significantly affect protein function; but, contrary to polypeptides, they are not directly encoded by genes, and the complex processes that regulate their assembly are poorly understood. A novel approach combining genome-wide association and high-throughput glycomics analysis of 2,705 individuals in three population cohorts showed that common variants in the Hepatocyte Nuclear Factor 1α (HNF1α and fucosyltransferase genes FUT6 and FUT8 influence N-glycan levels in human plasma. We show that HNF1α and its downstream target HNF4α regulate the expression of key fucosyltransferase and fucose biosynthesis genes. Moreover, we show that HNF1α is both necessary and sufficient to drive the expression of these genes in hepatic cells. These results reveal a new role for HNF1α as a master transcriptional regulator of multiple stages in the fucosylation process. This mechanism has implications for the regulation of immunity, embryonic development, and protein folding, as well as for our understanding of the molecular mechanisms underlying cancer, coronary heart disease, and metabolic and inflammatory disorders.

  16. Modular synthesis of N-glycans and arrays for the hetero-ligand binding analysis of HIV antibodies

    Science.gov (United States)

    Shivatare, Sachin S.; Chang, Shih-Huang; Tsai, Tsung-I.; Tseng, Susan Yu; Shivatare, Vidya S.; Lin, Yih-Shyan; Cheng, Yang-Yu; Ren, Chien-Tai; Lee, Chang-Chun David; Pawar, Sujeet; Tsai, Charng-Sheng; Shih, Hao-Wei; Zeng, Yi-Fang; Liang, Chi-Hui; Kwong, Peter D.; Burton, Dennis R.; Wu, Chung-Yi; Wong, Chi-Huey

    2016-04-01

    A new class of broadly neutralizing antibodies (bNAbs) from HIV donors has been reported to target the glycans on gp120—a glycoprotein found on the surface of the virus envelope—thus renewing hope of developing carbohydrate-based HIV vaccines. However, the version of gp120 used in previous studies was not from human T cells and so the glycosylation pattern could be somewhat different to that found in the native system. Moreover, some antibodies recognized two different glycans simultaneously and this cannot be detected with the commonly used glycan microarrays on glass slides. Here, we have developed a glycan microarray on an aluminium-oxide-coated glass slide containing a diverse set of glycans, including homo- and mixed N-glycans (high-mannose, hybrid and complex types) that were prepared by modular chemo-enzymatic methods to detect the presence of hetero-glycan binding behaviours. This new approach allows rapid screening and identification of optimal glycans recognized by neutralizing antibodies, and could speed up the development of HIV-1 vaccines targeting cell surface glycans.

  17. A vacuolar carboxypeptidase mutant of Arabidopsis thaliana is degraded by the ERAD pathway independently of its N-glycan

    Energy Technology Data Exchange (ETDEWEB)

    Yamamoto, Masaya; Kawanabe, Mitsuyoshi; Hayashi, Yoko; Endo, Toshiya [Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602 (Japan); Nishikawa, Shuh-ichi, E-mail: shuh@biochem.chem.nagoya-u.ac.jp [Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602 (Japan)

    2010-03-12

    Misfolded proteins produced in the endoplasmic reticulum (ER) are degraded by a mechanism, the ER-associated degradation (ERAD). Here we report establishment of the experimental system to analyze the ERAD in plant cells. Carboxypeptidase Y (CPY) is a vacuolar enzyme and its mutant CPY* is degraded by the ERAD in yeast. Since Arabidopsis thaliana has AtCPY, an ortholog of yeast CPY, we constructed and expressed fusion proteins consisting of AtCPY and GFP and of AtCPY*, which carries a mutation homologous to yeast CPY*, and GFP in A. thaliana cells. While AtCPY-GFP was efficiently transported to the vacuole, AtCPY*-GFP was retained in the ER to be degraded in proteasome- and Cdc48-dependent manners. We also found that AtCPY*-GFP was degraded by the ERAD in yeast cells, but that its single N-glycan did not function as a degradation signal in yeast or plant cells. Therefore, AtCPY*-GFP can be used as a marker protein to analyze the ERAD pathway, likely for nonglycosylated substrates, in plant cells.

  18. A dual approach for improving homogeneity of a human-type N-glycan structure in Saccharomyces cerevisiae.

    Science.gov (United States)

    Piirainen, Mari A; Boer, Harry; de Ruijter, Jorg C; Frey, Alexander D

    2016-04-01

    N-glycosylation is an important feature of therapeutic and other industrially relevant proteins, and engineering of the N-glycosylation pathway provides opportunities for developing alternative, non-mammalian glycoprotein expression systems. Among yeasts, Saccharomyces cerevisiae is the most established host organism used in therapeutic protein production and therefore an interesting host for glycoengineering. In this work, we present further improvements in the humanization of the N-glycans in a recently developed S. cerevisiae strain. In this strain, a tailored trimannosyl lipid-linked oligosaccharide is formed and transferred to the protein, followed by complex-type glycan formation by Golgi apparatus-targeted human N-acetylglucosamine transferases. We improved the glycan pattern of the glycoengineered strain both in terms of glycoform homogeneity and the efficiency of complex-type glycosylation. Most of the interfering structures present in the glycoengineered strain were eliminated by deletion of the MNN1 gene. The relative abundance of the complex-type target glycan was increased by the expression of a UDP-N-acetylglucosamine transporter from Kluyveromyces lactis, indicating that the import of UDP-N-acetylglucosamine into the Golgi apparatus is a limiting factor for efficient complex-type N-glycosylation in S. cerevisiae. By a combination of the MNN1 deletion and the expression of a UDP-N-acetylglucosamine transporter, a strain forming complex-type glycans with a significantly improved homogeneity was obtained. Our results represent a further step towards obtaining humanized glycoproteins with a high homogeneity in S. cerevisiae.

  19. FX knockout CHO hosts can express desired ratios of fucosylated or afucosylated antibodies with high titers and comparable product quality.

    Science.gov (United States)

    Louie, Salina; Haley, Benjamin; Marshall, Brett; Heidersbach, Amy; Yim, Mandy; Brozynski, Martina; Tang, Danming; Lam, Cynthia; Petryniak, Bronislawa; Shaw, David; Shim, Jeongsup; Miller, Aaron; Lowe, John B; Snedecor, Brad; Misaghi, Shahram

    2017-03-01

    During antibody dependent cell cytotoxicity (ADCC) the target cells are killed by monocytes and natural killer cells. ADCC is enhanced when the antibody heavy chain's core N-linked glycan lacks the fucose molecule(s). Several strategies have been utilized to generate fully afucosylated antibodies. A commonly used and efficient approach has been knocking out the FUT8 gene of the Chinese hamster ovary (CHO) host cells, which results in expression of antibody molecules with fully afucosylated glycans. However, a major drawback of the FUT8-KO host is the requirement for undertaking two separate cell line development (CLD) efforts in order to obtain both primarily fucosylated and fully afucosylated antibody species for comparative studies in vitro and in vivo. Even more challenging is obtaining primarily fucosylated and FUT8-KO clones with similar enough product quality attributes to ensure that any observed ADCC advantage(s) can be strictly attributed to afucosylation. Here, we report generation and use of a FX knockout (FXKO) CHO host cell line that is capable of expressing antibody molecules with either primarily fucosylated or fully afucosylated glycan profiles with otherwise similar product quality attributes, depending on addition of fucose to the cell culture media. Hence, the FXKO host not only obviates the requirement for undertaking two separate CLD efforts, but it also averts the need for screening many colonies to identify clones with comparable product qualities. Finally, FXKO clones can express antibodies with the desired ratio of primarily fucosylated to afucosylated glycans when fucose is titrated into the production media, to allow achieving intended levels of FcγRIII-binding and ADCC for an antibody. Biotechnol. Bioeng. 2017;114: 632-644. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  20. The role of single N-glycans in proteolytic processing and cell surface transport of the Lassa virus glycoprotein GP-C

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    Lenz Oliver

    2006-05-01

    Full Text Available Abstract Lassa virus glycoprotein is synthesised as a precursor (preGP-C into the lumen of the endoplasmic reticulum. After cotranslational cleavage of the signal peptide, the immature GP-C is posttranslationally processed into the N-terminal subunit GP-1 and the C-terminal subunit GP-2 by the host cell subtilase SKI-1/S1P. The glycoprotein precursor contains eleven potential N-glycosylation sites. In this report, we investigated the effect of each N-glycan on proteolytic cleavage and cell surface transport by disrupting the consensus sequences of eleven potential N-glycan attachment sites individually. Five glycoprotein mutants with disrupted N-glycosylation sites were still proteolytically processed, whereas the remaining N-glycosylation sites are necessary for GP-C cleavage. Despite the lack of proteolytic processing, all cleavage-defective mutants were transported to the cell surface and remained completely endo H-sensitive. The findings indicate that N-glycans are needed for correct conformation of GP-C in order to be cleaved by SKI-1/S1P.

  1. Effect of Bu-Zhong-Yi-Qi-Tang on deficiency of N-glycan/nitric oxide and islet damage induced by streptozotocin in diabetic rats

    Institute of Scientific and Technical Information of China (English)

    Xiao-Qiu Liu; Ling Wu; Xue-Jun Guo

    2009-01-01

    AIM: To investigate the effect of Bu-Zhong-Yi-Qi-Tang (Decoction for Reinforcing Middle Jiao and Replenishing Qi) on deficiency of N-glycan/nitric oxide (NO) and islet damage induced by injecting two medium doses of streptozotocin (STZ). METHODS: Diabetes was induced by intraperitoneal injection of STZ at 55 mg/kg on day 1 and day 8. Islet damage was evaluated using a scoring system. Nitrite, nitrate, α-mannosidase and amylase activities were measured by colorimetry. N-glycan patterns of amylase were determined with lectin [ConA, pisum sativum agglutinin (PSA), peanut agglutinin (PNA), and lens culinaris agglutinin (LCA)] affinity precipitation method. RESULTS: Severe islet necrosis and mild islet atrophy were observed in diabetic rats. The number and size of islets, the activities of α-mannosidase, amylase and nitrite were decreased, while the binding of PNA and LCA to amylase was increased. All of which were improved after treatment with Bu-Zhong-Yi-Qi-Tang. Islet damage was significantly correlated with nitrite, nitrate, α-mannosidase, amylase and the binding of LCA, PNA, and PSA to amylase. PNA, and PSA to amylase.CONCLUSION: STZ- induced i s let damage i s related to N-glycan def iciency in proteins by blocking α-mannosidase activity and no deficiency, accumulation of unfolded proteins, and endoplasmic reticulum stress and activation of cellular signals, all of which are improved after treatment with Bu-Zhong-Yi- Qi-Tang.

  2. State-of-the-art technologies for rapid and high-throughput sample preparation and analysis of N-glycans from antibodies.

    Science.gov (United States)

    Aich, Udayanath; Lakbub, Jude; Liu, Aston

    2016-06-01

    Glycosylation is a PTM that occurs during production of many protein-based biologic drugs and can have a profound impact on their biological, clinical, and pharmacological properties. Quality by design, process optimization, and advance in manufacturing technology create a demand for robust, sensitive, and accurate profiling and quantification of antibody glycosylation. Potential drawbacks in antibody glycosylation profiling include the high hands-on time required for sample preparation and several hours for data acquisition and analysis. Rapid and high-throughput (HTP) N-glycan profiling and characterization along with automation for sample preparation and analysis are essential for extensive antibody glycosylation analysis due to the substantial improvement of turnaround time. The first part of this review article will focus on the recent progress in rapid and HTP sample preparation and analysis of antibody glycosylation. Subsequently, the article will cover a brief overview of various separation and mass spectrometric methods for the rapid and HTP analysis of N-glycans in antibodies. Finally, we will discuss the recent developments in process analytical technologies for the screening and quantification of N-glycans in antibodies.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Yusuke Takeuchi

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

  5. Inactivation of thrombin by a fucosylated chondroitin sulfate from echinoderm.

    Science.gov (United States)

    Mourão, P A; Boisson-Vidal, C; Tapon-Bretaudière, J; Drouet, B; Bros, A; Fischer, A

    2001-04-15

    A polysaccharide extracted from the sea cucumber body wall has the same backbone structure as the mammalian chondroitin sulfate, but some of the glucuronic acid residues display sulfated fucose branches. These branches confer high anticoagulant activity to the polysaccharide. Since the sea cucumber chondroitin sulfate has analogy in structure with mammalian glycosaminoglycans and sulfated fucans from brown algae, we compared its anticoagulant action with that of heparin and of a homopolymeric sulfated fucan with approximately the same level of sulfation as the sulfated fucose branches found in the sea cucumber polysaccharide. These various compounds differ not only in their anticoagulant potencies but also in the mechanisms of thrombin inhibition. Fucosylated chondroitin sulfate, like heparin, requires antithrombin or heparin cofactor II for thrombin inhibition. Sulfated fucans from brown algae have an antithrombin effect mediated by antithrombin and heparin cofactor II, plus a direct antithrombin effect more pronounced for some fractions. But even in the case of these two polysaccharides, we observed some differences. In contrast with heparin, total inhibition of thrombin in the presence of antithrombin is not achieved with fucosylated chondroitin sulfate, possibly reflecting a less specific interaction. Fucosylated chondroitin sulfate is able to inhibit thrombin generation after stimulation by both contact-activated and thromboplastin-activated systems. It delayed only the contact-induced thrombin generation, as expected for an anticoagulant without direct thrombin inhibition. Overall, the specific spatial array of the sulfated fucose branches in the fucosylated chondroitin sulfate not only confer high anticoagulant activity to the polysaccharide but also determine differences in the way it inhibits thrombin.

  6. Co-administration of the Campylobacter jejuni N-glycan based vaccine with probiotics improves vaccine performance in broiler chickens.

    Science.gov (United States)

    Nothaft, H; Perez-Muñoz, M E; Gouveia, G J; Duar, R M; Wanford, J J; Lango-Scholey, L; Panagos, C G; Srithayakumar, V; Plastow, G S; Coros, C; Bayliss, C D; Edison, A S; Walter, J; Szymanski, C M

    2017-09-22

    Source attribution studies report that consumption of contaminated poultry is the primary source for acquiring human campylobacteriosis. Oral administration of an engineered Escherichia coli strain expressing the Campylobacter jejuni N-glycan reduces bacterial colonization in specific-pathogen-free leghorn chickens, but only a fraction of birds respond to vaccination. Optimizing the vaccine for commercial broiler chickens has great potential to prevent pathogen entry into the food chain. Here, we tested the same vaccination approach in broilers and observed similar efficacy in pathogen load reduction, stimulation of host IgY response, lack of C. jejuni resistance development, uniformity in microbial gut composition, and bimodal response to treatment. Gut microbiota analysis of leghorn and broiler vaccine responders identified one member of the Clostridiales XIVa cluster, Anaerosporobacter mobilis, significantly more abundant in responder birds. In broilers, co-administration of the live vaccine with A. mobilis or Lactobacillus reuteri, a commonly used probiotic, resulted in increased vaccine efficacy, antibody response, and weight gain. To investigate whether the responder/non-responder effect was due to selection of a C. jejuni 'super colonizer mutant' with altered phase-variable genes, we analysed all polyG-containing loci of the input strain compared to non-responder colony isolates and found no evidence of phase state selection. However, untargeted NMR-based metabolomics identified a potential biomarker negatively correlated with C. jejuni colonization levels possibly linked to the increased microbial diversity in this subgroup. The comprehensive methods used to examine the vaccine response bimodality provide several opportunities to improve the C. jejuni vaccine and the efficacy of any vaccination strategy.ImportanceCampylobacter jejuni is a common cause of human diarrheal disease worldwide and listed by the World Health Organization as a high priority

  7. Targeting N-Glycan Cryptic Sugar Moieties for Broad-Spectrum Virus Neutralization: Progress in Identifying Conserved Molecular Targets in Viruses of Distinct Phylogenetic Origins

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    Denong Wang

    2015-03-01

    Full Text Available Identifying molecular targets for eliciting broadly virus-neutralizing antibodies is one of the key steps toward development of vaccines against emerging viral pathogens. Owing to genomic and somatic diversities among viral species, identifying protein targets for broad-spectrum virus neutralization is highly challenging even for the same virus, such as HIV-1. However, viruses rely on host glycosylation machineries to synthesize and express glycans and, thereby, may display common carbohydrate moieties. Thus, exploring glycan-binding profiles of broad-spectrum virus-neutralizing agents may provide key information to uncover the carbohydrate-based virus-neutralizing epitopes. In this study, we characterized two broadly HIV-neutralizing agents, human monoclonal antibody 2G12 and Galanthus nivalis lectin (GNA, for their viral targeting activities. Although these agents were known to be specific for oligomannosyl antigens, they differ strikingly in virus-binding activities. The former is HIV-1 specific; the latter is broadly reactive and is able to neutralize viruses of distinct phylogenetic origins, such as HIV-1, severe acute respiratory syndrome coronavirus (SARS-CoV, and human cytomegalovirus (HCMV. In carbohydrate microarray analyses, we explored the molecular basis underlying the striking differences in the spectrum of anti-virus activities of the two probes. Unlike 2G12, which is strictly specific for the high-density Man9GlcNAc2Asn (Man9-clusters, GNA recognizes a number of N-glycan cryptic sugar moieties. These include not only the known oligomannosyl antigens but also previously unrecognized tri-antennary or multi-valent GlcNAc-terminating N-glycan epitopes (Tri/m-Gn. These findings highlight the potential of N-glycan cryptic sugar moieties as conserved targets for broad-spectrum virus neutralization and suggest the GNA-model of glycan-binding warrants focused investigation.

  8. Paper analytical devices for dynamic evaluation of cell surface N-glycan expression via a bimodal biosensor based on multibranched hybridization chain reaction amplification.

    Science.gov (United States)

    Liang, Linlin; Lan, Feifei; Li, Li; Ge, Shenguang; Yu, Jinghua; Ren, Na; Liu, Haiyun; Yan, Mei

    2016-12-15

    A novel colorimetric/fluorescence bimodal lab-on-paper cyto-device was fabricated based on concanavalin A (Con A)-integrating multibranched hybridization chain reaction (mHCR). The product of mHCR was modified PtCu nanochains (colorimetric signal label) and graphene quantum dot (fluorescence signal label) for in situ and dynamically evaluating cell surface N-glycan expression. In this strategy, preliminary detection was carried out through colorimetric method, if needed, then the fluorescence method was applied for a precise determination. Au-Ag-paper devices increased the surface areas and active sites for immobilizing larger amount of aptamers, and then specifically and efficiently captured more cancer cells. Moreover, it could effectively reduce the paper background fluorescence. Due to the specific recognition of Con A with mannose and the effective signal amplification of mHCR, the proposed strategy exhibited excellent high sensitivity for the cytosensing of MCF-7 cells ranging from 100 to 1.0×10(7) and 80-5.0×10(7) cellsmL(-1) with the detection limit of 33 and 26 cellsmL(-1) for colorimetric and fluorescence, respectively. More importantly, this strategy was successfully applied to dynamically monitor cell-surface multi-glycans expression on living cells under external stimuli of inhibitors as well as for N-glycan expression inhibitor screening. These results implied that this biosensor has potential in studying complex native glycan-related biological processes and elucidating the N-glycan-related diseases in biological and physiological processes.

  9. Modulation of GDP-fucose level for generating proteins with reduced rate of fucosylation (WO2010141855).

    Science.gov (United States)

    Taupin, Philippe

    2011-09-01

    The application (WO2010141855) is in the field of glycobiology, and involves the control of the rate of fucosylation of proteins by exogenous factors. It aims at controlling the rate of protein fucosylation with inhibitors (drugs or nucleic acid antagonists) of enzymes involved in the synthesis of GDP-fucose. Mammalian cell lines were cultured in the presence of inhibitors, for example, siRNA. The rates of GDP-fucose in cells and during protein fucosylation were characterized. The level of protein fucosylation decreases rapidly in response to a decrease in GDP-fucose level. The relationship between the rate of fucosylation of proteins and the level of GDP-fucose in a cell is non-linear. Reduction in the rate of protein fucosylation can be achieved with a minimal reduction of the level of GDP-fucose in cells. The paradigm may be used to synthesize proteins and antibodies, with a reduced rate of fucosylation. The application claims that the use of drugs or nucleic acid antagonists that inhibit the enzymes involved in GDP-fucose biosynthesis optimizes the level of GDP-fucose present in cells, and reduces the rate of fucosylation of glycoproteins.

  10. Growth phase-dependent expression of proteins with decreased plant-specific N-glycans and immunogenicity in tobacco BY2 cells

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Plants possess some desirable characteristics to synthesize recombinant glycoproteins for pharma-ceutical application.However,the mammalian glycoproteins produced in plants are somewhat different from their natural counterparts in terms of N-glycoforms.The immunogenicity of plant-specific glyco-epitopes is the major concern in human therapy.Here,the distribution of N-glycans in different growth phases of tobacco BY2 cells and their immunogenicity in mice were determined.It was ob-served that the percentage of β1,2-xylose and α1,3-fucose in proteins of growing cells increased and the corresponding protein extracts caused accelerated immune response in mice.Based on this observation,the recombinant erythropoietin in BY2 cells was expressed and characterized,and Western blot analysis showed that the recombinant erythropoietin contained a relatively small amount of plant-specific glyco-epitopes in the early phase of culture growth.This study may provide a simple but effective strategy for the production of therapeutic glycoproteins with human-like N-glycan structures in plant hosts to avoid a great allergenic risk.

  11. Anomalous N-glycan structures with an internal fucose branched to GlcA and GlcN residues isolated from a mollusk shell-forming fluid.

    Science.gov (United States)

    Zhou, Hui; Hanneman, Andrew J; Chasteen, N Dennis; Reinhold, Vernon N

    2013-10-01

    This report describes the structural details of a unique N-linked valence epitope on the major protein within the extrapallial (EP) fluid of the mollusk, Mytilus edulis. Fluids from this area are considered to be responsible for shell expansion by a self-assembly process that provides an organic framework for the growth of CaCO3 crystals. Previous reports from our laboratories have described the purification and amino acid sequence of this EP protein, which was found to be a glycoprotein (EPG) of approximately 28 KDa with 14.3% carbohydrate on a single N-linked consensus site. Described herein is the de novo sequence of the major glycan and its glycomers. The sequence was determined by ion trap sequential mass spectrometry (ITMS(n)) resolving structure by tracking precursor-product relationships through successive rounds of collision induced disassociation (CID), thereby spatially resolving linkage and branching details within the confines of the ion trap. Three major glycomers were detected, each possessing a 6-linked fucosylated N-linked core. Two glycans possessed four and five identical antennae, while the third possessed four antennas, but with an additional methylfucose 2-linked to the glucuronic acid moiety, forming a pentasaccharide. The tetrasaccharide structure was: 4-O-methyl-GlcA(1-4)[GlcNAc(1-3)]Fuc(1-4)GlcNAc, while the pentasaccharide was shown to be as follows: mono-O-methyl-Fuc(1-2)-4-O-methyl-GlcA(1-4)[GlcNAc(1-3)]Fuc(1-4)GlcNAc. Samples were differentially deuteriomethylated (CD3/CH3) to localize indigenous methylation, further analyzed by high resolution mass spectrometry (HRMS) to confirm monomer compositions, and finally gas chromatography mass spectrometry (GC-MS) to assign structural and stereoisomers. The interfacial shell surface location of this major extrapallial glycoprotein, its calcium and heavy metal binding properties and unique structure suggests a probable role in shell formation and possibly metal ion detoxification. A closely

  12. Fucosylation and protein glycosylation create functional receptors for cholera toxin

    Science.gov (United States)

    Wands, Amberlyn M; Fujita, Akiko; McCombs, Janet E; Cervin, Jakob; Dedic, Benjamin; Rodriguez, Andrea C; Nischan, Nicole; Bond, Michelle R; Mettlen, Marcel; Trudgian, David C; Lemoff, Andrew; Quiding-Järbrink, Marianne; Gustavsson, Bengt; Steentoft, Catharina; Clausen, Henrik; Mirzaei, Hamid; Teneberg, Susann; Yrlid, Ulf; Kohler, Jennifer J

    2015-01-01

    Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors using its B subunit (CTB). The ganglioside (glycolipid) GM1 is thought to be the sole CT receptor; however, the mechanism by which CTB binding to GM1 mediates internalization of CT remains enigmatic. Here we report that CTB binds cell surface glycoproteins. Relative contributions of gangliosides and glycoproteins to CTB binding depend on cell type, and CTB binds primarily to glycoproteins in colonic epithelial cell lines. Using a metabolically incorporated photocrosslinking sugar, we identified one CTB-binding glycoprotein and demonstrated that the glycan portion of the molecule, not the protein, provides the CTB interaction motif. We further show that fucosylated structures promote CTB entry into a colonic epithelial cell line and subsequent host cell intoxication. CTB-binding fucosylated glycoproteins are present in normal human intestinal epithelia and could play a role in cholera. DOI: http://dx.doi.org/10.7554/eLife.09545.001 PMID:26512888

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

    Science.gov (United States)

    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.

  14. A Modular Approach to a Library of Semi-Synthetic Fucosylated Chondroitin Sulfate Polysaccharides with Different Sulfation and Fucosylation Patterns.

    Science.gov (United States)

    Laezza, Antonio; Iadonisi, Alfonso; Pirozzi, Anna V A; Diana, Paola; De Rosa, Mario; Schiraldi, Chiara; Parrilli, Michelangelo; Bedini, Emiliano

    2016-12-12

    Fucosylated chondroitin sulfate (fCS)-a glycosaminoglycan (GAG) found in sea cucumbers-has recently attracted much attention owing to its biological properties. In particular, a low molecular mass fCS polysaccharide has very recently been suggested as a strong candidate for the development of an antithrombotic drug that would be safer and more effective than heparin. To avoid the use of animal sourced drugs, here we present the chemical transformation of a microbial sourced unsulfated chondroitin polysaccharide into a small library of fucosylated (and sulfated) derivatives thereof. To this aim, a modular approach based on the different combination of only five reactions was employed, with an almost unprecedented polysaccharide branching by O-glycosylation as the key step. The library was differentiated for sulfation patterns and/or positions of the fucose branches, as confirmed by detailed 2D NMR spectroscopic analysis. These semi-synthetic polysaccharides will allow a wider and more accurate structure-activity relationship study with respect to those reported in literature to date.

  15. N-Glycans on the Rift Valley Fever Virus Envelope Glycoproteins Gn and Gc Redundantly Support Viral Infection via DC-SIGN.

    Science.gov (United States)

    Phoenix, Inaia; Nishiyama, Shoko; Lokugamage, Nandadeva; Hill, Terence E; Huante, Matthew B; Slack, Olga A L; Carpio, Victor H; Freiberg, Alexander N; Ikegami, Tetsuro

    2016-05-23

    Rift Valley fever is a mosquito-transmitted, zoonotic disease that infects humans and ruminants. Dendritic cell specific intercellular adhesion molecule 3 (ICAM-3) grabbing non-integrin (DC-SIGN) acts as a receptor for members of the phlebovirus genus. The Rift Valley fever virus (RVFV) glycoproteins (Gn/Gc) encode five putative N-glycan sequons (asparagine (N)-any amino acid (X)-serine (S)/threonine (T)) at positions: N438 (Gn), and N794, N829, N1035, and N1077 (Gc). The N-glycosylation profile and significance in viral infection via DC-SIGN have not been elucidated. Gc N-glycosylation was first evaluated by using Gc asparagine (N) to glutamine (Q) mutants. Subsequently, we generated a series of recombinant RVFV MP-12 strain mutants, which encode N-to-Q mutations, and the infectivity of each mutant in Jurkat cells stably expressing DC-SIGN was evaluated. Results showed that Gc N794, N1035, and N1077 were N-glycosylated but N829 was not. Gc N1077 was heterogeneously N-glycosylated. RVFV Gc made two distinct N-glycoforms: "Gc-large" and "Gc-small", and N1077 was responsible for "Gc-large" band. RVFV showed increased infection of cells expressing DC-SIGN compared to cells lacking DC-SIGN. Infection via DC-SIGN was increased in the presence of either Gn N438 or Gc N1077. Our study showed that N-glycans on the Gc and Gn surface glycoproteins redundantly support RVFV infection via DC-SIGN.

  16. Introduction of an N-glycan sequon into HEXA enhances human beta-hexosaminidase cellular uptake in a model of Sandhoff disease.

    Science.gov (United States)

    Matsuoka, Kazuhiko; Tsuji, Daisuke; Aikawa, Sei-Ichi; Matsuzawa, Fumiko; Sakuraba, Hitoshi; Itoh, Kohji

    2010-08-01

    Human lysosomal beta-hexosaminidase A is a heterodimer composed of alpha- and beta-subunits encoded by HEXA and HEXB, respectively. We genetically introduced an additional N-glycosylation sequon into HEXA, which caused amino acid substitutions (S51 to N and A53 to T) at homologous positions to N84 and T86 in the beta-subunit. The mutant HexA (NgHexA) obtained from a Chinese hamster ovary (CHO) cell line co-expressing the mutated HEXA and wild-type HEXB complementary DNAs was demonstrated to contain an additional mannose-6-phosphate (M6P)-type-N-glycan. NgHexA was more efficiently taken up than the wild-type HexA and delivered to lysosomes, where it degraded accumulated substrates including GM2 ganglioside (GM2) when administered to cultured fibroblasts derived from a Sandhoff disease (SD) patient. On intracerebroventricular (i.c.v.) administration of NgHexA to SD model mice, NgHexA more efficiently restored the HexA activity and reduced the GM2 and GA2 (asialoGM2) accumulated in neural cells of the brain parenchyma than the wild-type HexA. These findings indicate that i.c.v. administration of the modified human HexA with an additional M6P-type N-glycan is applicable for enzyme replacement therapy (ERT) involving an M6P-receptor as a molecular target for HexA deficiencies including Tay-Sachs disease and SD.

  17. Introduction of an N-Glycan Sequon Into HEXA Enhances Human β-Hexosaminidase Cellular Uptake in a Model of Sandhoff Disease.

    Science.gov (United States)

    Matsuoka, Kazuhiko; Tsuji, Daisuke; Aikawa, Sei-Ichi; Matsuzawa, Fumiko; Sakuraba, Hitoshi; Itoh, Kohji

    2010-08-01

    Human lysosomal β-hexosaminidase A is a heterodimer composed of α- and β-subunits encoded by HEXA and HEXB, respectively. We genetically introduced an additional N-glycosylation sequon into HEXA, which caused amino acid substitutions (S51 to N and A53 to T) at homologous positions to N84 and T86 in the β-subunit. The mutant HexA (NgHexA) obtained from a Chinese hamster ovary (CHO) cell line co-expressing the mutated HEXA and wild-type HEXB complementary DNAs was demonstrated to contain an additional mannose-6-phosphate (M6P)-type-N-glycan. NgHexA was more efficiently taken up than the wild-type HexA and delivered to lysosomes, where it degraded accumulated substrates including GM2 ganglioside (GM2) when administered to cultured fibroblasts derived from a Sandhoff disease (SD) patient. On intracerebroventricular (i.c.v.) administration of NgHexA to SD model mice, NgHexA more efficiently restored the HexA activity and reduced the GM2 and GA2 (asialoGM2) accumulated in neural cells of the brain parenchyma than the wild-type HexA. These findings indicate that i.c.v. administration of the modified human HexA with an additional M6P-type N-glycan is applicable for enzyme replacement therapy (ERT) involving an M6P-receptor as a molecular target for HexA deficiencies including Tay-Sachs disease and SD.

  18. Serum fucosyl transferase activity and serum fucose levels as diagnostic tools in malignancy.

    Directory of Open Access Journals (Sweden)

    Sen,Umi

    1983-12-01

    Full Text Available Glycoproteins play a significant role in neoplastic transformations. Both the levels of fucose and the activity of fucosyl transferase, which mediates the assembly of the oligosaccharide moieties of the glycoprotein chains, have been found to be elevated in neoplastic conditions. Since these elevations are common features of a variety of neoplastic cells, these two have been designated as non-specific markers of malignancy. In the present study, the fucose level and fucosyl transferase activity were determined in the sera of cancer patients and an attempt was made to establish a relationship between the two. It was found that both the fucose levels and fucosyl transferase activities showed considerable elevation in the five cancer groups studied, establishing them as useful diagnostic parameters. However, it was also observed that the rate of increased fucosyl transferase activity was not fully reflected in the resulting serum fucose levels in a few cases.

  19. Highly specific purification of N-glycans using phosphate-based derivatization as an affinity tag in combination with Ti{sup 4+}-SPE enrichment for mass spectrometric analysis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Ying [Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032 (China); Key Laboratory of Glycoconjugates Research Ministry of Public Health, Fudan University, Shanghai 200032 (China); Peng, Ye; Bin, Zhichao [Department of Chemistry, Fudan University, Shanghai 200032 (China); Wang, Huijie [Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032 (China); Lu, Haojie, E-mail: luhaojie@fudan.edu.cn [Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032 (China); Department of Chemistry, Fudan University, Shanghai 200032 (China); Key Laboratory of Glycoconjugates Research Ministry of Public Health, Fudan University, Shanghai 200032 (China)

    2016-08-31

    N-linked protein glycosylation is involved in regulation of a wide variety of cellular processes and associated with numerous diseases. Highly specific identification of N-glycome remains a challenge while its biological significance is acknowledged. The relatively low abundance of glycan in complex biological mixtures, lack of basic sites for protonation, and suppression by other highly abundant proteins/peptides lead to the particularly poor detection sensitivity of N-glycans in the MS analysis. Therefore, the highly specific purification procedure becomes a crucial step prior to MS analysis of the N-glycome. Herein, a novel N-glycans enrichment approach based on phosphate derivatization combined with Ti{sup 4+}-SPE (solid phase extraction) was developed. Briefly, in this strategy, N-glycans were chemically labeled with a phospho-group at their reducing ends, such that the Ti{sup 4+}-SPE microspheres were able to capture the phospho-containing glycans. The enrichment method was developed and optimized using model oligosaccharides (maltoheptaose DP7 and sialylated glycan A1) and also glycans from a standard glycoprotein (asialofetuin, ASF). This method experimentally showed high derivatization efficiency (almost 100%), excellent selectivity (analyzing DP7 in the digests of bovine serum albumin at a mass ratio of 1:100), high enriching recovery (90%), good reproducibility (CV<15%) as well as high sensitivity (LOD at fmol level). At last, the proposed method was successfully applied in the profiling of N-glycome in human serum, in which a total of 31 N-glycan masses were identified. - Graphical abstract: A selective enrichment method for the N-glycome is reported. N-glycans were chemically labeled with a phosphate derivatization reagent (AMS), then the phospho-containing glycans were enriched using Ti{sup 4+}-microspheres. - Highlights: • A highly specific N-glycans purification method based on phosphate derivatization combined with Ti{sup 4+}-SPE was developed

  20. Different immunity elicited by recombinant H5N1 hemagglutinin proteins containing pauci-mannose, high-mannose, or complex type N-glycans.

    Directory of Open Access Journals (Sweden)

    Shih-Chang Lin

    Full Text Available Highly pathogenic avian influenza H5N1 viruses can result in poultry and occasionally in human mortality. A safe and effective H5N1 vaccine is urgently needed to reduce the pandemic potential. Hemagglutinin (HA, a major envelope protein accounting for approximately 80% of spikes in influenza virus, is often used as a major antigen for subunit vaccine development. In this study, we conducted a systematic study of the immune response against influenza virus infection following immunization with recombinant HA proteins expressed in insect (Sf9 cells, insect cells that contain exogenous genes for elaborating N-linked glycans (Mimic and mammalian cells (CHO. While the antibody titers are higher with the insect cell derived HA proteins, the neutralization and HA inhibition titers are much higher with the mammalian cell produced HA proteins. Recombinant HA proteins containing tri- or tetra-antennary complex, terminally sialylated and asialyated-galactose type N-glycans induced better protective immunity in mice to lethal challenge. The results are highly relevant to issues that should be considered in the production of fragment vaccines.

  1. MALDI Imaging Mass Spectrometry Profiling of N-Glycans in Formalin-Fixed Paraffin Embedded Clinical Tissue Blocks and Tissue Microarrays

    Science.gov (United States)

    Powers, Thomas W.; Neely, Benjamin A.; Shao, Yuan; Tang, Huiyuan; Troyer, Dean A.; Mehta, Anand S.; Haab, Brian B.; Drake, Richard R.

    2014-01-01

    A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers. PMID:25184632

  2. Evolution of protein N-glycosylation process in Golgi apparatus which shapes diversity of protein N-glycan structures in plants, animals and fungi

    Science.gov (United States)

    Wang, Peng; Wang, Hong; Gai, Jiangtao; Tian, Xiaoli; Zhang, Xiaoxiao; Lv, Yongzhi; Jian, Yi

    2017-01-01

    Protein N-glycosylation (PNG) is crucial for protein folding and enzymatic activities, and has remarkable diversity among eukaryotic species. Little is known of how unique PNG mechanisms arose and evolved in eukaryotes. Here we demonstrate a picture of onset and evolution of PNG components in Golgi apparatus that shaped diversity of eukaryotic protein N-glycan structures, with an emphasis on roles that domain emergence and combination played on PNG evolution. 23 domains were identified from 24 known PNG genes, most of which could be classified into a single clan, indicating a single evolutionary source for the majority of the genes. From 153 species, 4491 sequences containing the domains were retrieved, based on which we analyzed distribution of domains among eukaryotic species. Two domains in GnTV are restricted to specific eukaryotic domains, while 10 domains distribute not only in species where certain unique PNG reactions occur and thus genes harboring these domains are supoosed to be present, but in other ehkaryotic lineages. Notably, two domains harbored by β-1,3 galactosyltransferase, an essential enzyme in forming plant-specific Lea structure, were present in separated genes in fungi and animals, suggesting its emergence as a result of domain shuffling. PMID:28074929

  3. MALDI imaging mass spectrometry profiling of N-glycans in formalin-fixed paraffin embedded clinical tissue blocks and tissue microarrays.

    Science.gov (United States)

    Powers, Thomas W; Neely, Benjamin A; Shao, Yuan; Tang, Huiyuan; Troyer, Dean A; Mehta, Anand S; Haab, Brian B; Drake, Richard R

    2014-01-01

    A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers.

  4. MALDI imaging mass spectrometry profiling of N-glycans in formalin-fixed paraffin embedded clinical tissue blocks and tissue microarrays.

    Directory of Open Access Journals (Sweden)

    Thomas W Powers

    Full Text Available A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers.

  5. N-Glycans on secretory component: mediators of the interaction between secretory IgA and gram-positive commensals sustaining intestinal homeostasis.

    Science.gov (United States)

    Mathias, Amandine; Corthésy, Blaise

    2011-09-01

    Human beings live in symbiosis with billions of microorganisms colonizing mucosal surfaces. The understanding of the mechanisms underlying this fine-tuned intestinal balance has made significant processes during the last decades. We have recently demonstrated that the interaction of SIgA with Gram-positive bacteria is essentially based on Fab-independent, glycan-mediated recognition. Results obtained using mouse hybridoma- and colostrum-derived secretory IgA (SIgA) consistently show that N-glycans present on secretory component (SC) play a crucial role in the process. Natural coating may involve specific Gram-positive cell wall components, which may explain selective recognition at the molecular level. More widely, the existence of these complexes is involved in the modulation of intestinal epithelial cell (IEC) responses in vitro and the formation of intestinal biofilms. Thus, SIgA may act as one of the pillars in homeostatic maintenance of the microbiota in the gut, adding yet another facet to its multiple roles in the mucosal environment.

  6. Chemo-Enzymatic Synthesis of (13)C Labeled Complex N-Glycans As Internal Standards for the Absolute Glycan Quantification by Mass Spectrometry.

    Science.gov (United States)

    Echeverria, Begoña; Etxebarria, Juan; Ruiz, Nerea; Hernandez, Álvaro; Calvo, Javier; Haberger, Markus; Reusch, Dietmar; Reichardt, Niels-Christian

    2015-11-17

    Methods for the absolute quantification of glycans are needed in glycoproteomics, during development and production of biopharmaceuticals and for the clinical analysis of glycan disease markers. Here we present a strategy for the chemo-enzymatic synthesis of (13)C labeled N-glycan libraries and provide an example for their use as internal standards in the profiling and absolute quantification of mAb glycans by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. A synthetic biantennary glycan precursor was (13)C-labeled on all four amino sugar residues and enzymatically derivatized to produce a library of 15 glycan isotopologues with a mass increment of 8 Da over the natural products. Asymmetrically elongated glycans were accessible by performing enzymatic reactions on partially protected UV-absorbing intermediates, subsequent fractionation by preparative HPLC, and final hydrogenation. Using a preformulated mixture of eight internal standards, we quantified the glycans in a monoclonal therapeutic antibody with excellent precision and speed.

  7. High mannose N-glycan binding lectin from Remusatia vivipara (RVL) limits cell growth, motility and invasiveness of human breast cancer cells.

    Science.gov (United States)

    Sindhura, B R; Hegde, Prajna; Chachadi, Vishwanath B; Inamdar, Shashikala R; Swamy, Bale M

    2017-09-01

    Breast cancer known for its high metastatic potential is responsible for large mortality rate amongst women; hence it is imperative to search for effective anti-metastatic molecules despite anticancer drugs. The current study describes the potential of Remusatia vivipara lectin (RVL), inducing apoptosis in breast cancer cells there by limiting motility and invasiveness. RVL binds to the cell surface glycans of MDA-MB-468 and MCF-7 cells, exhibiting strong glycan mediated cytotoxic effect, but show marginal effect on non-tumorigenic MCF-10A cells. RVL elicits increased cellular stress, apoptotic vacuoles and nuclear disintegration in both MDA-MB-468 and MCF-7 cells accompanied by depletion of G0/G1, S and G2/M phases. Lectin interaction induced production of reactive oxygen species through altering mitochondrial membrane potential progressing to apoptosis. Further, RVL strongly elicited reproductive cell death in MDA-MB-468 cells and showed strong inhibitory effect on neovascularization demonstrated in chorioallantoic membrane assay. Treatment of MDA-MB-468 cells with RVL, suppress the motility and invasive property as shown by scratch wound heal and Boyden chamber transwell assays respectively. These results provide an insight into significance of interaction of RVL with specific cell surface high mannose N-glycans resulting in curtailing the metastatic ability of cancer cells. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  8. Profiling and characterization of sialylated N-glycans by 2D-HPLC (HIAX/PGC) with online orbitrap MS/MS and offline MSn.

    Science.gov (United States)

    Hanneman, Andrew J S; Strand, James; Huang, Chi-Ting

    2014-02-01

    Glycosylation is a critical parameter used to evaluate protein quality and consistency. N-linked glycan profiling is fundamental to the support of biotherapeutic protein manufacturing from early stage process development through drug product commercialization. Sialylated glycans impact the serum half-life of receptor-Fc fusion proteins (RFPs), making their quality and consistency a concern during the production of fusion proteins. Here, we describe an analytical approach providing both quantitative profiling and in-depth mass spectrometry (MS)-based structural characterization of sialylated RFP N-glycans. Aiming to efficiently link routine comparability studies with detailed structural characterization, an integrated workflow was implemented employing fluorescence detection, online positive and negative ion tandem mass spectrometry (MS/MS), and offline static nanospray ionization-sequential mass spectrometry (NSI-MS(n)). For routine use, high-performance liquid chromatography profiling employs established fluorescence detection of 2-aminobenzoic acid derivatives (2AA) and hydrophilic interaction anion-exchange chromatography (HIAX) charge class separation. Further characterization of HIAX peak fractions is achieved by online (-) ion orbitrap MS/MS, offering the advantages of high mass accuracy and data-dependent MS/MS. As required, additional characterization uses porous graphitized carbon in the second chromatographic dimension to provide orthogonal (+) ion MS/MS spectra and buffer-free liquid chromatography peak eluants that are optimum for offline (+)/(-) NSI-MS(n) investigations to characterize low-abundance species and specific moieties including O-acetylation and sulfation.

  9. Novel α-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides.

    Directory of Open Access Journals (Sweden)

    Mateusz Lezyk

    Full Text Available This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil-derived metagenome library and expressed in E. coli as recombinant 6xHis-tagged proteins. All seven fucosidases belong to glycosyl hydrolase family 29 (GH 29. Six of the seven α-L-fucosidases were substrate-inhibited, moderately thermostable and most hydrolytically active in the pH range 6-7, when tested with para-nitrophenyl-α-L-fucopyranoside (pNP-Fuc as the substrate. In contrast, one fucosidase (Mfuc6 exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self-condensation or to lactose. With the α-L-fucosidase from Thermotoga maritima and the metagenome-derived Mfuc5, different fucosyllactose variants including the principal fucosylated HMO 2'-fucosyllactose were synthesised in yields of up to ~6.4%. Mfuc5 was able to release fucose from xyloglucan and could also use it as a fucosyl-donor for synthesis of fucosyllactose. This is the first study describing the use of glycosyl hydrolases for the synthesis of genuine fucosylated human milk oligosaccharides.

  10. Synthesis of the Oligosaccharides Related to Branching Sites of Fucosylated Chondroitin Sulfates from Sea Cucumbers

    Directory of Open Access Journals (Sweden)

    Nadezhda E. Ustyuzhanina

    2015-02-01

    Full Text Available Natural anionic polysaccharides fucosylated chondroitin sulfates (FCS from sea cucumbers attract great attention nowadays due to their ability to influence various biological processes, such as blood coagulation, thrombosis, angiogenesis, inflammation, bacterial and viral adhesion. To determine pharmacophore fragments in FCS we have started systematic synthesis of oligosaccharides with well-defined structure related to various fragments of these polysaccharides. In this communication, the synthesis of non-sulfated and selectively O-sulfated di- and trisaccharides structurally related to branching sites of FCS is described. The target compounds are built up of propyl β-d-glucuronic acid residue bearing at O-3 α-l-fucosyl or α-l-fucosyl-(1→3-α-l-fucosyl substituents. O-Sulfation pattern in the fucose units of the synthetic targets was selected according to the known to date holothurian FCS structures. Stereospecific α-glycoside bond formation was achieved using 2-O-benzyl-3,4-di-O-chloroacetyl-α-l-fucosyl trichloroacetimidate as a donor. Stereochemical outcome of the glycosylation was explained by the remote participation of the chloroacetyl groups with the formation of the stabilized glycosyl cations, which could be attacked by the glycosyl acceptor only from the α-side. The experimental results were in good agreement with the SCF/MP2 calculated energies of such participation. The synthesized oligosaccharides are regarded as model compounds for the determination of a structure-activity relationship in FCS.

  11. Novel α-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides.

    Science.gov (United States)

    Lezyk, Mateusz; Jers, Carsten; Kjaerulff, Louise; Gotfredsen, Charlotte H; Mikkelsen, Maria D; Mikkelsen, Jørn D

    2016-01-01

    This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil-derived metagenome library and expressed in E. coli as recombinant 6xHis-tagged proteins. All seven fucosidases belong to glycosyl hydrolase family 29 (GH 29). Six of the seven α-L-fucosidases were substrate-inhibited, moderately thermostable and most hydrolytically active in the pH range 6-7, when tested with para-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as the substrate. In contrast, one fucosidase (Mfuc6) exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self-condensation) or to lactose. With the α-L-fucosidase from Thermotoga maritima and the metagenome-derived Mfuc5, different fucosyllactose variants including the principal fucosylated HMO 2'-fucosyllactose were synthesised in yields of up to ~6.4%. Mfuc5 was able to release fucose from xyloglucan and could also use it as a fucosyl-donor for synthesis of fucosyllactose. This is the first study describing the use of glycosyl hydrolases for the synthesis of genuine fucosylated human milk oligosaccharides.

  12. Novel interactions of complex carbohydrates with peanut (PNA), Ricinus communis (RCA-I), Sambucus nigra (SNA-I) and wheat germ (WGA) agglutinins as revealed by the binding specificities of these lectins towards mucin core-2 O-linked and N-linked glycans and related structures.

    Science.gov (United States)

    Chandrasekaran, E V; Xue, Jun; Xia, Jie; Khaja, Siraj D; Piskorz, Conrad F; Locke, Robert D; Neelamegham, Sriram; Matta, Khushi L

    2016-10-01

    Plant lectins through their multivalent quaternary structures bind intrinsically flexible oligosaccharides. They recognize fine structural differences in carbohydrates and interact with different sequences in mucin core 2 or complex-type N-glycan chain and also in healthy and malignant tissues. They are used in characterizing cellular and extracellular glycoconjugates modified in pathological processes. We study here, the complex carbohydrate-lectin interactions by determining the effects of substituents in mucin core 2 tetrasaccharide Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAcα-O-R and fetuin glycopeptides on their binding to agarose-immobilized lectins PNA, RCA-I, SNA-I and WGA. Briefly, in mucin core 2 tetrasaccharide (i) structures modified by α2-3/6-Sialyl LacNAc, LewisX and α1-3-Galactosyl LacNAc resulted in regular binding to PNA whereas compounds with 6-sulfo LacNAc displayed no-binding; (ii) strucures bearing α2-6-sialyl 6-sulfo LacNAc, or 6-sialyl LacdiNAc carbohydrates displayed strong binding to SNA-I; (iii) structures with α2-3/6-sialyl, α1-3Gal LacNAc or LewisX were non-binder to RCA-I and compounds with 6-sulfo LacNAc only displayed weak binding; (iv) structures containing LewisX, 6-Sulfo LewisX, α2-3/6-sialyl LacNAc, α2-3/6-sialyl 6-sulfo LacNAc and GalNAc Lewis-a were non-binding to WGA, those with α1-2Fucosyl, α1-3-Galactosyl LacNAc, α2-3-sialyl T-hapten plus 3'/6'sulfo LacNAc displayed weak binding, and compounds with α2-3-sialyl T-hapten, α2.6-Sialyl LacdiNAc, α2-3-sialyl D-Fucβ1-3 GalNAc and Fucα-1-2 D-Fucβ-1-3GalNAc displaying regular binding and GalNAc LewisX and LacdiNAc plus D-Fuc β-1-3 GalNAcα resulting in tight binding. RCA-I binds Fetuin triantennary asialoglycopeptide 100 % after α-2-3 and 25 % after α-2-6 sialylation, 30 % after α-1-2 and 100 % after α-1-3 fucosylation, and 50 % after α-1-3 galactosylation. WGA binds 3-but not 6-Fucosyl chitobiose core. Thus, information on the influence of complex carbohydrate

  13. Travelling-wave ion mobility mass spectrometry and negative ion fragmentation of hybrid and complex N-glycans.

    Science.gov (United States)

    Harvey, David J; Scarff, Charlotte A; Edgeworth, Matthew; Pagel, Kevin; Thalassinos, Konstantinos; Struwe, Weston B; Crispin, Max; Scrivens, James H

    2016-11-01

    Nitrogen collisional cross sections (CCSs) of hybrid and complex glycans released from the glycoproteins IgG, gp120 (from human immunodeficiency virus), ovalbumin, α1-acid glycoprotein and thyroglobulin were measured with a travelling-wave ion mobility mass spectrometer using dextran as the calibrant. The utility of this instrument for isomer separation was also investigated. Some isomers, such as Man3 GlcNAc3 from chicken ovalbumin and Man3 GlcNAc3 Fuc1 from thyroglobulin could be partially resolved and identified by their negative ion fragmentation spectra obtained by collision-induced decomposition (CID). Several other larger glycans, however, although existing as isomers, produced only asymmetric rather than separated arrival time distributions (ATDs). Nevertheless, in these cases, isomers could often be detected by plotting extracted fragment ATDs of diagnostic fragment ions from the negative ion CID spectra obtained in the transfer cell of the Waters Synapt mass spectrometer. Coincidence in the drift times of all fragment ions with an asymmetric ATD profile in this work, and in the related earlier paper on high-mannose glycans, usually suggested that separations were because of conformers or anomers, whereas symmetrical ATDs of fragments showing differences in drift times indicated isomer separation. Although some significant differences in CCSs were found for the smaller isomeric glycans, the differences found for the larger compounds were usually too small to be analytically useful. Possible correlations between CCSs and structural types were also investigated, and it was found that complex glycans tended to have slightly smaller CCSs than high-mannose glycans of comparable molecular weight. In addition, biantennary glycans containing a core fucose and/or a bisecting GlcNAc residue fell on different mobility-m/z trend lines to those glycans not so substituted with both of these substituents contributing to larger CCSs. Copyright © 2016 John Wiley & Sons

  14. CORE

    DEFF Research Database (Denmark)

    Krigslund, Jeppe; Hansen, Jonas; Hundebøll, Martin

    2013-01-01

    different flows. Instead of maintaining these approaches separate, we propose a protocol (CORE) that brings together these coding mechanisms. Our protocol uses random linear network coding (RLNC) for intra- session coding but allows nodes in the network to setup inter- session coding regions where flows...... intersect. Routes for unicast sessions are agnostic to other sessions and setup beforehand, CORE will then discover and exploit intersecting routes. Our approach allows the inter-session regions to leverage RLNC to compensate for losses or failures in the overhearing or transmitting process. Thus, we...... increase the benefits of XORing by exploiting the underlying RLNC structure of individual flows. This goes beyond providing additional reliability to each individual session and beyond exploiting coding opportunistically. Our numerical results show that CORE outperforms both forwarding and COPE...

  15. CORE

    DEFF Research Database (Denmark)

    Krigslund, Jeppe; Hansen, Jonas; Hundebøll, Martin

    2013-01-01

    different flows. Instead of maintaining these approaches separate, we propose a protocol (CORE) that brings together these coding mechanisms. Our protocol uses random linear network coding (RLNC) for intra- session coding but allows nodes in the network to setup inter- session coding regions where flows...... intersect. Routes for unicast sessions are agnostic to other sessions and setup beforehand, CORE will then discover and exploit intersecting routes. Our approach allows the inter-session regions to leverage RLNC to compensate for losses or failures in the overhearing or transmitting process. Thus, we...... increase the benefits of XORing by exploiting the underlying RLNC structure of individual flows. This goes beyond providing additional reliability to each individual session and beyond exploiting coding opportunistically. Our numerical results show that CORE outperforms both forwarding and COPE...

  16. Insights into transcriptional regulation of β-D-N-acetylhexosaminidase, an N-glycan-processing enzyme involved in ripening-associated fruit softening.

    Science.gov (United States)

    Irfan, Mohammad; Ghosh, Sumit; Kumar, Vinay; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asis

    2014-11-01

    Tomato (Solanum lycopersicum) fruit ripening-specific N-glycan processing enzyme, β-D-N-acetylhexosaminidase (β-Hex), plays an important role in the ripening-associated fruit-softening process. However, the regulation of fruit ripening-specific expression of β-Hex is not well understood. We have identified and functionally characterized the fruit ripening-specific promoter of β-Hex and provided insights into its transcriptional regulation during fruit ripening. Our results demonstrate that RIPENING INHIBITOR (RIN), a global fruit ripening regulator, and ABSCISIC ACID STRESS RIPENING 1 (SlASR1), a poorly characterized ripening-related protein, are the transcriptional regulators of β-Hex. Both RIN and SlASR1 directly bound to the β-Hex promoter fragments containing CArG and C₂₋₃(C/G)A cis-acting elements, the binding sites for RIN and SlASR1, respectively. Moreover, β-Hex expression/promoter activity in tomato fruits was downregulated once expression of either RIN or SlASR1 was suppressed; indicating that RIN and SlASR1 positively regulate the transcription of β-Hex during fruit ripening. Interestingly, RIN could also bind to the SlASR1 promoter, which contains several CArG cis-acting elements, and SlASR1 expression was suppressed in rin mutant fruits, indicating that RIN also acts as a positive regulator of SlASR1 expression during fruit ripening. Taken together, these results suggest that RIN, both directly and indirectly, through SlASR1, regulates the transcription of β-Hex during fruit ripening. The fruit ripening-specific promoter of β-Hex could be a useful tool in regulating gene expression during fruit ripening.

  17. Fasciola hepatica Surface Coat Glycoproteins Contain Mannosylated and Phosphorylated N-glycans and Exhibit Immune Modulatory Properties Independent of the Mannose Receptor.

    Science.gov (United States)

    Ravidà, Alessandra; Aldridge, Allison M; Driessen, Nicole N; Heus, Ferry A H; Hokke, Cornelis H; O'Neill, Sandra M

    2016-04-01

    Fascioliasis, caused by the liver fluke Fasciola hepatica, is a neglected tropical disease infecting over 1 million individuals annually with 17 million people at risk of infection. Like other helminths, F. hepatica employs mechanisms of immune suppression in order to evade its host immune system. In this study the N-glycosylation of F. hepatica's tegumental coat (FhTeg) and its carbohydrate-dependent interactions with bone marrow derived dendritic cells (BMDCs) were investigated. Mass spectrometric analysis demonstrated that FhTeg N-glycans comprised mainly of oligomannose and to a lesser extent truncated and complex type glycans, including a phosphorylated subset. The interaction of FhTeg with the mannose receptor (MR) was investigated. Binding of FhTeg to MR-transfected CHO cells and BMDCs was blocked when pre-incubated with mannan. We further elucidated the role played by MR in the immunomodulatory mechanism of FhTeg and demonstrated that while FhTeg's binding was significantly reduced in BMDCs generated from MR knockout mice, the absence of MR did not alter FhTeg's ability to induce SOCS3 or suppress cytokine secretion from LPS activated BMDCs. A panel of negatively charged monosaccharides (i.e. GlcNAc-4P, Man-6P and GalNAc-4S) were used in an attempt to inhibit the immunoregulatory properties of phosphorylated oligosaccharides. Notably, GalNAc-4S, a known inhibitor of the Cys-domain of MR, efficiently suppressed FhTeg binding to BMDCs and inhibited the expression of suppressor of cytokine signalling (SOCS) 3, a negative regulator the TLR and STAT3 pathway. We conclude that F. hepatica contains high levels of mannose residues and phosphorylated glycoproteins that are crucial in modulating its host's immune system, however the role played by MR appears to be limited to the initial binding event suggesting that other C-type lectin receptors are involved in the immunomodulatory mechanism of FhTeg.

  18. Knockdown of β3GnT8 reverses 5-fluorouracil resistance in human colorectal cancer cells via inhibition the biosynthesis of polylactosamine-type N-glycans.

    Science.gov (United States)

    Shen, Li; Yu, Meiyun; Xu, Xu; Gao, Liping; Ni, Jianlong; Luo, Zhiguo; Wu, Shiliang

    2014-12-01

    Aberrant glycosylation is known to be associated with cancer chemoresistance. β-1,3-N-acetyl-glucosaminyltransferase (β3GnT)8, which synthesizes polylactosamine on β1-6 branched N-glycans, is dramatically upregulated in colorectal cancer (CRC). 5-Fluorouracil (5-FU) resistance remains a major obstacle to the chemotherapy of CRC. However, little is known with regard to the correlation between 5‑FU resistance and the expression of β3GnT8 in CRC. In this study, a 5-FU‑resistant cell line (SW620/5-FU) was generated, and 50% inhibition concentration (IC50) of 5-FU was determined by MTT assay. Flow cytometry and lectin blot analysis were performed to detect the alteration of polylactosamine structures. Quantitative RT-‑PCR and western blot analysis were used to identify and evaluate candidate genes involved in the synthesis of polylactosamine in SW620/5-FU cells. We found polylactosamine chains were significantly increased in SW620/5-FU cells. Inhibition of the biosynthesis of polylactosamine by 3'-azidothymidine (AZT) was able to reduce 5-FU tolerance. Further studies showed that β3GnT8 expression was also upregulated in 5-FU‑resistant cancer cells, and knockdown of β3GnT8 by RNA interference reversed 5-FU resistance through, at least partly, by suppressing the formation of polylactosamine. In conclusion, the alteration of β3GnT8 in CRC cells correlates with tumor sensitivity to the chemotherapeutic drug and has significant implication for the development of new treatment strategies.

  19. Terminal sialic acids on CD44 N-glycans can block hyaluronan binding by forming competing intramolecular contacts with arginine sidechains

    Science.gov (United States)

    Faller, Christina E.; Guvench, Olgun

    2014-01-01

    Specific sugar residues and their linkages form the basis of molecular recognition for interactions of glycoproteins with other biomolecules. Seemingly small changes, like the addition of a single monosaccharide in the covalently attached glycan component of glycoproteins, can greatly affect these interactions. For instance, the sialic acid capping of glycans affects protein-ligand binding involved in cell-cell and cell-matrix interactions. CD44 is a single-pass transmembrane glycoprotein whose binding with its carbohydrate ligand hyaluronan (HA), an extracellular matrix component, mediates processes such as leukocyte homing, cell adhesion, and tumor metastasis. This binding is highly regulated by glycosylation of the N-terminal extracellular hyaluronan-binding domain (HABD); specifically, sialic acid capped N-glycans of HABD inhibit ligand binding. However, the molecular mechanism behind this sialic acid mediated regulation has remained unknown. Two of the five N-glycosyation sites of HABD have been previously identified as having the greatest inhibitory effect on HA binding, but only if the glycans contain terminal sialic acid residues. These two sites, Asn25 and Asn120, were chosen for in silico glycosylation in this study. Here, from extensive standard molecular dynamics simulations and biased simulations, we propose a molecular mechanism for this behavior based on spontaneously-formed charge-paired hydrogen bonding interactions between the negatively-charged sialic acid residues and positively-charged Arg sidechains known to be critically important for binding to HA, which itself is negatively charged. Such intramolecular hydrogen bonds would preclude associations critical to hyaluronan binding. This observation suggests how CD44 and related glycoprotein binding is regulated by sialylation as cellular environments fluctuate. PMID:25116630

  20. Fucosylated high molecular mass but not non-fucosylated low molecular mass xyloglucans undergo an extensive depolymerization in cell walls of azuki bean epicotyls.

    Science.gov (United States)

    Arai, Kuninori; Wakabayashi, Kazuyuki; Soga, Kouichi; Hoson, Takayuki

    2010-07-01

    Epicotyl cuttings of azuki bean were incubated with [14C]-glucose (Glc) or [3H]-fucose (Fuc), and the metabolism of radiolabeled polymers in the 24% KOH-extractable cell wall fraction was investigated. Applied 14C-Glc and (3)H-Fuc were predominantly incorporated into the glucan backbone and Fuc residue of xyloglucan molecules, respectively. On gel permeation chromatography, 14C-polymers consisted of a main peak (0.7-1.0 MDa) and shoulder peak (30 kDa). The pattern was similar to that of iodine-reactive xyloglucans in the fraction. On the other hand, 3H-polymers consisted of a single peak eluted around 0.7-1.0 MDa. The elution patterns of 14C- and 3H-polymers were constant during the incubation period, although incorporated radioactivity increased with time. In the pulse-chase experiment, the high molecular mass peaks (0.7-1.0 MDa) of both 14C- and 3H-polymers showed an extensive molecular mass downshift, but not the shoulder peak of 14C-polymers. These results indicate that xyloglucans in the fraction consist of two types of molecules; fucosylated high molecular mass polymers and non-fucosylated low molecular mass polymers. Azuki bean epicotyls may synthesize both types of xyloglucans independently, but only fucosylated xyloglucans undergo an active depolymerization in the cell wall.

  1. New serum markers for small-cell lung cancer. I. The ganglioside fucosyl-GM1

    DEFF Research Database (Denmark)

    Vangsted, A; Drivsholm, L; Andersen, E

    1994-01-01

    The ganglioside fucosyl-GM1 (FucGM1) has been suggested as a marker for small-cell lung cancer (SCLC). Immunohistochemical analyses have shown the expression of the ganglioside in tumors in 75 to 90% of patients with SCLC. We have demonstrated that the ganglioside is shedded from SCLC cells both...

  2. Mesothelin-MUC16 binding is a high affinity, N-glycan dependent interaction that facilitates peritoneal metastasis of ovarian tumors

    Directory of Open Access Journals (Sweden)

    Sathyanarayana Bangalore K

    2006-10-01

    Full Text Available Abstract Background The mucin MUC16 and the glycosylphosphatidylinositol anchored glycoprotein mesothelin likely facilitate the peritoneal metastasis of ovarian tumors. The biochemical basis and the kinetics of the binding between these two glycoproteins are not clearly understood. Here we have addressed this deficit and provide further evidence supporting the role of the MUC16-mesothelin interaction in facilitating cell-cell binding under conditions that mimic the peritoneal environment. Results In this study we utilize recombinant-Fc tagged human mesothelin to measure the binding kinetics of this glycoprotein to MUC16 expressed on the ovarian tumor cell line OVCAR-3. OVCAR-3 derived sublines that did not express MUC16 showed no affinity for mesothelin. In a flow cytometry-based assay mesothelin binds with very high affinity to the MUC16 on the OVCAR-3 cells with an apparent Kd of 5–10 nM. Maximum interaction occurs within 5 mins of incubation of the recombinant mesothelin with the OVCAR-3 cells and significant binding is observed even after 10 sec. A five-fold molar excess of soluble MUC16 was unable to completely inhibit the binding of mesothelin to the OVCAR-3 cells. Oxidation of the MUC16 glycans, removal of its N-linked oligosaccharides, and treatment of the mucin with wheat germ agglutinin and erythroagglutinating phytohemagglutinin abrogates its binding to mesothelin. These observations suggest that at least a subset of the MUC16-asscociated N-glycans is required for binding to mesothelin. We also demonstrate that MUC16 positive ovarian tumor cells exhibit increased adherence to A431 cells transfected with mesothelin (A431-Meso+. Only minimal adhesion is observed between MUC16 knockdown cells and A431-Meso+ cells. The binding between the MUC16 expressing ovarian tumor cells and the A431-Meso+ cells occurs even in the presence of ascites from patients with ovarian cancer. Conclusion The strong binding kinetics of the mesothelin-MUC16

  3. Fut8基因通过促进VLA-4/VCAM-1表达影响B细胞发育%Fut8 gene affects the development of B cells by regulating the core fucosylation of VLA-4/VCAM-1

    Institute of Scientific and Technical Information of China (English)

    焦鑫艳; 马彪; 王旭; 刘庆平; 李文哲

    2012-01-01

    The aim of this study is to explore the role of fucosyltransferase VIII (Fut8) in VLA -4/VCAM-1 expression and B cell development. Firstly, Fut8 -knockdown 70Z/3 cells and Fut8 -knockdown ST2 cells were established by RNA inference using 70Z/3 cells (pre-B cell line) and ST2 cells. Then Fut8 gene-silencing effects were detected by Western blot. In cell adhesion assays and colony forming tests, we found that the loss of a core fucose in both very late antigen 4 (VLA-4) and vascular cell adhesion molecule 1 (VCAM-1) would lead to a decreased binding between pre-B cells and stromal cells, followed the impaired pre-B cells generation in Fut8+ mice. Flow cytometry analysis revealed that Fut8 expression was extremely required for regulating pre-B cell reproduction. These findings clearly demonstrated that the Fut8 could regulate the VLA-4/VCAM-1 interaction and the proper transition from pro-B to pre-B cells, showing the important role of Fut8 in B cell differentiation and development%目的 探讨核心岩藻糖基化修饰对VLA-4/VCAM-1表达及B细胞发育的影响.方法 通过RNA干扰技术使前B细胞(70Z/3)和基质细胞(ST2)的Fut8基因沉默,免疫沉淀和Western blot检测Fut8基因沉默效率,以细胞黏附实验和克隆形成实验分别研究Fut8基因对VLA-4/VCAM-1之间亲和力和对B细胞分化发育的影响.结果 Fut8基因沉默使VLA-4/VCAM-1之间的亲和力以及前B细胞和基质细胞之间的黏附作用降低,并使前B细胞的克隆形成能力明显下降.流式细胞仪分析结果也表明,Fut8祖B细胞向前B细胞分化过程受阻.结论 本实验揭示了Fut8基因调节VLA-4/VCAM-1之间结合能力以及前B细胞克隆形成的机理,为B细胞分化发育研究提供理论依据.

  4. Novel α-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides

    DEFF Research Database (Denmark)

    Lezyk, Mateusz Jakub; Jers, Carsten; Kjaerulff, Louise

    2016-01-01

    -nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as the substrate. In contrast, one fucosidase (Mfuc6) exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self......-condensation) or to lactose. With the α-L-fucosidase from Thermotoga maritima and the metagenome-derived Mfuc5, different fucosyllactose variants including the principal fucosylated HMO 2'-fucosyllactose were synthesised in yields of up to ~6.4%. Mfuc5 was able to release fucose from xyloglucan and could also use...

  5. Effects of fucosylated milk of goat and mouse on Helicobacter pylori binding to Lewis b antigen

    Institute of Scientific and Technical Information of China (English)

    Hong-Tao Xu; Ning Li; Lennart Hammarstr(o)m; Thomas Borén; Rolf Sj(o)str(o)m; Yao-Feng Zhao; Zheng-Xing Lian; Bao-Liang Fan; Zhi-Hui Zhao; Shu-Yang Yu; Yun-Ping Dai; Li-Li Wang; Hui-Ling Niu

    2004-01-01

    AIM: To evaluate the effects of animal milk containing fucosylated antigens on Helicobacter pylori (Hpylori) binding to Lewis b antigen.METHODS: A mammary gland expression vector containing human α1-3/4-fucosyltransferase cDNA sequences was constructed. Transient expression of human α1-3/4-fucosyltransferase cDNA in goat mammary cell and establishment of transgenic mice were performed. The adhesion inhibitory properties of milk samples were analyzed by using H pylori RESULTS: Goat milk samples were found to inhibit bacterial binding to Lewis b antigen. The highest inhibition was observed 42 h after injection of the plasmid. The binding activity of Hpylori to Lewis b antigen reduced mostly, by 83%, however milk samples from transgenic mice did not inhibit H pylori binding to Lewis b antigen.CONCLUSION: The use of "humanized" animal milk produced by the transgenic introduction of fucosylated antigen can perhaps provide an alternative therapy and preventive measure for H pylori infection.

  6. Serum Fucosylated Haptoglobin as a Novel Diagnostic Biomarker for Predicting Hepatocyte Ballooning and Nonalcoholic Steatohepatitis.

    Directory of Open Access Journals (Sweden)

    Yoshihiro Kamada

    Full Text Available Nonalcoholic fatty liver disease (NAFLD is a growing medical problem around the world. NAFLD patients with nonalcoholic steatohepatitis (NASH can develop cirrhosis and hepatocellular carcinoma. The ability to distinguish NASH from simple steatosis would be of great clinical significance. Ballooning hepatocytes are characteristic of typical pathological NASH; here, the polarized secretion of proteins is disrupted due to destruction of the cytoskeleton. We previously reported that fucosylated glycoproteins are secreted into bile, but not into sera in normal liver. Therefore, we hypothesized that the fucosylation-based sorting machinery would be disrupted in ballooning hepatocytes, and serum fucosylated glycoproteins would increase in NASH patients. To confirm our hypothesis, we evaluated serum fucosylated haptoglobin (Fuc-Hpt levels in biopsy-proven NAFLD patients (n = 126 using a lectin-antibody ELISA kit. Fuc-Hpt levels were significantly increased in NASH patients compared with non-NASH (NAFLD patients without NASH patients. Interestingly, Fuc-Hpt levels showed a significant stepwise increase with increasing hepatocyte ballooning scores. Multiple logistic regression analysis showed that Fuc-Hpt levels were independent and significant determinants of the presence of ballooning hepatocytes. Moreover, Fuc-Hpt levels were useful in monitoring liver fibrosis staging. Next, to investigate the significance of serum Fuc-Hpt in a larger population, we measured Fuc-Hpt levels in ultrasound-diagnosed NAFLD subjects (n = 870 who received a medical health checkup. To evaluate NAFLD disease severity, we used the FIB-4 index (based on age, serum AST and ALT levels, and platelet counts. Fuc-Hpt levels increased stepwise with increasing FIB-4 index.Measurement of serum Fuc-Hpt levels can distinguish NASH from non-NASH patients, and predict the presence of ballooning hepatocytes in NAFLD patients with sufficient accuracy. These results support the

  7. Reverse lectin ELISA for detecting fucosylated forms of α1-acid glycoprotein associated with hepatocellular carcinoma

    Science.gov (United States)

    Stål, Per; Zenlander, Robin; Edenvik, Pia; Alexandersson, Catharina; Haglund, Mats; Rydén, Ingvar; Påhlsson, Peter

    2017-01-01

    Altered fucosylation of glycoproteins is associated with development of hepatocellular carcinoma (HCC). Lectins have been commonly used to assay changes in fucosylation of plasma glycoproteins. In the present study a recombinantly engineered form of the fucose binding lectin Aleuria aurantia (AAL) consisting of a single binding site for fucose (S2), was used to construct a reverse lectin ELISA method. Microtiter plates coated with the S2 lectin were used to capture glycoproteins from plasma samples followed by antibody detection of S2-bound fucosylated α1-acid glycoprotein (S2-bound AGP). The method was used to compare the level of S2-bound AGP in serum samples from a small cohort of patients with hepatitis, cirrhosis or HCC. Using the reverse S2 lectin ELISA it was shown that the levels of S2-bound AGP was significantly higher in HCC patients compared to non-cancer patients and that there was also a significant elevation of S2-bound AGP in HCC patients compared to cirrhosis patients. There was no correlation between the level of S2-bound AGP and total AGP concentration. The performance of S2-bound AGP in differentiating HCC from cirrhosis samples or hepatitis samples were compared to other markers. A combination of S2-bound AGP, α-fetoprotein and AGP concentration showed performances giving area under receiver operating curves of 0.87 and 0.95 respectively. PMID:28296934

  8. Fucosyl neoglycoprotein binds to mouse epididymal spermatozoa and inhibits sperm binding to the egg zona pellucida.

    Science.gov (United States)

    Oh, Y S; Ahn, H S; Gye, M C

    2013-12-01

    Glycan epitopes of cellular glycoconjugates act as versatile biochemical signals, and this sugar coding plays an important role in cell-to-cell recognition processes. In this study, our aims were to determine the distribution of sperm receptors with activity for fucosyl- and galactosyl glycans and to address whether monosugar neoglycoproteins functionally mimic the binding between zona pellucida (ZP) glycoproteins and spermatozoa. In mouse epididymal spermatozoa with intact acrosomes, fucopyranosyl bovine serum albumin (BSA-Fuc) bound to the segment of the acrosome, the equatorial segment, and the postacrosome region of the sperm head. Galactosyl BSA (BSA-Gal) binding activity was similar to that of BSA-Fuc, but was weaker. In acrosome-reacted spermatozoa treated with the Ca(2+) ionophore A23187, BSA-zuc binding was lost in the apical segment of the acrosome but remained in the equatorial segment and postacrosome regions. BSA-Gal binding to the equatorial region was increased. In the presence of 2.5 μg ml(-1) BSA-Fuc, in vitro sperm-ZP binding was significantly decreased, indicating that fucosyl BSA functionally mimics ZP glycoproteins during sperm-egg ZP interactions. At the same concentration, BSA-Gal was not effective. Fucosyl BSA that efficiently inhibited the sperm-ZP binding can mimic the ZP glycoconjugate and has potential for use as a sperm fertility control agent in mouse.

  9. A novel gene cluster allows preferential utilization of fucosylated milk oligosaccharides in Bifidobacterium longum subsp. longum SC596

    Science.gov (United States)

    Garrido, Daniel; Ruiz-Moyano, Santiago; Kirmiz, Nina; Davis, Jasmine C.; Totten, Sarah M.; Lemay, Danielle G.; Ugalde, Juan A.; German, J. Bruce; Lebrilla, Carlito B.; Mills, David A.

    2016-01-01

    The infant intestinal microbiota is often colonized by two subspecies of Bifidobacterium longum: subsp. infantis (B. infantis) and subsp. longum (B. longum). Competitive growth of B. infantis in the neonate intestine has been linked to the utilization of human milk oligosaccharides (HMO). However, little is known how B. longum consumes HMO. In this study, infant-borne B. longum strains exhibited varying HMO growth phenotypes. While all strains efficiently utilized lacto-N-tetraose, certain strains additionally metabolized fucosylated HMO. B. longum SC596 grew vigorously on HMO, and glycoprofiling revealed a preference for consumption of fucosylated HMO. Transcriptomes of SC596 during early-stage growth on HMO were more similar to growth on fucosyllactose, transiting later to a pattern similar to growth on neutral HMO. B. longum SC596 contains a novel gene cluster devoted to the utilization of fucosylated HMO, including genes for import of fucosylated molecules, fucose metabolism and two α-fucosidases. This cluster showed a modular induction during early growth on HMO and fucosyllactose. This work clarifies the genomic and physiological variation of infant-borne B. longum to HMO consumption, which resembles B. infantis. The capability to preferentially consume fucosylated HMO suggests a competitive advantage for these unique B. longum strains in the breast-fed infant gut. PMID:27756904

  10. Comparative analysis of the N-glycans of rat, mouse and human Thy-1. Site-specific oligosaccharide patterns of neural Thy-1, a member of the immunoglobulin superfamily.

    Science.gov (United States)

    Williams, A F; Parekh, R B; Wing, D R; Willis, A C; Barclay, A N; Dalchau, R; Fabre, J W; Dwek, R A; Rademacher, T W

    1993-08-01

    Protein structure and tissue type are known to influence glycosylation of proteins. We have previously investigated the N-glycans at each of the three glycosylation sites of the cell surface glycoprotein Thy-1 when isolated from rat brain and thymocytes. Here we report a comparative analysis of the site-specific N-glycosylation patterns from rat (Asn 23, 74, 98), mouse (Asn 23, 75, 99) and human (Asn 23, 60, 100) neural Thy-1. Despite considerable differences in amino acid sequence, the results show a remarkable conservation of the pattern of N-glycans at corresponding sites between the three species, as judged by chromatographic comparisons and glycosidase susceptibility. This is particularly marked for sites at Asn 74/75 in rat/mouse and the equivalent site at 60 in human Thy-1, as well as for sites at Asn 98/99 and 100, respectively. The sites at Asn 23 in rat/mouse also contained almost identical glycosylation patterns, but at this site human Thy-1 showed significantly different glycosylation patterns. These site glycosylation patterns are discussed in relation to the likely accessibility of the oligosaccharides for processing. It is known that within a species, the glycosylation of Thy-1 is tissue specific; therefore, this degree of conservation of glycosylation of Thy-1 expressed in the same tissue in different species is all the more striking, given the known variation between species in the amino acid sequence of Thy-1. It is therefore proposed that neural cells have a particular requirement for specific surface carbohydrates and that the Thy-1 polypeptide serves as an appropriate carrier for these structures.

  11. A study of the prognostic role of serum fucose and fucosyl transferase in cancer of the uterine cervix.

    Directory of Open Access Journals (Sweden)

    Sen,Urmi

    1985-04-01

    Full Text Available Serum fucose levels and fucosyl transferase activities have been designated as nonspecific markers of malignancy, and play an important role in the diagnosis of different types of malignancies. In the present study, attempts were made to determine the prognostic significance of these markers in patients with cancer of the uterine cervix after therapy. It was found that both serum fucose and fucosyl transferase, which were elevated in untreated patients declined significantly in patients responsive to therapy at different follow-up intervals, but not in patients unresponsive to therapy.

  12. Novel α-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides

    DEFF Research Database (Denmark)

    Lezyk, Mateusz Jakub; Jers, Carsten; Kjaerulff, Louise;

    2016-01-01

    This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil......-derived metagenome library and expressed in E. coli as recombinant 6xHis-tagged proteins. All seven fucosidases belong to glycosyl hydrolase family 29 (GH 29). Six of the seven α-L-fucosidases were substrate-inhibited, moderately thermostable and most hydrolytically active in the pH range 6-7, when tested with para......-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as the substrate. In contrast, one fucosidase (Mfuc6) exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self...

  13. In vitro grown pollen tubes of Nicotiana alata actively synthesise a fucosylated xyloglucan.

    Directory of Open Access Journals (Sweden)

    Edwin R Lampugnani

    Full Text Available Nicotiana alata pollen tubes are a widely used model for studies of polarized tip growth and cell wall synthesis in plants. To better understand these processes, RNA-Seq and de novo assembly methods were used to produce a transcriptome of N. alata pollen grains. Notable in the reconstructed transcriptome were sequences encoding proteins that are involved in the synthesis and remodelling of xyloglucan, a cell wall polysaccharide previously not thought to be deposited in Nicotiana pollen tube walls. Expression of several xyloglucan-related genes in actively growing pollen tubes was confirmed and xyloglucan epitopes were detected in the wall with carbohydrate-specific antibodies: the major xyloglucan oligosaccharides found in N. alata pollen grains and tubes were fucosylated, an unusual structure for the Solanaceae, the family to which Nicotiana belongs. Finally, carbohydrate linkages consistent with xyloglucan were identified chemically in the walls of N. alata pollen grains and pollen tubes grown in culture. The presence of a fucosylated xyloglucan in Nicotiana pollen tube walls was thus confirmed. The consequences of this discovery to models of pollen tube growth dynamics and more generally to polarised tip-growing cells in plants are discussed.

  14. Biotechnological production of fucosylated human milk oligosaccharides: Prokaryotic fucosyltransferases and their use in biocatalytic cascades or whole cell conversion systems.

    Science.gov (United States)

    Petschacher, Barbara; Nidetzky, Bernd

    2016-10-10

    Human milk oligosaccharides (HMOs) constitute a class of complex carbohydrates unique to mother's milk and are strongly correlated to the health benefits of breastfeeding in infants. HMOs are important as functional ingredients of advanced infant formula and have attracted broad interest for use in health-related human nutrition. About 50% of the HMOs structures contain l-fucosyl residues, which are introduced into nascent oligosaccharides by enzymatic transfer from GDP-l-fucose. To overcome limitation in the current availability of fucosylated HMOs, biotechnological approaches for their production have been developed. Functional expression of the fucosyltransferase(s) and effective supply of GDP-l-fucose, respectively, are both bottlenecks of the biocatalytic routes of synthesis. Strategies of in vitro and in vivo production of fucosylated HMOs are reviewed here. Besides metabolic engineering for enhanced HMO production in whole cells, the focus is on the characteristics and the heterologous overexpression of prokaryotic α1,2- and α1,3/4-fucosyltransferases. Up to 20g/L of fucosylated HMOs were obtained in optimized production systems. Optimized expression enabled recovery of purified fucosyltransferases in a yield of up to 45mg/L culture for α1,2-fucosyltransferases and of up to 200mg protein/L culture for α1,3/4-fucosyltransferases.

  15. Structural, Mutagenic and In Silico Studies of Xyloglucan Fucosylation in Arabidopsis thaliana Suggest a Water-Mediated Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Crowley, Michael F [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Alahuhta, Petri M [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Lunin, Vladimir V [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Bomble, Yannick J [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Himmel, Michael E [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Urbanowicz, Breeanna R. [Univerisity of Georgia; Bharadwaj, Vivek [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Pena, Maria J. [University of Georgia; Wang, Shuo [University of Georgia; Yang, Jeong-Yeh [University of Georgia; Tuomivaara, Sami [University of Georgia; Moremen, Kelley W. [University of Georgia; York, William S. [University of Georgia

    2017-07-03

    The mechanistic underpinnings of the complex process of plant polysaccharide biosynthesis are poorly understood, largely due to the resistance of glycosyltransferase (GT) enzymes to structural characterization. In Arabidopsis thaliana, a glycosyl transferase family 37 (GT37) fucosyltransferase-1 (AtFUT1) catalyzes the regiospecific transfer of terminal 1,2-fucosyl residues to xyloglucan side chains - a key step in the biosynthesis of fucosylated sidechains of galactoxyloglucan. We unravel the mechanistic basis for fucosylation by AtFUT1 with a multipronged approach involving protein expression, X-ray crystallography, mutagenesis experiments and molecular simulations. Mammalian cell culture expressions enable sufficient production of the enzyme for X-ray crystallography, which reveals the structural architecture of AtFUT1 in complex with bound donor and acceptor substrate analogs. The lack of an appropriately positioned active site residue as a catalytic base leads us to propose an atypical water-mediated fucosylation mechanism facilitated by an H-bonded network, which is corroborated by mutagenesis experiments as well as detailed atomistic simulations.

  16. An Automated Micro-Total Immunoassay System for Measuring Cancer-Associated α2,3-linked Sialyl N-Glycan-Carrying Prostate-Specific Antigen May Improve the Accuracy of Prostate Cancer Diagnosis

    Science.gov (United States)

    Ishikawa, Tomokazu; Yoneyama, Tohru; Tobisawa, Yuki; Hatakeyama, Shingo; Kurosawa, Tatsuo; Nakamura, Kenji; Narita, Shintaro; Mitsuzuka, Koji; Duivenvoorden, Wilhelmina; Pinthus, Jehonathan H.; Hashimoto, Yasuhiro; Koie, Takuya; Habuchi, Tomonori; Arai, Yoichi; Ohyama, Chikara

    2017-01-01

    The low specificity of the prostate-specific antigen (PSA) for early detection of prostate cancer (PCa) is a major issue worldwide. The aim of this study to examine whether the serum PCa-associated α2,3-linked sialyl N-glycan-carrying PSA (S2,3PSA) ratio measured by automated micro-total immunoassay systems (μTAS system) can be applied as a diagnostic marker of PCa. The μTAS system can utilize affinity-based separation involving noncovalent interaction between the immunocomplex of S2,3PSA and Maackia amurensis lectin to simultaneously determine concentrations of free PSA and S2,3PSA. To validate quantitative performance, both recombinant S2,3PSA and benign-associated α2,6-linked sialyl N-glycan-carrying PSA (S2,6PSA) purified from culture supernatant of PSA cDNA transiently-transfected Chinese hamster ovary (CHO)-K1 cells were used as standard protein. Between 2007 and 2016, fifty patients with biopsy-proven PCa were pair-matched for age and PSA levels, with the same number of benign prostatic hyperplasia (BPH) patients used to validate the diagnostic performance of serum S2,3PSA ratio. A recombinant S2,3PSA- and S2,6PSA-spiked sample was clearly discriminated by μTAS system. Limit of detection of S2,3PSA was 0.05 ng/mL and coefficient variation was less than 3.1%. The area under the curve (AUC) for detection of PCa for the S2,3PSA ratio (%S2,3PSA) with cutoff value 43.85% (AUC; 0.8340) was much superior to total PSA (AUC; 0.5062) using validation sample set. Although the present results are preliminary, the newly developed μTAS platform for measuring %S2,3PSA can achieve the required assay performance specifications for use in the practical and clinical setting and may improve the accuracy of PCa diagnosis. Additional validation studies are warranted. PMID:28241428

  17. An Automated Micro-Total Immunoassay System for Measuring Cancer-Associated α2,3-linked Sialyl N-Glycan-Carrying Prostate-Specific Antigen May Improve the Accuracy of Prostate Cancer Diagnosis

    Directory of Open Access Journals (Sweden)

    Tomokazu Ishikawa

    2017-02-01

    Full Text Available The low specificity of the prostate-specific antigen (PSA for early detection of prostate cancer (PCa is a major issue worldwide. The aim of this study to examine whether the serum PCa-associated α2,3-linked sialyl N-glycan-carrying PSA (S2,3PSA ratio measured by automated micro-total immunoassay systems (μTAS system can be applied as a diagnostic marker of PCa. The μTAS system can utilize affinity-based separation involving noncovalent interaction between the immunocomplex of S2,3PSA and Maackia amurensis lectin to simultaneously determine concentrations of free PSA and S2,3PSA. To validate quantitative performance, both recombinant S2,3PSA and benign-associated α2,6-linked sialyl N-glycan-carrying PSA (S2,6PSA purified from culture supernatant of PSA cDNA transiently-transfected Chinese hamster ovary (CHO-K1 cells were used as standard protein. Between 2007 and 2016, fifty patients with biopsy-proven PCa were pair-matched for age and PSA levels, with the same number of benign prostatic hyperplasia (BPH patients used to validate the diagnostic performance of serum S2,3PSA ratio. A recombinant S2,3PSA- and S2,6PSA-spiked sample was clearly discriminated by μTAS system. Limit of detection of S2,3PSA was 0.05 ng/mL and coefficient variation was less than 3.1%. The area under the curve (AUC for detection of PCa for the S2,3PSA ratio (%S2,3PSA with cutoff value 43.85% (AUC; 0.8340 was much superior to total PSA (AUC; 0.5062 using validation sample set. Although the present results are preliminary, the newly developed μTAS platform for measuring %S2,3PSA can achieve the required assay performance specifications for use in the practical and clinical setting and may improve the accuracy of PCa diagnosis. Additional validation studies are warranted.

  18. Pregnancy-Associated Changes of IgG and Serum N-Glycosylation in Camel (Camelus dromedarius).

    Science.gov (United States)

    Adamczyk, Barbara; Albrecht, Simone; Stöckmann, Henning; Ghoneim, Ibrahim M; Al-Eknah, Marzook; Al-Busadah, Khalid A S; Karlsson, Niclas G; Carrington, Stephen D; Rudd, Pauline M

    2016-09-01

    The dromedary camel (Camelus dromedarius) is an agriculturally important species of high economic value but of low reproductive efficiency. Serum and IgG N-glycosylation are affected by physiological and pathogenic changes and might therefore be a useful diagnostic tool in camel livestock management. This study presents the first comprehensive annotation of the N-glycome from dromedary camel serum as well as their single-domain and conventional antibodies and its subsequent application for camel pregnancy diagnostics. N-glycans were released by PNGaseF, labeled with 2-aminobenzamide (2-AB), and analyzed by hydrophilic interaction liquid chromatography with fluorescent detection (HILIC-UPLC-FLD), enzymatic sequencing and mass spectrometry (MS). The use of a high-throughput robotic platform for sample preparation allowed the rapid generation of glycomics data from pregnant (n = 8) and nonpregnant (n = 8) camels of the Majaheem and Wadha breed. IgG N-glycans dominate the glycan profile of camel serum and present a mixture of core-fucosylated and noncore-fucosylated N-glycans which can contain N-glycolylneuraminic and N-acetylneuraminic acid. Significant pregnancy-associated but breed-independent increases in galactosylation, core-fucosylation, sialylation, and decreases in serum O-acetylation were observed. The monitoring of IgG and serum N-glycosylation presents an attractive complementary test for camel pregnancy diagnostics and presents an interesting tool for biomarker discovery in camel health and breeding.

  19. Site specific N-glycan profiling of NeuAc(α2-6)-Gal/GalNAc-binding bark Sambucus nigra agglutinin using LC-MS(n) revealed differential glycosylation.

    Science.gov (United States)

    Gnanesh Kumar, B S; Surolia, Avadhesha

    2016-12-01

    The bark of Sambucus nigra contains a complex mixture of glycoproteins that are characterized as chimeric lectins known as type II ribosome inactivating proteins and holo lectins. These type II ribosome inactivating proteins possess RNA N-glycosidase activity in subunit A and lectin activity associated with subunit B exhibiting distinct sugar specificities to NeuAc(α2-6)-Gal/GalNAc and Gal/GalNAc. In the present study we have determined the N-glycosylation pattern of type II ribosome inactivating protein specific to NeuAc(α2-6)-Gal/GalNAc (Sambucus nigra agglutinin I) by subjecting it to digestion with multiple proteases. The resulting mixture of peptides and N-glycopeptides were analyzed on liquid chromatography coupled to electro spray ionization-iontrap mass spectrometry in MS(n) mode. MS(2) of precursor ions was carried out using CID which provided information on glycan sequence. In subsequent MS(3) of Y1/Y1α ions (peptide + HexNAc)(+n) of corresponding N-glycopeptides, resulted in the fragmentation of peptide backbone confirming the site of attachment. We observed microheterogeneity in each glycan occupied site with subunit A possessing four N-glycans out of six sites with complex and paucimannose types while subunit B comprises occupancy of two sites with a paucimannose and a high mannose type. The differential N-glycosylation of subunits in SNA is discussed in the context of other type II RIPs glycans.

  20. Reducing Macro- and Microheterogeneity of N-Glycans Enables the Crystal Structure of the Lectin and EGF-Like Domains of Human L-Selectin To Be Solved at 1.9 Å Resolution.

    Science.gov (United States)

    Wedepohl, Stefanie; Dernedde, Jens; Vahedi-Faridi, Ardeschir; Tauber, Rudolf; Saenger, Wolfram; Bulut, Haydar

    2017-07-04

    L-Selectin, a cell-adhesion receptor on the surface of most leukocytes, contains seven N-glycosylation sites. In order to obtain the crystal structure of human L-selectin, we expressed a shortened version of L-selectin comprising the C-type lectin and EGF-like domains (termed LE) and systematically analysed mutations of the three glycosylation sites (Asn22, Asn66 and Asn139) in order to reduce macroheterogeneity. After we further removed microheterogeneity, we obtained crystals that diffracted X-rays up to 1.9 Å from a variant (LE010) with exchanges N22Q and N139Q and one GlcNAc2 Man5 N-glycan chain attached to Asn66. Crystal-structure analysis showed that the terminal mannose of GlcNAc2 Man5 of one LE010 molecule was coordinated to Ca(2+) in the binding site of a symmetry-related LE010. The orientation of the lectin and EGF-like domain was similar to the described "bent" conformation of E- and P-selectins. The Ca(2+) -binding site reflects the binding mode seen in E- and P-selectin structures co-crystallised with ligands. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. 2-Fluoro-L-Fucose Is a Metabolically Incorporated Inhibitor of Plant Cell Wall Polysaccharide Fucosylation.

    Science.gov (United States)

    Villalobos, Jose A; Yi, Bo R; Wallace, Ian S

    2015-01-01

    The monosaccharide L-fucose (L-Fuc) is a common component of plant cell wall polysaccharides and other plant glycans, including the hemicellulose xyloglucan, pectic rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II), arabinogalactan proteins, and N-linked glycans. Mutations compromising the biosynthesis of many plant cell wall polysaccharides are lethal, and as a result, small molecule inhibitors of plant cell wall polysaccharide biosynthesis have been developed because these molecules can be applied at defined concentrations and developmental stages. In this study, we characterize novel small molecule inhibitors of plant fucosylation. 2-fluoro-L-fucose (2F-Fuc) analogs caused severe growth phenotypes when applied to Arabidopsis seedlings, including reduced root growth and altered root morphology. These phenotypic defects were dependent upon the L-Fuc salvage pathway enzyme L-Fucose Kinase/ GDP-L-Fucose Pyrophosphorylase (FKGP), suggesting that 2F-Fuc is metabolically converted to the sugar nucleotide GDP-2F-Fuc, which serves as the active inhibitory molecule. The L-Fuc content of cell wall matrix polysaccharides was reduced in plants treated with 2F-Fuc, suggesting that this molecule inhibits the incorporation of L-Fuc into these polysaccharides. Additionally, phenotypic defects induced by 2F-Fuc treatment could be partially relieved by the exogenous application of boric acid, suggesting that 2F-Fuc inhibits RG-II biosynthesis. Overall, the results presented here suggest that 2F-Fuc is a metabolically incorporated inhibitor of plant cellular fucosylation events, and potentially suggest that other 2-fluorinated monosaccharides could serve as useful chemical probes for the inhibition of cell wall polysaccharide biosynthesis.

  2. Rapid and sensitive screening of N-glycans as 9-fluorenylmethyl derivatives by high-performance liquid chromatography: a method which can recover free oligosaccharides after analysis.

    Science.gov (United States)

    Kamoda, Satoru; Nakano, Miyako; Ishikawa, Rika; Suzuki, Shigeo; Kakehi, Kazuaki

    2005-01-01

    There are a large number of labeling methods for asparagine-type oligosaccharides with fluorogenic and chromophoric reagents. We have to choose the most appropriate labeling method based on the purposes such as mass spectrometry, high-performance liquid chromatography and capillary electrophoresis. Asparagine-type glycans are released from core proteins as N-glycosylamine at the initial step of the releasing reaction when glycoamidase F is employed as the enzyme. The N-glycosylamine-type oligosaccharides thus released by the enzyme are subjected to hydrolysis or mutarotation to form free-form oligosaccharides. In the detailed studies on the enzyme reaction, we found a condition in which the released N-glycosylamine-type oligosaccharides were exclusively present at least during the course of enzyme reaction, and developed a method for in situ derivatization of the glycosylamine-type oligosaccharides with 9-fluorenylmethyl chloroformate (Fmoc-Cl). The Fmoc labeled sialo- and asialo- (or high-mannose and hybrid) oligosaccharides were successfully analyzed on an amine-bonded polymer column and amide-silica column, respectively. The present method showed approximately 5 times higher sensitivities than that using 2-aminobenzoic acid (2-AA). The separation profile was similar to that observed using 2-AA method as examined by the analyses of carbohydrate chains derived from several glycoproteins including complex-type, high-mannose type and hybrid type of N-linked oligosaccharides. The labeled oligosaccharides were stable at least for several months when stored at -20 degrees C. Furthermore, it should be emphasized that the Fmoc-derivatized oligosaccharides could be easily recovered as free reducing oligosaccharides simply by incubation with morpholine in dimethylformamide solution. We obtained a pure triantennary oligosaccharide with 3 sialic acid residues as a free reducing form from fetuin in good yield after isolation of the corresponding Fmoc oligosaccharide followed

  3. In Vivo Anti-Cancer Mechanism of Low-Molecular-Weight Fucosylated Chondroitin Sulfate (LFCS from Sea Cucumber Cucumaria frondosa

    Directory of Open Access Journals (Sweden)

    Xiaoxiao Liu

    2016-05-01

    Full Text Available The low-molecular-weight fucosylated chondroitin sulfate (LFCS was prepared from native fucosylated chondroitin sulfate (FCS, which was extracted and isolated from sea cucumber Cucumaria frondosa, and the anti-cancer mechanism of LFCS on mouse Lewis lung carcinoma (LLC was investigated. The results showed that LFCS remarkably inhibited LLC growth and metastasis in a dose-dependent manner. LFCS induced cell cycle arrest by increasing p53/p21 expression and apoptosis through activation of caspase-3 activity in LLC cells. Meanwhile, LFCS suppressed the expression of vascular endothelial growth factor (VEGF, increased the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1 and downregulated the matrix metalloproteinases (MMPs level. Furthermore, LFCS significantly suppressed the activation of ERK1/2/p38 MAPK/NF-κB pathway, which played a prime role in expression of MMPs. All of these data indicate LFCS may be used as anti-cancer drug candidates and deserve further study.

  4. Utilization of Natural Fucosylated Oligosaccharides by Three Novel α-l-Fucosidases from a Probiotic Lactobacillus casei Strain ▿

    Science.gov (United States)

    Rodríguez-Díaz, Jesús; Monedero, Vicente; Yebra, María J.

    2011-01-01

    Three putative α-l-fucosidases encoded in the Lactobacillus casei BL23 genome were cloned and purified. The proteins displayed different abilities to hydrolyze natural fucosyloligosaccharides like 2′-fucosyllactose, H antigen disaccharide, H antigen type II trisaccharide, and 3′-, 4′-, and 6′-fucosyl-GlcNAc. This indicated a possible role in the utilization of oligosaccharides present in human milk and intestinal mucosa. PMID:21097595

  5. Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose.

    Science.gov (United States)

    Ferrara, Claudia; Grau, Sandra; Jäger, Christiane; Sondermann, Peter; Brünker, Peter; Waldhauer, Inja; Hennig, Michael; Ruf, Armin; Rufer, Arne Christian; Stihle, Martine; Umaña, Pablo; Benz, Jörg

    2011-08-02

    Antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism, relies on the binding of antigen-antibody complexes to Fcγ receptors expressed on immune cells. Antibodies lacking core fucosylation show a large increase in affinity for FcγRIIIa leading to an improved receptor-mediated effector function. Although afucosylated IgGs exist naturally, a next generation of recombinant therapeutic, glycoenginereed antibodies is currently being developed to exploit this finding. In this study, the crystal structures of a glycosylated Fcγ receptor complexed with either afucosylated or fucosylated Fc were determined allowing a detailed, molecular understanding of the regulatory role of Fc-oligosaccharide core fucosylation in improving ADCC. The structures reveal a unique type of interface consisting of carbohydrate-carbohydrate interactions between glycans of the receptor and the afucosylated Fc. In contrast, in the complex structure with fucosylated Fc, these contacts are weakened or nonexistent, explaining the decreased affinity for the receptor. These findings allow us to understand the higher efficacy of therapeutic antibodies lacking the core fucose and also suggest a unique mechanism by which the immune system can regulate antibody-mediated effector functions.

  6. Single-chain antibody-fragment M6P-1 possesses a mannose 6-phosphate monosaccharide-specific binding pocket that distinguishes N-glycan phosphorylation in a branch-specific manner†

    Science.gov (United States)

    Blackler, Ryan J; Evans, Dylan W; Smith, David F; Cummings, Richard D; Brooks, Cory L; Braulke, Thomas; Liu, Xinyu; Evans, Stephen V; Müller-Loennies, Sven

    2016-01-01

    The acquisition of mannose 6-phosphate (Man6P) on N-linked glycans of lysosomal enzymes is a structural requirement for their transport from the Golgi apparatus to lysosomes mediated by the mannose 6-phosphate receptors, 300 kDa cation-independent mannose 6-phosphate receptor (MPR300) and 46 kDa cation-dependent mannose 6-phosphate receptor (MPR46). Here we report that the single-chain variable domain (scFv) M6P-1 is a unique antibody fragment with specificity for Man6P monosaccharide that, through an array-screening approach against a number of phosphorylated N-glycans, is shown to bind mono- and diphosphorylated Man6 and Man7 glycans that contain terminal αMan6P(1 → 2)αMan(1 → 3)αMan. In contrast to MPR300, scFv M6P-1 does not bind phosphodiesters, monophosphorylated Man8 or mono- or diphosphorylated Man9 structures. Single crystal X-ray diffraction analysis to 2.7 Å resolution of Fv M6P-1 in complex with Man6P reveals that specificity and affinity is achieved via multiple hydrogen bonds to the mannose ring and two salt bridges to the phosphate moiety. In common with both MPRs, loss of binding was observed for scFv M6P-1 at pH values below the second pKa of Man6P (pKa = 6.1). The structures of Fv M6P-1 and the MPRs suggest that the change of the ionization state of Man6P is the main driving force for the loss of binding at acidic lysosomal pH (e.g. lysosome pH ∼ 4.6), which provides justification for the evolution of a lysosomal enzyme transport pathway based on Man6P recognition. PMID:26503547

  7. A novel fucosyl glycosphingolipid of brine shrimp that is highly sensitive to endoglycoceramidase.

    Science.gov (United States)

    Xu, Xu; Horibata, Yasuhiro; Inagaki, Masanori; Hama, Yoichiro; Sakaguchi, Keishi; Goda, Hatsumi M; Okino, Nozomu; Ito, Makoto

    2009-12-01

    Endoglycoceramidase (EGCase; EC 3.2.1.123) is a glycohydrolase that hydrolyzes the glycosidic linkage between the oligosaccharide and ceramide of various glycosphingolipids. We previously reported that hydra produced EGCase to digest glycosphingolipids of brine shrimp (Artemia salina), a type of aquatic crustacean used as a diet for the culture of hydra (Horibata Y, Sakaguchi K, Okino N, Iida H, Inagaki M, Fujisawa T, Hama Y, Ito M. 2004. J Biol Chem. 279:33379-33389). We report here that a major glycosphingolipid of brine shrimp is unique in structure and highly sensitive to EGCase. The glycosphingolipid was extracted from freshly hatched brine shrimp by Folch's partition, followed by mild alkaline hydrolysis and purification with a Sep-Pak plus silica cartridge. The structure of brine shrimp glycosphingolipid was determined by gas chromatography, gas chromatography-mass spectrometry, fast-atom bombardment mass spectrometry, and (1)H-NMR spectrometry to be GlcNAcalpha1-2Fucalpha1-3Manbeta1-4Glcbeta1-1'Cer. Two major molecular species of the glycosphingolipid were identified; the sugar and sphingoid base of each were the same but the major fatty acid was C22:0 and 2-hydroxy C22:0, respectively. This is the first report describing the glycosphingolipid that has an internal fucosyl residue substituted with alpha1-2 N-acetylglucosaminyl residue. This study also suggests the biological relevance of the glycosphingolipid as a dietary source of hydra which possesses EGCase as a digestion enzyme.

  8. Partial Hydrolysis of the Fucosylated Chondroitin Sulfate from Sea Cucumber Isostichopus badionotus and Its Mechanism

    Institute of Scientific and Technical Information of China (English)

    CHEN Shi-Guo; LI Guo-Yun; YE Xing-Qian; XUE Chang-Hu

    2012-01-01

    The method for preparing low molecular weight fucosylated chondroitin sulfate from sea cucumber lsostichopus badionotus using partial acid hydrolysis was reported, and its hydrolysis mechanism was also investigated. The sea cucumber chondroitin sulfate FCS was hydrolyzed under different conditions (80℃3 h and 6 h), then isolated and purified on a Bio-P-4 geltration to prepare low molecular weight fractions (LMWF-FCS). The chemical compositions of LMWF-FCS showed the branched fucose (Fuc) was cleaved during acid hydrolysis process, whereas the mole ratio of acetyl-galactosamine (GalNAc) and glucuronic acid (GlcA) in the backbone remained the same, which indicated the backbone was a typical chondroitin sulfate structure. The disaccharide composition analysis of LMWF-FCS suggested that the sulfation patterns of GalNAc in the backbone chain changed and the substitution value was reduced. Furthermore, the 1D NMR analysis illustrated the branched-Fuc was cleaved during acid hydrolysis, but their substitution patterns were not influenced, which was distinct from the previous reports that the substitutions of branched-Fuc in FCS were easy to change. Simultaneously, the sulfation pattern of GalNAc in backbone chain changed obviously in the acid hydrolysis process. The anticoagulant activity in vitro illuminated the anticoagulant activity of the degradation products over time in the acid hydrolysis are gradually declined, but still kept good. Therefore, the LMWF-FCS prepared could be developed as a new anticoagulant and antithrombotic drug like low molecular weight heparin.

  9. Conformational Analysis of the Oligosaccharides Related to Side Chains of Holothurian Fucosylated Chondroitin Sulfates

    Directory of Open Access Journals (Sweden)

    Alexey G. Gerbst

    2015-02-01

    Full Text Available Anionic polysaccharides fucosylated chondroitin sulfates (FCS from holothurian species were shown to affect various biological processes, such as metastasis, angiogenesis, clot formation, thrombosis, inflammation, and some others. To understand the mechanism of FCSs action, knowledge about their spatial arrangement is required. We have started the systematic synthesis, conformational analysis, and study of biological activity of the oligosaccharides related to various fragments of these types of natural polysaccharides. In this communication, five molecules representing distinct structural fragments of chondroitin sulfate have been studied by means of molecular modeling and NMR. These are three disaccharides and two trisaccharides containing fucose and glucuronic acid residues with one sulfate group per each fucose residue or without it. Long-range C–H coupling constants were used for the verification of the theoretical models. The presence of two conformers for both linkage types was revealed. For the Fuc–GlA linkage, the dominant conformer was the same as described previously in a literature as the molecular dynamics (MD average in a dodechasaccharide FCS fragment representing the backbone chain of the polysaccharide including GalNAc residues. This shows that the studied oligosaccharides, in addition to larger ones, may be considered as reliable models for Quantitative Structure-Activity Relationship (QSAR studies to reveal pharmacophore fragments of FCS.

  10. Study on correlation between speciifc N-Glycans in hepatocarcinoma serum and the expression of GnT-Ⅴin hepatocar­ cinoma tissue%肝癌血清特异糖链与癌组织中GnT-Ⅴ表达的相关性研究

    Institute of Scientific and Technical Information of China (English)

    孙建民; 张玉宝

    2014-01-01

    be further defined. Methods To find Glycomic specific tumor markers of hepatocarcinoma, DSA-FACE was performed in 40 preoperation serum and post operation serum of hepatocarcinoma patients and 50 healthy human. And the same 40 primary hepatocarcinoma tissues and cancer nearby tissues were detected for the expression levels of GnT-Ⅴwith immunohistochemistry staining. Results The serum N-glycan profiles of hepatocarcinoma was identified by the DSA-FACE technique. The results indicated that there were significant differences in N-linked glycans between hepatocarcinoma patients and healthy persons. The Peak 3,6 and Peak 9 were the most significant peaks which were picked as a new tumor marker of hepatocarcinoma. Taking log (p9/p3*p6) as indicator of the ROC curve analysis, the area under ROC curve was 0.811±0.034 with 84%sensitivity and 67%specificity. The positive expression of N-acetylglucosaminyltransferase V(GnT-Ⅴ), which adds N-acetylglu-cosamine (GlcNAc) to the mannose of the trimannosyl core in aβ-1,6 linkage, in clinical cancer was 65%which is much higher than that in cancer nearby tissues 7.5%(P<0.001).Conclusion The Peak 9, 3 and Peak 6 were the most significant peaks which were picked as a new tumor marker of hepatocarcinoma. And the changes of theβ-1,6 branches was mainly caused by the expression of GnT-Ⅴin the prime hepatocarcinoma tissues.

  11. The single N-glycan deletion mutant of soluble ErbB3 protein attenuates heregulin β1-induced tumor progression by blocking of the HIF-1 and Nrf2 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Takamiya, Rina, E-mail: rinataka0429@gmail.com; Takahashi, Motoko; Uehara, Yasuaki; Ariki, Shigeru; Hashimoto, Jiro; Hasegawa, Yoshihiro; Kuroki, Yoshio

    2014-11-21

    Highlights: • The sErbB3 N418Q mutant blocks heregulin β1 induced nuclear accumulation of HIF-1α. • The sErbB3 N418Q mutant attenuates cancer cell migration induced by heregulin β1. • The sErbB3 N418Q mutant blocks heregulin β1 induced nuclear accumulation of Nrf2. • The sErbB3 N418Q mutant may be a potential therapeutic application for tumor. - Abstract: It has been well documented that activation of the ErbB3–PI3K–Akt pathway is implicated in tumor survival and progression. We previously demonstrated that the single N-glycan deletion mutant of soluble ErbB3 protein (sErbB3 N418Q) attenuates heregulin β1-induced ErbB3 signaling. The active PI3K–Akt pathway augments the nuclear accumulation of hypoxia inducible factor (HIF)-1α, which activates the transcription of many target genes and drives cancer progression. In this study, we focused on the effects of sErbB3 N418Q mutant on nuclear accumulation of HIF-1α. Pretreatment with the sErbB3 N418Q mutant suppressed heregulin β1-induced HIF-1α activation in MCF7 cells. Similar results were also obtained in other breast cancer cell lines, T47D and BT474. Interestingly, these suppressive effects were not observed with the sErbB3 wild type. In addition, pretreatment with the sErbB3 N418Q mutant suppressed the cell migration of MCF7 cells induced by heregulin β1. Furthermore, incubation with heregulin β1 also induced the nuclear accumulation of Nrf2, and this effect was also reduced by the sErbB3 N418Q mutant, but not the sErbB3 wild type. These findings indicated that the sErbB3 N418Q mutant suppressed malignant formation of cancer cells by blocking of the HIF-1α and Nrf2 pathways.

  12. Galactosylated fucose epitopes in nematodes: increased expression in a Caenorhabditis mutant associated with altered lectin sensitivity and occurrence in parasitic species.

    Science.gov (United States)

    Yan, Shi; Bleuler-Martinez, Silvia; Plaza, David Fernando; Künzler, Markus; Aebi, Markus; Joachim, Anja; Razzazi-Fazeli, Ebrahim; Jantsch, Verena; Geyer, Rudolf; Wilson, Iain B H; Paschinger, Katharina

    2012-08-17

    The modification of α1,6-linked fucose residues attached to the proximal (reducing-terminal) core N-acetylglucosamine residue of N-glycans by β1,4-linked galactose ("GalFuc" epitope) is a feature of a number of invertebrate species including the model nematode Caenorhabditis elegans. A pre-requisite for both core α1,6-fucosylation and β1,4-galactosylation is the presence of a nonreducing terminal N-acetylglucosamine; however, this residue is normally absent from the final glycan structure in invertebrates due to the action of specific hexosaminidases. Previously, we have identified two hexosaminidases (HEX-2 and HEX-3) in C. elegans, which process N-glycans. In the present study, we have prepared a hex-2;hex-3 double mutant, which possesses a radically altered N-glycomic profile. Whereas in the double mutant core α1,3-fucosylation of the proximal N-acetylglucosamine was abolished, the degree of galactosylation of core α1,6-fucose increased, and a novel Galα1,2Fucα1,3 moiety attached to the distal core N-acetylglucosamine residue was detected. Both galactosylated fucose moieties were also found in two parasitic nematodes, Ascaris suum and Oesophagostomum dentatum. As core modifications of N-glycans are known targets for fungal nematotoxic lectins, the sensitivity of the C. elegans double hexosaminidase mutant was assessed. Although this mutant displayed hypersensitivity to the GalFuc-binding lectin CGL2 and the N-acetylglucosamine-binding lectin XCL, the mutant was resistant to CCL2, which binds core α1,3-fucose. Thus, the use of C. elegans mutants aids the identification of novel N-glycan modifications and the definition of in vivo specificities of nematotoxic lectins with potential as anthelmintic agents.

  13. Sulfation pattern of fucose branches affects the anti-hyperlipidemic activities of fucosylated chondroitin sulfate.

    Science.gov (United States)

    Wu, Nian; Zhang, Yu; Ye, Xingqian; Hu, Yaqin; Ding, Tian; Chen, Shiguo

    2016-08-20

    Fucosylated chondroitin sulfates (fCSs) are glycosaminoglycans extracted from sea cucumbers, consisting of chondroitin sulfate E (CSE) backbones and sulfated fucose branches. The biological properties of fCSs could be affected by the sulfation pattern of their fucose branches. In the present study, two fCSs were isolated from sea cucumbers Isostichopus badionotus (fCS-Ib) and Pearsonothuria graeffei (fCS-Pg). Their monosaccharide compositions of glucuronic acid (GlcA), N-acetylgalactosamine (GalNAc), fucose (Fuc) and sulfate were at similar molar ratio with 1.0/0.7/0.9/3.1 for fCS-Ib and 1.0/0.8/1.5/2.6 for fCS-Pg. The two fCSs have different sulfation patterns on their fucose branches, fCS-Pg with 3,4-O-disulfation while fCS-Ib with 2,4-O-disulfation. Their antihyperlipidemic effects were compared using a high-fat high-fructose diet (HFFD)-fed C57BL/6J mice model. Both fCS-Ib and fCS-Pg had significant effects on lipid profile improvement, liver protection, blood glucose diminution and hepatic glycogen synthesis. Specifically, fCS-Pg with 3,4-O-disulfation fucose branches was more effective in reduction of blood cholesterol (TC), low density lipoprotein (LDL) and atherogenic index (AI). Our results indicate that both fCSs, especially fCS-Pg, could be used as a potential anti-hyperlipidemic drug.

  14. Development of a Novel System for Mass Spectrometric Analysis of Cancer-Associated Fucosylation in Plasma α1-Acid Glycoprotein

    Directory of Open Access Journals (Sweden)

    Takayuki Asao

    2013-01-01

    Full Text Available Human plasma α1-acid glycoprotein (AGP from cancer patients and healthy volunteers was purified by sequential application of ion-exchange columns, and N-linked glycans enzymatically released from AGP were labeled and applied to a mass spectrometer. Additionally, a novel software system for use in combination with a mass spectrometer to determine N-linked glycans in AGP was developed. A database with 607 glycans including 453 different glycan structures that were theoretically predicted to be present in AGP was prepared for designing the software called AGPAS. This AGPAS was applied to determine relative abundance of each glycan in the AGP molecules based on mass spectra. It was found that the relative abundance of fucosylated glycans in tri- and tetra-antennary structures (FUCAGP was significantly higher in cancer patients as compared with the healthy group (P<0.001. Furthermore, extremely elevated levels of FUCAGP were found specifically in patients with a poor prognosis but not in patients with a good prognosis. In conclusion, the present software system allowed rapid determination of the primary structures of AGP glycans. The fucosylated glycans as novel tumor markers have clinical relevance in the diagnosis and assessment of cancer progression as well as patient prognosis.

  15. Protein expression and fucosylated glycans of the serum haptoglobin-β subunit in hepatitis B virus-based liver diseases

    Institute of Scientific and Technical Information of China (English)

    Hong Shu; Shu Zhang; Xiaonan Kang; Shan Li; Xue Qin; Chun Sun; Haojie Lu; Yinkun Liu

    2011-01-01

    Glycosylation, which regulates the configuration and function of glycoproteins, is the most important post-translational modification. The aim of this study was to observe the differential patterns in glycan and protein parts of the serum haptoglobin-β subunit (Hp-β) purified from patients with hepatitis B virus (HBV) infection, liver cirrhosis (LC), or hepatocellular carcinoma (HCC). 2-D gel electrophoresis and multiplexed proteomics staining technique were employed to investigate whether the Hp-β glycan level was proportional to the protein level. Multilectin blot, high-performance liquid chromatography (HPLC), and western blot analysis were carried out to identify the glycoform of Hp-β quantitatively. Our experiments showed that the ratio of total serum Hp-β to the glycosylated form of Hp-β varied among the patients with different liver diseases. The total Hp-β protein expression level was much higher in HCC than LC, while an incremental proportion of fucosylated Hp-β was also observed in LC and HCC patients compared with that in HBV and healthy controls. Differential fucosylation was firther identified as a Lewis X structure by HPLC and antihuman SialyI-Lewis X antibody. In conclusion, the aberrant alternation of Hp-β glycan and total protein expression may be a promising biomarker for early hepatocarcinogenesis.

  16. Metabolism and transportation pathways of GDP-fucose that are required for the O-fucosylation of Notch.

    Science.gov (United States)

    Yamakawa, Tomoko; Ayukawa, Tomonori; Matsuno, Kenji

    2012-01-01

    Notch is a single-pass transmembrane receptor that mediates the local cell-cell interactions necessary for many cell-fate decisions. The extra cellular domain of Notch contains a tandem array of epidermal growth factor-like (EGF-like) repeats. Some of these EGF-like repeats are O-fucosylated by protein O-fucosyltransferase 1 (O-fut1), which is essential for Notch signaling in Drosophila and mouse. This O-fucose is further modified by Fringe, a GlcNAc transferase and other glycosyltransferases (O-fut1 in Drosophila and Pofut1 in mouse), to form an O-linked tetrasaccharide, which modulates Notch's selective binding to its ligands.

  17. Partial characterization of the N-linked oligosaccharide structures on Pselectin glycoprotein ligand-1 (PSGL-1)

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    PSGL-1,a specific ligand for P-,E- and L-selectin,was isolated from in vivo [3H]-glucosamine labeled HL-60 cells by a combination of wheat germ agglutinin-agarose and P- or E-selectin-agarose chromatography.N-linked oligosaccharides were released from the purified,denatured ligand molecule by peptide: N-glycosidase F treatment and,following separation by Sephacryl S-200 chromatography,partially characterized using lectin,ion-exchange and size-exclusion chromatography in combination with glycosidase digestions.The data obtained suggest that the N-glycans on PSGL-1 are predominantly core-fucosylated,multiantennary complex type structures with extended,poly-N-acetyllactosamine containing outer chains.A portion of the outer chains appears to be substituted with fucose indicating that the N-glycans,in addition to the O-glycans on PSGL-1,may be involved in selectin binding.

  18. One-step non-chromatography purification of a low abundant fucosylated protein from complex plant crude extract

    Directory of Open Access Journals (Sweden)

    Lindsay Arnold

    2015-08-01

    Full Text Available Effective methods for isolation and purification of glycoproteins and other glycoconjugates are important to biopharmaceutical industry and diagnostic industry. They are also critical to an emerging field of glycoproteomics. In this work, we applied the newly-developed affinity ligand, a fusion protein of elastic like polymer (ELP and a bacterial lectin, in an affinity precipitation process to purify soybean peroxidase (SBP based on the presence of fucoseon the protein surface. We addressed, in particular, the challenge of purifying a low abundant protein from a complex dilute crude plant extract. The novel affinity precipitation developed in this work was very promising. One step binding and precipitation resulted in >95% recovery yield directly from crude extract and a 22.7 fold purification, giving a specific activity of 420 U/mg. The SBP isolated using this affinity precipitation meets or exceeds the quality specifications of reagent grade products by Sigma. We showed that the recovery yield had a strong dependence on the molar ratio of ligand to target fucosylated protein, with a ratio of three giving nearly full recovery, which could be predicted based on the total fucose content per protein molecule and the number of binding site per ligand molecule. We additionally developed a method of ligand regeneration and investigated its reuse. A simple wash with pH buffer was shown to be effective to regenerate the binding capacity for the ligand, and the ligand could be used for 10 times, giving an averaged 80% isolation yield based on initial input of soybean peroxidase. Taken together, an effective method of affinity precipitation was developed, which could be used to enrich a low abundant target glycoprotein from a complex mixture with a high recovery yield. The high selectivity for fucosylated protein and its ease of operation make this method particularly useful for purification of low abundant glycoprotein from natural sources. This work

  19. A highly regular fucosylated chondroitin sulfate from the sea cucumber Massinium magnum: Structure and effects on coagulation.

    Science.gov (United States)

    Ustyuzhanina, Nadezhda E; Bilan, Maria I; Dmitrenok, Andrey S; Borodina, Elizaveta Yu; Stonik, Valentin A; Nifantiev, Nikolay E; Usov, Anatolii I

    2017-07-01

    A fucosylated chondroitin sulfate MM was isolated from the sea cucumber Massinium magnum. Structure of this polysaccharide was determined using chemical and NMR spectroscopic methods. The backbone of MM was shown to consist mainly of chondroitin sulfate E units with a small portion (about 10%) of chondroitin sulfate A fragments. Practically one type of branches Fuc3S4S attached to O-3 of GlcA residues was found in the polysaccharide molecules. The main repeating units of MM are →4)-[α-l-Fuc3S4S-(1→3)]-β-d-GlcA-(1→3)-β-d-GalNAc4S6S-(1→, whereas the minor repeating units are →4)-[α-l-Fuc3S4S-(1→3)]-β-d-GlcA-(1→3)-β-d-GalNAc4S-(1→. Anticoagulant activity of MM determined in APTT and TT tests was shown to be lower than that of heparin, but higher than that of enoxaparin. In the experiments with purified proteins MM effectively potentiated inhibition of thrombin and factor Xa by ATIII. Besides, MM did not induce platelets aggregation in platelets rich plasma. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. FleA Expression in Aspergillus fumigatus Is Recognized by Fucosylated Structures on Mucins and Macrophages to Prevent Lung Infection.

    Science.gov (United States)

    Kerr, Sheena C; Fischer, Gregory J; Sinha, Meenal; McCabe, Orla; Palmer, Jonathan M; Choera, Tsokyi; Lim, Fang Yun; Wimmerova, Michaela; Carrington, Stephen D; Yuan, Shaopeng; Lowell, Clifford A; Oscarson, Stefan; Keller, Nancy P; Fahy, John V

    2016-04-01

    The immune mechanisms that recognize inhaled Aspergillus fumigatus conidia to promote their elimination from the lungs are incompletely understood. FleA is a lectin expressed by Aspergillus fumigatus that has twelve binding sites for fucosylated structures that are abundant in the glycan coats of multiple plant and animal proteins. The role of FleA is unknown: it could bind fucose in decomposed plant matter to allow Aspergillus fumigatus to thrive in soil, or it may be a virulence factor that binds fucose in lung glycoproteins to cause Aspergillus fumigatus pneumonia. Our studies show that FleA protein and Aspergillus fumigatus conidia bind avidly to purified lung mucin glycoproteins in a fucose-dependent manner. In addition, FleA binds strongly to macrophage cell surface proteins, and macrophages bind and phagocytose fleA-deficient (∆fleA) conidia much less efficiently than wild type (WT) conidia. Furthermore, a potent fucopyranoside glycomimetic inhibitor of FleA inhibits binding and phagocytosis of WT conidia by macrophages, confirming the specific role of fucose binding in macrophage recognition of WT conidia. Finally, mice infected with ΔfleA conidia had more severe pneumonia and invasive aspergillosis than mice infected with WT conidia. These findings demonstrate that FleA is not a virulence factor for Aspergillus fumigatus. Instead, host recognition of FleA is a critical step in mechanisms of mucin binding, mucociliary clearance, and macrophage killing that prevent Aspergillus fumigatus pneumonia.

  1. Depolymerization of Fucosylated Chondroitin Sulfate with a Modified Fenton-System and Anticoagulant Activity of the Resulting Fragments

    Science.gov (United States)

    Li, Jun-hui; Li, Shan; Zhi, Zi-jian; Yan, Lu-feng; Ye, Xing-qian; Ding, Tian; Yan, Lei; Linhardt, Robert John; Chen, Shi-guo

    2016-01-01

    Fucosylated chondroitin sulfate (fCS) from sea cucumber Isostichopus badionotus (fCS-Ib) with a chondroitin sulfate type E (CSE) backbone and 2,4-O-sulfo fucose branches has shown excellent anticoagulant activity although has also show severe adverse effects. Depolymerization represents an effective method to diminish this polysaccharide’s side effects. The present study reports a modified controlled Fenton system for degradation of fCS-Ib and the anticoagulant activity of the resulting fragments. Monosaccharides and nuclear magnetic resonance (NMR) analysis of the resulting fragments indicate that no significant chemical changes in the backbone of fCS-Ib and no loss of sulfate groups take place during depolymerization. A reduction in the molecular weight of fCS-Ib should result in a dramatic decrease in prolonging activated partial thromboplastin time and thrombin time. A decrease in the inhibition of thrombin (FIIa) by antithromin III (AT III) and heparin cofactor II (HCII), and the slight decrease of the inhibition of factor X activity, results in a significant increase of anti-factor Xa (FXa)/anti-FIIa activity ratio. The modified free-radical depolymerization method enables preparation of glycosaminoglycan (GAG) oligosaccharides suitable for investigation of clinical anticoagulant application. PMID:27657094

  2. Drosophila sperm surface alpha-L-fucosidase interacts with the egg coats through its core fucose residues.

    Science.gov (United States)

    Intra, Jari; Concetta, Veltri; Daniela, De Caro; Perotti, Maria Elisa; Pasini, Maria Enrica

    2015-08-01

    Sperm-oocyte interaction during fertilization is multiphasic, with multicomponent events, taking place between egg's glycoproteins and sperm surface receptors. Protein-carbohydrate complementarities in gamete recognition have observed in cases throughout the whole evolutionary scale. Sperm-associated α-L-fucosidases have been identified in various organisms. Their wide distribution and known properties reflect the hypothesis that fucose and α-L-fucosidases have fundamental function(s) during gamete interactions. An α-L-fucosidase has been detected as transmembrane protein on the surface of spermatozoa of eleven species across the genus Drosophila. Immunofluorescence labeling showed that the protein is localized in the sperm plasma membrane over the acrosome and the tail, in Drosophila melanogaster. In the present study, efforts were made to analyze with solid phase assays the oligosaccharide recognition ability of fruit fly sperm α-L-fucosidase with defined carbohydrate chains that can functionally mimic egg glycoconjugates. Our results showed that α-L-fucosidase bound to fucose residue and in particular it prefers N-glycans carrying core α1,6-linked fucose and core α1,3-linked fucose in N-glycans carrying only a terminal mannose residue. The ability of sperm α-L-fucosidase to bind to the micropylar chorion and to the vitelline envelope was examined in in vitro assays in presence of α-L-fucosidase, either alone or in combination with molecules containing fucose residues. No binding was detected when α-L-fucosidase was pre-incubated with fucoidan, a polymer of α-L-fucose and the monosaccharide fucose. Furthermore, egg labeling with anti-horseradish peroxidase, that recognized only core α1,3-linked fucose, correlates with α-L-fucosidase micropylar binding. Collectively, these data support the hypothesis of the potential role of this glycosidase in sperm-egg interactions in Drosophila.

  3. Distinct structures of the α-fucose branches in fucosylated chondroitin sulfates do not affect their anticoagulant activity.

    Science.gov (United States)

    Santos, Gustavo R C; Glauser, Bianca F; Parreiras, Luane A; Vilanova, Eduardo; Mourão, Paulo A S

    2015-10-01

    Fucosylated chondroitin sulfate (FCS) is a glycosaminoglycan found in sea cucumbers. It has a backbone like that of mammalian chondroitin sulfate (4-β-d-GlcA-1→3-β-d-GalNAc-1)n but substituted at the 3rd position of the β-d-glururonic acid residues with α-fucose branches. The structure of these branches varies among FCSs extracted from different species of sea cucumbers, as revealed by solution NMR spectroscopy. Some species (Isostichopus badionotus and Patalus mollis) contain branches formed by single α-fucose residues but with variable sulfation patterns (2,4-, 3,4- and 4-sulfation). FCS from Ludwigothurea grisea is distinguished because it contains preponderant branches formed by disaccharide units containing non-sulfated and 3-sulfated α-fucose units at the reducing and non-reducing ends, respectively. Despite the structural variability on their α-fucose branches, these FCSs have similar anticoagulant action on assays using purified reagents. They have serpin-dependent and serpin-independent effects. Pharmacological assays using experimental animals showed that the three types of FCSs have similar antithrombotic effect and bleeding tendency. They also activate factor XII on the same range of concentration. Based on these observations, we proposed that only few sulfated α-fucose branches along the FCS chain are enough to assure the binding of this glycosaminoglycan to proteins of the coagulation system. Substitution with additional sulfated α-fucose does not increase further the activity. Overall, the use of FCSs with marked variability on their branches of α-fucose allowed us to establish correlations between structures vs biological effects of these glycosaminoglycans on a more refined basis. It opens new avenues for therapeutic intervention using FCSs.

  4. Full-length core sequence dependent complex-type glycosylation of hepatitis C virus E2 glycoprotein

    Institute of Scientific and Technical Information of China (English)

    Li-Xin Zhu; Jing Liu; Ying-Chun Li; Yu-Ying Kong; Caroline Staib; Gerd Sutter; Yuan Wang; Guang-Di Li

    2002-01-01

    AIM: To study HCV polyprotein processing is important forthe understanding of the natural history of HCV and thedesign of vaccines against HCV. The purpose of this studyis to investigate the affection of context sequences onhepatitis C virus (HCV) E2 processingMETHODS: HCV genes of different lengths were expressedand compared in vaccinia virus/T7 system with homologouspatient serum S94 and mouse anti-serum ME2116 raisedagainst E. coli-derived E2 peptide, respectively.Deglycosylation analysis and GNA (Galanthus nivalus )lectin binding assay were performed to study the post-translational processing of the expressed products.RESULTS: E2 glycoproteins with different molecular weights( ~ 75kDa end ~ 60kDa) were detected using S94 and ME2116,respectively. Deglycosylation analysis showed that thisdifference was mainly due to different glycosylation. Endo Hresistance and its failure to bind to GNA lectin demonstratedthat the higher molecular weight form (75kDa) of E2 wascomplex-type glycosylated, which was readily recognized byhomologous patient serum S94. Expression of complex-typeglycosylated E2 could not be detected in all of the core-truncated constructs tested, but readily detected inconstructs encoding full-length core sequences.CONCLUSION: The upstream conserved full-length corecoding sequence was required for the production of E2glycoproteins carrying complex-type N-glycans whichreacted strongly with homologous patient serum andtherefore possibly represented more mature forms of E2. Ascomplex-type N-glycans indicated modification by Golgienzymes, the results suggest that the presence of full-lengthcore might be critical for E1/E2 complex to leave ER. Ourdata may contribute to a better understanding of theprocessing of HCV structural proteins as well as HCVmorphogenesis.

  5. Serum N-glycome biomarker for monitoring development of DENA-induced hepatocellular carcinoma in rat

    Directory of Open Access Journals (Sweden)

    Fang Meng

    2010-08-01

    Full Text Available Abstract Background There is a demand for serum markers for the routine assessment of the progression of liver cancer. We previously found that serum N-linked sugar chains are altered in hepatocellular carcinoma (HCC. Here, we studied glycomic alterations during development of HCC in a rat model. Results Rat HCC was induced by the hepatocarcinogen, diethylnitrosamine (DENA. N-glycans were profiled using the DSA-FACE technique developed in our laboratory. In comparison with control rats, DENA rats showed a gradual but significant increase in two glycans (R5a and R5b in serum total N-glycans during progression of liver cirrhosis and cancer, and a decrease in a biantennary glycan (P5. The log of the ratio of R5a to P1 (NGA2F and R5b to P1 [log(R5a/P1 and log(R5b/P1] were significantly (p Conclusions: We found an increase in core-α-1,6-fucosylated glycoproteins in serum and liver of rats with HCC, which demonstrates that fucosylation is altered during progression of HCC. Our GlycoTest model can be used to monitor progression of HCC and to follow up treatment of liver tumors in the DENA rat. This GlycoTest model is particularly important because a rapid non-invasive diagnostic procedure for tumour progression in this rat model would greatly facilitate the search for anticancer drugs.

  6. Proteomics Core

    Data.gov (United States)

    Federal Laboratory Consortium — Proteomics Core is the central resource for mass spectrometry based proteomics within the NHLBI. The Core staff help collaborators design proteomics experiments in a...

  7. Proteomics Core

    Data.gov (United States)

    Federal Laboratory Consortium — Proteomics Core is the central resource for mass spectrometry based proteomics within the NHLBI. The Core staff help collaborators design proteomics experiments in...

  8. Structure and anticoagulant activity of a fucosylated chondroitin sulfate from echinoderm. Sulfated fucose branches on the polysaccharide account for its high anticoagulant action.

    Science.gov (United States)

    Mourão, P A; Pereira, M S; Pavão, M S; Mulloy, B; Tollefsen, D M; Mowinckel, M C; Abildgaard, U

    1996-09-27

    A polysaccharide isolated from the body wall of the sea cucumber Ludwigothurea grisea has a backbone like that of mammalian chondroitin sulfate: [4-beta-D-GlcA-1-->3-beta-D-GalNAc-1]n but substituted at the 3-position of the beta--glucuronic acid residues with sulfated alpha--fucopyranosyl branches (Vieira, R. P., Mulloy, B., and Mourão, P. A. S. (1991) J. Biol. Chem. 266, 13530-13536). Mild acid hydrolysis removes the sulfated alpha--fucose branches, and cleaved residues have been characterized by 1H NMR spectroscopy; the most abundant species is fucose 4-O-monosulfate, but 2,4- and 3, 4-di-O-sulfated residues are also present. Degradation of the remaining polysaccharide with chondroitin ABC lyase shows that the sulfated alpha-L-fucose residues released by mild acid hydrolysis are concentrated toward the non-reducing end of the polysaccharide chains; enzyme-resistant polysaccharide material includes the reducing terminal and carries acid-resistant -fucose substitution. The sulfated alpha-L-fucose branches confer anticoagulant activity on the polysaccharide. The specific activity of fucosylated chondroitin sulfate in the activated partial thromboplastin time assay is greater than that of a linear homopolymeric alpha-L-fucan with about the same level of sulfation; this activity is lost on defucosylation or desulfation but not on carboxyl-reduction of the polymer. Assays with purified reagents show that the fucosylated chondroitin sulfate can potentiate the thrombin inhibition activity of both antithrombin and heparin cofactor II.

  9. Inhibition of fucosylation of cell wall components by 2-fluoro 2-deoxy-L-fucose induces defects in root cell elongation.

    Science.gov (United States)

    Dumont, Marie; Lehner, Arnaud; Bardor, Muriel; Burel, Carole; Vauzeilles, Boris; Lerouxel, Olivier; Anderson, Charles T; Mollet, Jean-Claude; Lerouge, Patrice

    2015-12-01

    Screening of commercially available fluoro monosaccharides as putative growth inhibitors in Arabidopsis thaliana revealed that 2-fluoro 2-l-fucose (2F-Fuc) reduces root growth at micromolar concentrations. The inability of 2F-Fuc to affect an Atfkgp mutant that is defective in the fucose salvage pathway indicates that 2F-Fuc must be converted to its cognate GDP nucleotide sugar in order to inhibit root growth. Chemical analysis of cell wall polysaccharides and glycoproteins demonstrated that fucosylation of xyloglucans and of N-linked glycans is fully inhibited by 10 μm 2F-Fuc in Arabidopsis seedling roots, but genetic evidence indicates that these alterations are not responsible for the inhibition of root development by 2F-Fuc. Inhibition of fucosylation of cell wall polysaccharides also affected pectic rhamnogalacturonan-II (RG-II). At low concentrations, 2F-Fuc induced a decrease in RG-II dimerization. Both RG-II dimerization and root growth were partially restored in 2F-Fuc-treated seedlings by addition of boric acid, suggesting that the growth phenotype caused by 2F-Fuc was due to a deficiency of RG-II dimerization. Closer investigation of the 2F-Fuc-induced growth phenotype demonstrated that cell division is not affected by 2F-Fuc treatments. In contrast, the inhibitor suppressed elongation of root cells and promoted the emergence of adventitious roots. This study further emphasizes the importance of RG-II in cell elongation and the utility of glycosyltransferase inhibitors as new tools for studying the functions of cell wall polysaccharides in plant development. Moreover, supplementation experiments with borate suggest that the function of boron in plants might not be restricted to RG-II cross-linking, but that it might also be a signal molecule in the cell wall integrity-sensing mechanism.

  10. Molecular clock regulates daily α1-2-fucosylation of the neural cell adhesion molecule (NCAM) within mouse secondary olfactory neurons.

    Science.gov (United States)

    Kondoh, Daisuke; Tateno, Hiroaki; Hirabayashi, Jun; Yasumoto, Yuki; Nakao, Reiko; Oishi, Katsutaka

    2014-12-26

    The circadian clock regulates various behavioral and physiological rhythms in mammals. Circadian changes in olfactory functions such as neuronal firing in the olfactory bulb (OB) and olfactory sensitivity have recently been identified, although the underlying molecular mechanisms remain unknown. We analyzed the temporal profiles of glycan structures in the mouse OB using a high-density microarray that includes 96 lectins, because glycoconjugates play important roles in the nervous system such as neurite outgrowth and synaptogenesis. Sixteen lectin signals significantly fluctuated in the OB, and the intensity of all three that had high affinity for α1-2-fucose (α1-2Fuc) glycan in the microarray was higher during the nighttime. Histochemical analysis revealed that α1-2Fuc glycan is located in a diurnal manner in the lateral olfactory tract that comprises axon bundles of secondary olfactory neurons. The amount of α1-2Fuc glycan associated with the major target glycoprotein neural cell adhesion molecule (NCAM) varied in a diurnal fashion, although the mRNA and protein expression of Ncam1 did not. The mRNA and protein expression of Fut1, a α1-2-specific fucosyltransferase gene, was diurnal in the OB. Daily fluctuation of the α1-2Fuc glycan was obviously damped in homozygous Clock mutant mice with disrupted diurnal Fut1 expression, suggesting that the molecular clock governs rhythmic α1-2-fucosylation in secondary olfactory neurons. These findings suggest the possibility that the molecular clock is involved in the diurnal regulation of olfaction via α1-2-fucosylation in the olfactory system.

  11. Ice cores

    DEFF Research Database (Denmark)

    Svensson, Anders

    2014-01-01

    Ice cores from Antarctica, from Greenland, and from a number of smaller glaciers around the world yield a wealth of information on past climates and environments. Ice cores offer unique records on past temperatures, atmospheric composition (including greenhouse gases), volcanism, solar activity......, dustiness, and biomass burning, among others. In Antarctica, ice cores extend back more than 800,000 years before present (Jouzel et al. 2007), whereas. Greenland ice cores cover the last 130,000 years...

  12. Ice cores

    DEFF Research Database (Denmark)

    Svensson, Anders

    2014-01-01

    Ice cores from Antarctica, from Greenland, and from a number of smaller glaciers around the world yield a wealth of information on past climates and environments. Ice cores offer unique records on past temperatures, atmospheric composition (including greenhouse gases), volcanism, solar activity......, dustiness, and biomass burning, among others. In Antarctica, ice cores extend back more than 800,000 years before present (Jouzel et al. 2007), whereas. Greenland ice cores cover the last 130,000 years...

  13. Comparison of the human and bovine milk N-glycome via high-performance microfluidic chip liquid chromatography and tandem mass spectrometry.

    Science.gov (United States)

    Nwosu, Charles C; Aldredge, Danielle L; Lee, Hyeyoung; Lerno, Larry A; Zivkovic, Angela M; German, J Bruce; Lebrilla, Carlito B

    2012-05-04

    The isolation of whey proteins from human and bovine milks followed by profiling of their entire N-glycan repertoire is described. Whey proteins resulting from centrifugation and ethanol precipitation of milk were treated with PNGase F to release protein-bound N-glycans. Once released, N-glycans were analyzed via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry following chromatographic separation on a porous graphitized carbon chip. In all, 38 N-glycan compositions were observed in the human milk sample while the bovine milk sample revealed 51 N-glycan compositions. These numbers translate to over a hundred compounds when isomers are considered and point to the complexity of the mixture. High mannose, neutral, and sialylated complex/hybrid glycans were observed in both milk sources. Although NeuAc sialylation was observed in both milk samples, the NeuGc residue was only observed in bovine milk and marks a major difference between human and bovine milks. To the best of our knowledge, this study is the first MS based confirmation of NeuGc in milk protein bound glycans as well as the first comprehensive N-glycan profile of bovine milk proteins. Tandem MS was necessary for resolving complications presented by the fact that (NeuGc:Fuc) corresponds to the exact mass of (NeuAc:Hex). Comparison of the relative distribution of the different glycan types in both milk sources was possible via their abundances. While the human milk analysis revealed a 6% high mannose, 57% sialylation, and 75% fucosylation distribution, a 10% high mannose, 68% sialylation, and 31% fucosylation distribution was observed in the bovine milk analysis. Comparison with the free milk oligosaccharides yielded low sialylation and high fucosylation in human, while high sialylation and low fucosylation are found in bovine. The results suggest that high fucosylation is a general trait in human, while high sialylation and low fucosylation are general features of

  14. Lactobacillus casei Ferments the N-Acetylglucosamine Moiety of Fucosyl-α-1,3-N-Acetylglucosamine and Excretes l-Fucose

    Science.gov (United States)

    Rodríguez-Díaz, Jesús; Rubio-del-Campo, Antonio

    2012-01-01

    We have previously characterized from Lactobacillus casei BL23 three α-l-fucosidases, AlfA, AlfB, and AlfC, which hydrolyze in vitro natural fucosyl-oligosaccharides. In this work, we have shown that L. casei is able to grow in the presence of fucosyl-α-1,3-N-acetylglucosamine (Fuc-α-1,3-GlcNAc) as a carbon source. Interestingly, L. casei excretes the l-fucose moiety during growth on Fuc-α-1,3-GlcNAc, indicating that only the N-acetylglucosamine moiety is being metabolized. Analysis of the genomic sequence of L. casei BL23 shows that downstream from alfB, which encodes the α-l-fucosidase AlfB, a gene, alfR, that encodes a transcriptional regulator is present. Divergently from alfB, three genes, alfEFG, that encode proteins with homology to the enzyme IIAB (EIIAB), EIIC, and EIID components of a mannose-class phosphoenolpyruvate:sugar phosphotransferase system (PTS) are present. Inactivation of either alfB or alfF abolishes the growth of L. casei on Fuc-α-1,3-GlcNAc. This proves that AlfB is involved in Fuc-α-1,3-GlcNAc metabolism and that the transporter encoded by alfEFG participates in the uptake of this disaccharide. A mutation in the PTS general component enzyme I does not eliminate the utilization of Fuc-α-1,3-GlcNAc, suggesting that the transport via the PTS encoded by alfEFG is not coupled to phosphorylation of the disaccharide. Transcriptional analysis with alfR and ccpA mutants shows that the two gene clusters alfBR and alfEFG are regulated by substrate-specific induction mediated by the inactivation of the transcriptional repressor AlfR and by carbon catabolite repression mediated by the catabolite control protein A (CcpA). This work reports for the first time the characterization of the physiological role of an α-l-fucosidase in lactic acid bacteria and the utilization of Fuc-α-1,3-GlcNAc as a carbon source for bacteria. PMID:22544237

  15. Fucosylation of LAMP-1 and LAMP-2 by FUT1 correlates with lysosomal positioning and autophagic flux of breast cancer cells.

    Science.gov (United States)

    Tan, Keng-Poo; Ho, Ming-Yi; Cho, Huan-Chieh; Yu, John; Hung, Jung-Tung; Yu, Alice Lin-Tsing

    2016-08-25

    Alpha1,2-fucosyltransferases, FUT1 and FUT2, which transfer fucoses onto the terminal galactose of N-acetyl-lactosamine via α1,2-linkage have been shown to be highly expressed in various types of cancers. A few studies have shown the involvement of FUT1 substrates in tumor cell proliferation and migration. Lysosome-associated membrane protein 1, LAMP-1, has been reported to carry alpha1,2-fucosylated Lewis Y (LeY) antigens in breast cancer cells, however, the biological functions of LeY on LAMP-1 remain largely unknown. Whether or not its family member, LAMP-2, displays similar modifications and functions as LAMP-1 has not yet been addressed. In this study, we have presented evidence supporting that both LAMP-1 and 2 are substrates for FUT1, but not FUT2. We have also demonstrated the presence of H2 and LeY antigens on LAMP-1 by a targeted nanoLC-MS(3) and the decreased levels of fucosylation on LAMP-2 by MALDI-TOF analysis upon FUT1 knockdown. In addition, we found that the expression of LeY was substantial in less invasive ER+/PR+/HER- breast cancer cells (MCF-7 and T47D) but negligible in highly invasive triple-negative MDA-MB-231 cells, of which LeY levels were correlated with the levels of LeY carried by LAMP-1 and 2. Intriguingly, we also observed a striking change in the subcellular localization of lysosomes upon FUT1 knockdown from peripheral distribution of LAMP-1 and 2 to a preferential perinuclear accumulation. Besides that, knockdown of FUT1 led to an increased rate of autophagic flux along with diminished activity of mammalian target of rapamycin complex 1 (mTORC1) and enhanced autophagosome-lysosome fusion. This may be associated with the predominantly perinuclear distribution of lysosomes mediated by FUT1 knockdown as lysosomal positioning has been reported to regulate mTOR activity and autophagy. Taken together, our results suggest that downregulation of FUT1, which leads to the perinuclear localization of LAMP-1 and 2, is correlated with increased

  16. Occurrence of sulfated fucose branches in fucosylated chondroitin sulfate are essential for the polysaccharide effect preventing muscle damage induced by toxins and crude venom from Bothrops jararacussu snake.

    Science.gov (United States)

    Monteiro-Machado, Marcos; Tomaz, Marcelo A; Fonseca, Roberto J C; Strauch, Marcelo A; Cons, Bruno L; Borges, Paula A; Patrão-Neto, Fernando C; Tavares-Henriques, Matheus S; Teixeira-Cruz, Jhonatha M; Calil-Elias, Sabrina; Cintra, Adélia C O; Martinez, Ana Maria B; Mourão, Paulo A S; Melo, Paulo A

    2015-05-01

    Snake envenoming is an important public health problem around the world, particularly in tropics. Beyond deaths, morbidity induced by snake venoms, such as myotoxicity, is of pivotal consequence to population. Bothrops jararacussu is the main venomous snake in southeast region of Brazil, and particularly presents strong myotoxic effect. The only available therapy, antibothropic antivenom, poorly affects venom-induced myotoxicity. The aim of this study is to assess the ability of fucosylated chondroitin sulfate (fucCS), a glycosaminoglycan with anticoagulant and antithrombotic properties, and its derivatives to inhibit toxic activities of B. jararacussu crude venom and its isolated toxins, named bothropstoxins (BthTX-I and BthTX-II). The in vitro myotoxic activities induced by crude venom, by BthTX-I alone and by toxins together were abolished by fucCS. Carboxyl reduction (fucCS-CR) kept this ability whereas defucosilation (defucCS) abrogates myoprotection. We observed the same pattern in the response of these polysaccharides in antagonizing the increase in plasma creatine kinase (CK) levels, the reduction of skeletal muscle CK content and the rise of myeloperoxidase (MPO) activity induced by crude venom and isolated toxins. FucCS inhibited edematogenic activity and partially prevented the reduction of total leukocytes in blood when pre-incubated with crude venom. Furthermore, the venom procoagulant effect was completely antagonized by increasing concentrations of fucCS, although this polyanion could stop neither the tail bleeding nor the skin hemorrhage induced by Bothrops jararaca venom. The B. jararacussu phospholipase, hyaluronidase, proteolytic and collagenase activities were inhibited in vitro. The results suggest that fucCS could be able to interact with both toxins, and it is able to inhibit BthTX-II phospholipase activity. Light microscopy of extensor digitorum longus muscle (EDL) muscle showed myoprotection by fucCS, once necrotic areas, edema and

  17. Transformer core

    NARCIS (Netherlands)

    Mehendale, A.; Hagedoorn, Wouter; Lötters, Joost Conrad

    2010-01-01

    A transformer core includes a stack of a plurality of planar core plates of a magnetically permeable material, which plates each consist of a first and a second sub-part that together enclose at least one opening. The sub-parts can be fitted together via contact faces that are located on either side

  18. Transformer core

    NARCIS (Netherlands)

    Mehendale, A.; Hagedoorn, Wouter; Lötters, Joost Conrad

    2008-01-01

    A transformer core includes a stack of a plurality of planar core plates of a magnetically permeable material, which plates each consist of a first and a second sub-part that together enclose at least one opening. The sub-parts can be fitted together via contact faces that are located on either side

  19. Ice Cores

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Records of past temperature, precipitation, atmospheric trace gases, and other aspects of climate and environment derived from ice cores drilled on glaciers and ice...

  20. Core BPEL

    DEFF Research Database (Denmark)

    Hallwyl, Tim; Højsgaard, Espen

    extensions. Combined with the fact that the language definition does not provide a formal semantics, it is an arduous task to work formally with the language (e.g. to give an implementation). In this paper we identify a core subset of the language, called Core BPEL, which has fewer and simpler constructs......, does not allow omissions, and does not contain ignorable elements. We do so by identifying syntactic sugar, including default values, and ignorable elements in WS-BPEL. The analysis results in a translation from the full language to the core subset. Thus, we reduce the effort needed for working...... formally with WS-BPEL, as one, without loss of generality, need only consider the much simpler Core BPEL. This report may also be viewed as an addendum to the WS-BPEL standard specification, which clarifies the WS-BPEL syntax and presents the essential elements of the language in a more concise way...

  1. Core BPEL

    DEFF Research Database (Denmark)

    Hallwyl, Tim; Højsgaard, Espen

    extensions. Combined with the fact that the language definition does not provide a formal semantics, it is an arduous task to work formally with the language (e.g. to give an implementation). In this paper we identify a core subset of the language, called Core BPEL, which has fewer and simpler constructs......, does not allow omissions, and does not contain ignorable elements. We do so by identifying syntactic sugar, including default values, and ignorable elements in WS-BPEL. The analysis results in a translation from the full language to the core subset. Thus, we reduce the effort needed for working...... formally with WS-BPEL, as one, without loss of generality, need only consider the much simpler Core BPEL. This report may also be viewed as an addendum to the WS-BPEL standard specification, which clarifies the WS-BPEL syntax and presents the essential elements of the language in a more concise way...

  2. Core benefits

    National Research Council Canada - National Science Library

    Keith, Brian W

    2010-01-01

    This SPEC Kit explores the core employment benefits of retirement, and life, health, and other insurance -benefits that are typically decided by the parent institution and often have significant governmental regulation...

  3. Gene expression of fucosyl- and sialyl-transferases which synthesize sialyl Lewisx, the carbohydrate ligands for E-selectin, in human breast cancer.

    Science.gov (United States)

    Matsuura, N; Narita, T; Hiraiwa, N; Hiraiwa, M; Murai, H; Iwase, T; Funahashi, H; Imai, T; Takagi, H; Kannagi, R

    1998-05-01

    The adhesion of circulating cancer cells to vascular endothelium is an important step in the hematogenous metastasis of cancer. Until recently, it has been believed that carbohydrate antigens are expressed on cancer cells, and E-selectin is expressed on endothelial cells to effect this adhesion. We investigated the gene expression of fucosyl-transferase (Fuc-T) and sialyltransferase (ST), which are involved in the synthesis of sialyl Lewisx (s-Lex) in breast cancer by using Northern blot analysis. The concentration of s-Lex in the cancerous portion was increased, compared to that in the adjacent non-cancerous portion. A correlation was found between the concentration of s-Lex and the amount of Fuc-T VI message in 9 cases of breast cancer tissue. Expression of the Fuc-T III message was found in only one case who expressed s-Lea. No expression of the Fuc-T V or VII message was observed. There was no relationship between the concentration of s-Lex and the amount of ST3N and ST4 transcripts. Similar findings were obtained from an analysis using cell lines derived from human breast cancer. When Fuc-T VI gene was transfected to MCF-7 cells, the expression of s-Lex was markedly induced on MCF-7 cells, and the attachment of cancer cells to endothelial cells was enhanced. These findings suggest that Fuc-T VI is chiefly involved in the synthesis of s-Lex on breast cancer cells.

  4. The fucosylation inhibitor, 2-fluorofucose, inhibits vaso-occlusion, leukocyte-endothelium interactions and NF-ĸB activation in transgenic sickle mice.

    Directory of Open Access Journals (Sweden)

    John D Belcher

    Full Text Available 2-Fluorofucose (2FF blocks the fucosylation and the tethering of sialyl-Lewisx tetrasaccharide and structural variants on leukocytes and red blood cells to P- and E-selectins on activated endothelial cell surfaces. Because P- and E-selectin are required for vaso-occlusion in murine sickle cell disease (SCD, we investigated whether 2FF would inhibit vaso-occlusion in SCD mice. Microvascular stasis was measured in subcutaneous venules in NY1DD and HbSS-Townes SCD mice with dorsal skin-fold chambers after infusion of hemoglobin or exposure to hypoxia/reoxygenation. 2FF in drinking water or administered by gavage inhibited stasis in sickle mice in a dose-responsive manner. Significant inhibitory effects on stasis were seen 1 day post-treatment. 2FF treatment of SCD mice also significantly reduced leukocyte rolling and adhesion along the vessel walls of SCD mice and the static adhesion of neutrophils and sickle red blood cells isolated from 2FF-treated SCD mice to resting and activated endothelial cells. Total white blood cell counts increased in response to 2FF. NF-ĸB activation and VCAM-1 and E-selectin expression were inhibited in the livers of SCD mice consistent with an overall decrease in vascular inflammation and ischemia-reperfusion physiology. Pretreatment with 2FF completely eliminated heme-induced lethality in HbSS-Townes mice, consistent with the observed anti-inflammatory and anti-adhesive properties of 2FF in SCD mice. These data suggest that 2FF may be beneficial for preventing or treating vaso-occlusive crises in SCD patients.

  5. Evaluation of Fucosylated Haptoglobin and Mac-2 Binding Protein as Serum Biomarkers to Estimate Liver Fibrosis in Patients with Chronic Hepatitis C.

    Science.gov (United States)

    Tawara, Seiichi; Tatsumi, Tomohide; Iio, Sadaharu; Kobayashi, Ichizou; Shigekawa, Minoru; Hikita, Hayato; Sakamori, Ryotaro; Hiramatsu, Naoki; Miyoshi, Eiji; Takehara, Tetsuo

    2016-01-01

    Fucosylated haptoglobin (Fuc-Hpt) and Mac-2 binding protein (Mac-2 bp) are identified as cancer biomarkers, based on the results from a glyco-proteomic analysis. Recently, we reported that these glyco-biomarkers were associated with liver fibrosis and/or ballooning hepatocytes in patients with nonalcoholic fatty liver disease (NAFLD). We evaluated the ability of these glycoproteins to estimate liver fibrosis in 317 patients with chronic hepatitis C. We measured the serum Fuc-Hpt and Mac-2 bp levels using a lectin-antibody ELISA and ELISA, respectively. The serum levels of both Fuc-Hpt and Mac-2 bp increased with the progression of liver fibrosis. The multivariate analysis revealed that Mac-2 bp was an independent factor associated with moderate liver fibrosis (F ≥ 2). In contrast, Fuc-Hpt was an independent factor associated with advanced liver fibrosis (F ≥ 3). In terms of evaluating liver fibrosis, the serum levels of these glycomarkers were correlated with well-known liver fibrosis indexes, such as the aspartate aminotransferase to platelet ratio index (APRI) and Fibrosis-4 (FIB4) index. An assay that combined the APRI or FIB4 index and the Fuc-Hpt or Mac-2 bp levels increased the AUC value for diagnosing hepatic fibrosis. Interestingly, the cumulative incidence of hepatocellular carcinoma (HCC) was significantly higher in the patients with elevated serum levels of Fuc-Hpt and Mac-2 bp. In conclusion, both Fuc-Hpt and Mac-2 bp could be useful glyco-biomarkers of liver fibrosis and predictors of HCC in patients with chronic hepatitis C.

  6. 6-alkynyl fucose is a bioorthogonal analog for O-fucosylation of epidermal growth factor-like repeats and thrombospondin type-1 repeats by protein O-fucosyltransferases 1 and 2.

    Science.gov (United States)

    Al-Shareffi, Esam; Chaubard, Jean-Luc; Leonhard-Melief, Christina; Wang, Sheng-Kai; Wong, Chi-Huey; Haltiwanger, Robert S

    2013-02-01

    Protein O-fucosyltransferase 1 (Pofut1) and protein O-fucosyltransferase 2 (Pofut2) add O-linked fucose at distinct consensus sequences in properly folded epidermal growth factor (EGF)-like repeats and thrombospondin type-1 (TSR) repeats, respectively. Glycan chain elongation past O-fucose can occur to yield a tetrasaccharide on EGF repeats and a disaccharide on TSRs. Elimination of Pofut1 in mice causes embryonic lethality with Notch-like phenotypes demonstrating that O-fucosylation of Notch is essential for its function. Similarly, elimination of Pofut2 results in an early embryonic lethal phenotype in mice, although the molecular mechanism for the lethality is unknown. The recent development of sugar analogs has revolutionized the study of glycans by providing a convenient method for labeling and tracking glycosylation. In order to study O-fucosylation, we took advantage of the recently developed reporter, 6-alkynyl fucose. Using the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), or "click" reaction, azido-biotin allows tagging and detection of 6AF-modified proteins. Here we examine whether proteins containing EGF repeats or TSRs with O-fucose consensus sequences are specifically modified with 6AF in cell culture. Using mass spectrometry (MS), we demonstrate that 6AF is efficiently incorporated onto the appropriate consensus sequences on EGF repeats and TSRs. Furthermore, the elongation of the O-fucose monosaccharide on EGF repeats and TSRs is not hampered when 6AF is used. These results show that 6AF is efficiently utilized in a truly bioorthogonal manner by Pofut1, Pofut2 and the enzymes that elongate O-fucose, providing evidence that 6AF is a significant new tool in the study of protein O-fucosylation.

  7. Altered glycosylation of complexed native IgG molecules is associated with disease activity of systemic lupus erythematosus.

    Science.gov (United States)

    Sjöwall, C; Zapf, J; von Löhneysen, S; Magorivska, I; Biermann, M; Janko, C; Winkler, S; Bilyy, R; Schett, G; Herrmann, M; Muñoz, L E

    2015-05-01

    In addition to the redundancy of the receptors for the Fc portion of immunoglobulins, glycans result in potential ligands for a plethora of lectin receptors found in immune effector cells. Here we analysed the exposure of glycans containing fucosyl residues and the fucosylated tri-mannose N-type core by complexed native IgG in longitudinal serum samples of well-characterized patients with systemic lupus erythematosus. Consecutive serum samples of a cohort of 15 patients with systemic lupus erythematosus during periods of increased disease activity and remission were analysed. All patients fulfilled the 1982 American College of Rheumatology classification criteria. Sera of 15 sex- and age-matched normal healthy blood donors served as controls. The levels and type of glycosylation of complexed random IgG was measured with lectin enzyme-immunosorbent assays. After specifically gathering IgG complexes from sera, biotinylated lectins Aleuria aurantia lectin and Lens culinaris agglutinin were employed to detect IgG-associated fucosyl residues and the fucosylated tri-mannose N-glycan core, respectively. In sandwich-ELISAs, IgG-associated IgM, IgA, C1q, C3c and C-reactive protein (CRP) were detected as candidates for IgG immune complex constituents. We studied associations of the glycan of complexed IgG and disease activity according to the physician's global assessment of disease activity and the systemic lupus erythematosus disease activity index 2000 documented at the moment of blood taking. Our results showed significantly higher levels of Aleuria aurantia lectin and Lens culinaris agglutinin binding sites exposed on IgG complexes of patients with systemic lupus erythematosus than on those of normal healthy blood donors. Disease activity in systemic lupus erythematosus correlated with higher exposure of Aleuria aurantia lectin-reactive fucosyl residues by immobilized IgG complexes. Top levels of Aleuria aurantia lectin-reactivity were found in samples taken during the

  8. Core Java

    CERN Document Server

    Horstmann, Cay S

    2013-01-01

    Fully updated to reflect Java SE 7 language changes, Core Java™, Volume I—Fundamentals, Ninth Edition, is the definitive guide to the Java platform. Designed for serious programmers, this reliable, unbiased, no-nonsense tutorial illuminates key Java language and library features with thoroughly tested code examples. As in previous editions, all code is easy to understand, reflects modern best practices, and is specifically designed to help jumpstart your projects. Volume I quickly brings you up-to-speed on Java SE 7 core language enhancements, including the diamond operator, improved resource handling, and catching of multiple exceptions. All of the code examples have been updated to reflect these enhancements, and complete descriptions of new SE 7 features are integrated with insightful explanations of fundamental Java concepts.

  9. The N-glycome of human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Olonen Anne

    2009-06-01

    Full Text Available Abstract Background Complex carbohydrate structures, glycans, are essential components of glycoproteins, glycolipids, and proteoglycans. While individual glycan structures including the SSEA and Tra antigens are already used to define undifferentiated human embryonic stem cells (hESC, the whole spectrum of stem cell glycans has remained unknown. We undertook a global study of the asparagine-linked glycoprotein glycans (N-glycans of hESC and their differentiated progeny using MALDI-TOF mass spectrometric and NMR spectroscopic profiling. Structural analyses were performed by specific glycosidase enzymes and mass spectrometric fragmentation analyses. Results The data demonstrated that hESC have a characteristic N-glycome which consists of both a constant part and a variable part that changes during hESC differentiation. hESC-associated N-glycans were downregulated and new structures emerged in the differentiated cells. Previously mouse embryonic stem cells have been associated with complex fucosylation by use of SSEA-1 antibody. In the present study we found that complex fucosylation was the most characteristic glycosylation feature also in undifferentiated hESC. The most abundant complex fucosylated structures were Lex and H type 2 antennae in sialylated complex-type N-glycans. Conclusion The N-glycan phenotype of hESC was shown to reflect their differentiation stage. During differentiation, hESC-associated N-glycan features were replaced by differentiated cell-associated structures. The results indicated that hESC differentiation stage can be determined by direct analysis of the N-glycan profile. These results provide the first overview of the N-glycan profile of hESC and form the basis for future strategies to target stem cell glycans.

  10. In silico analysis of the fucosylation-associated genome of the human blood fluke Schistosoma mansoni: cloning and characterization of the enzymes involved in GDP-L-fucose synthesis and Golgi import.

    Science.gov (United States)

    Peterson, Nathan A; Anderson, Tavis K; Wu, Xiao-Jun; Yoshino, Timothy P

    2013-07-09

    Carbohydrate structures of surface-expressed and secreted/excreted glycoconjugates of the human blood fluke Schistosoma mansoni are key determinants that mediate host-parasite interactions in both snail and mammalian hosts. Fucose is a major constituent of these immunologically important glycans, and recent studies have sought to characterize fucosylation-associated enzymes, including the Golgi-localized fucosyltransferases that catalyze the transfer of L-fucose from a GDP-L-fucose donor to an oligosaccharide acceptor. Importantly, GDP-L-fucose is the only nucleotide-sugar donor used by fucosyltransferases and its availability represents a bottleneck in fucosyl-glycotope expression. A homology-based genome-wide bioinformatics approach was used to identify and molecularly characterize the enzymes that contribute to GDP-L-fucose synthesis and Golgi import in S. mansoni. Putative functions were further investigated through molecular phylogenetic and immunocytochemical analyses. We identified homologs of GDP-D-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase (GMER), which constitute a de novo pathway for GDP-L-fucose synthesis, in addition to a GDP-L-fucose transporter (GFT) that putatively imports cytosolic GDP-L-fucose into the Golgi. In silico primary sequence analyses identified characteristic Rossman loop and short-chain dehydrogenase/reductase motifs in GMD and GMER as well as 10 transmembrane domains in GFT. All genes are alternatively spliced, generating variants of unknown function. Observed quantitative differences in steady-state transcript levels between miracidia and primary sporocysts may contribute to differential glycotope expression in early larval development. Additionally, analyses of protein expression suggest the occurrence of cytosolic GMD and GMER in the ciliated epidermal plates and tegument of miracidia and primary sporocysts, respectively, which is consistent with previous localization of highly

  11. Preparation of branched cyclomaltoheptaose with 3-O-α-L-fucopyranosyl-α-D-mannopyranose and changes in fucosylation of HCT116 cells treated with the fucose-modified cyclomaltoheptaose.

    Science.gov (United States)

    Kimura, Madoka; Masui, Yuki; Shirai, Yuko; Honda, Chie; Moriwaki, Kenta; Imai, Taku; Takagi, Uichiro; Kiryu, Takaaki; Kiso, Taro; Murakami, Hiromi; Nakano, Hirofumi; Kitahata, Sumio; Miyoshi, Eiji; Tanimoto, Toshiko

    2013-06-07

    From a mixture of 4-nitrophenyl α-L-fucopyranoside and D-mannopyranose, 3-O-α-L-fucopyranosyl-D-mannopyranose was synthesised through the transferring action of α-fucosidase (Sumizyme PHY). 6(I),6(IV)-Di-O-(3-O-α-L-fucopyranosyl-α-D-mannopyranosyl)-cyclomaltoheptaose {8, 6(I),6(IV)-di-O-[α-L-Fuc-(1→3)-α-D-Man]-βCD} was chemically synthesised using the trichloroacetimidate method. The structures were confirmed by MS and NMR spectroscopy. A cell-based assay using the fucosyl βCD derivatives, including the newly synthesised 8, showed that derivatives with two branches of the α-L-Fuc or α-L-Fuc-(1→3)-α-D-Man residues possessed slight growth-promoting effects and lower toxicity in HCT116 cells compared to those with one branch. These compounds may be useful as drug carriers in targeted drug delivery systems.

  12. Sialosyl-fucosyl Poly-LacNAc without the sialosyl-Lex epitope as the physiological myeloid cell ligand in E-selectin-dependent adhesion: studies under static and dynamic flow conditions.

    Science.gov (United States)

    Handa, K; Stroud, M R; Hakomori, S

    1997-10-14

    The majority of E- and P-selectin ligands in leukocytes and myelocytic or monocytic leukemia cells are carried by transmembrane glycoproteins having a tandem repeat mucin-like domain through which O-linked carbohydrate ligands are carried. However, determination of structure and adhesive function of carbohydrates in glycoproteins is extremely difficult because of the extensive structural heterogeneity and the scarcity of material for functional analysis. We have overcome this difficulty through use of poly-LacNAc gangliosides isolated from a large quantity of ( approximately 1.2 L packed) HL60 cells [Stroud, M. R., Handa, K., Salyan, M. E. K., Ito, K., Levery, S. B., Hakomori, S., Reinhold, B. B., & Reinhold, V. N. (1996) Biochemistry 35, 758-769, 770-778]. We identified two major types of poly-LacNAc gangliosides without the sialosyl-Lex epitope as being capable of binding to E-selectin: (i) those having a single alpha1-->3 fucosylation at internal GlcNAcs but not at the penultimate GlcNAc and (ii) those having double alpha1-->3 fucosylation at internal GlcNAcs, excluding the penultimate GlcNAc. Gangliosides from group i above did not show any adhesion under static conditions, but showed strong adhesion under dynamic flow conditions. Gangliosides from group ii above showed adhesion under both static and dynamic conditions, as did sialosyl-Lex (SLex)-containing structures in previous studies. However, SLex-containing poly-LacNAc gangliosides are virtually absent or present in only trace quantities in leukocytes and HL60 cells. Poly-LacNAc gangliosides from groups i and ii above, lacking SLex structure, are the major membrane components of leukocytes and HL60 cells. These carbohydrates, bound to lipid or to protein, may therefore be the physiological epitope for E-selectin-dependent binding of these cells, particularly under dynamic flow conditions.

  13. Dual-core antiresonant hollow core fibers.

    Science.gov (United States)

    Liu, Xuesong; Fan, Zhongwei; Shi, Zhaohui; Ma, Yunfeng; Yu, Jin; Zhang, Jing

    2016-07-25

    In this work, dual-core antiresonant hollow core fibers (AR-HCFs) are numerically demonstrated, based on our knowledge, for the first time. Two fiber structures are proposed. One is a composite of two single-core nested nodeless AR-HCFs, exhibiting low confinement loss and a circular mode profile in each core. The other has a relatively simple structure, with a whole elliptical outer jacket, presenting a uniform and wide transmission band. The modal couplings of the dual-core AR-HCFs rely on a unique mechanism that transfers power through the air. The core separation and the gap between the two cores influence the modal coupling strength. With proper designs, both of the dual-core fibers can have low phase birefringence and short modal coupling lengths of several centimeters.

  14. Animal MRI Core

    Data.gov (United States)

    Federal Laboratory Consortium — The Animal Magnetic Resonance Imaging (MRI) Core develops and optimizes MRI methods for cardiovascular imaging of mice and rats. The Core provides imaging expertise,...

  15. Differential expression of anti-glycan antibodies in schistosome-infected humans, rhesus monkeys and mice

    Science.gov (United States)

    Luyai, Anthony E; Heimburg-Molinaro, Jamie; Prasanphanich, Nina Salinger; Mickum, Megan L; Lasanajak, Yi; Song, Xuezheng; Nyame, A Kwame; Wilkins, Patricia; Rivera-Marrero, Carlos A; Smith, David F; Van Die, Irma; Secor, W Evan; Cummings, Richard D

    2014-01-01

    Schistosomiasis is a debilitating parasitic disease of humans, endemic in tropical areas, for which no vaccine is available. Evidence points to glycan antigens as being important in immune responses to infection. Here we describe our studies on the comparative humoral immune responses to defined schistosome-type glycan epitopes in Schistosoma mansoni-infected humans, rhesus monkeys and mice. Rhesus anti-glycan responses over the course of infection were screened on a defined glycan microarray comprising semi-synthetic glycopeptides terminating with schistosome-associated or control mammalian-type glycan epitopes, as well as a defined glycan microarray of mammalian-type glycans representing over 400 glycan structures. Infected rhesus monkeys generated a high immunoglobulin G (IgG) antibody response to the core xylose/core α3 fucose epitope of N-glycans, which peaked at 8–11 weeks post infection, coinciding with maximal ability to kill schistosomula in vitro. By contrast, infected humans generated low antibody levels to this epitope. At 18 months following praziquantel therapy to eliminate the parasite, antibody levels were negligible. Mice chronically infected with S. mansoni generated high levels of anti-fucosylated LacdiNAc (GalNAcβ1, 4(Fucα1, 3)GlcNAc) IgM antibodies, but lacked a robust response to the core xylose/core α3 fucose N-glycan antigens compared with other species studied, and their sera demonstrated an intermediate level of schistosomula killing in vitro. These differential responses to parasite glycan antigens may be related to the ability of rhesus monkeys to self-cure in contrast to the chronic infection seen in humans and mice. Our results validate defined glycan microarrays as a useful technology to evaluate diagnostic and vaccine antigens for schistosomiasis and perhaps other infections. PMID:24727442

  16. k-core covers and the core

    NARCIS (Netherlands)

    Sanchez-Rodriguez, E.; Borm, Peter; Estevez-Fernandez, A.; Fiestras-Janeiro, G.; Mosquera, M.A.

    2015-01-01

    This paper extends the notion of individual minimal rights for a transferable utility game (TU-game) to coalitional minimal rights using minimal balanced families of a specific type, thus defining a corresponding minimal rights game. It is shown that the core of a TU-game coincides with the core of

  17. Academic Rigor: The Core of the Core

    Science.gov (United States)

    Brunner, Judy

    2013-01-01

    Some educators see the Common Core State Standards as reason for stress, most recognize the positive possibilities associated with them and are willing to make the professional commitment to implementing them so that academic rigor for all students will increase. But business leaders, parents, and the authors of the Common Core are not the only…

  18. k-core covers and the core

    NARCIS (Netherlands)

    Sanchez-Rodriguez, E.; Borm, Peter; Estevez-Fernandez, A.; Fiestras-Janeiro, G.; Mosquera, M.A.

    This paper extends the notion of individual minimal rights for a transferable utility game (TU-game) to coalitional minimal rights using minimal balanced families of a specific type, thus defining a corresponding minimal rights game. It is shown that the core of a TU-game coincides with the core of

  19. Absence of lactobacilli containing glycolipids with the α-galactose epitope and the enhanced fucosylation of a receptor glycolipid GA1 in the digestive tracts of immune-deficient scid mice.

    Science.gov (United States)

    Iwamori, Masao; Tanaka, Kyoko; Adachi, Shigeki; Aoki, Daisuke; Nomura, Taisei

    2015-07-01

    The Lactobacillus species in the digestive tracts of immune-deficient scid mice was distinct from that in control mice, i.e. Lactobacillus murinus in scid and L. johnsonii in control mice, according to their 16S-rRNA, indicating that a symbiotic relationship between lactobacilli and a host is established under pressure from the immune system. The caecal and colonal contents rich in L. murinus of scid mice were loose with a strong sour smell, resulting in diarrhoea, and those with L. johnsonii in control mice included abundant solid materials. Lactobacillus glycolipids were revealed to be recognized by the immune system, and by TLC-immunostaining, LacTetH-DG (Galα1-6Galα1-6Galα1-2Glcα1-3'DG) of L. johnsonii was detected in the stomach, caecum and colon of control mice, but not in those of scid ones, in which fucosylation of a receptor GA1 for L. johnsonii was enhanced more than 4-fold compared with in the control mice. Thus, structural modification of receptor glycolipids was revealed to occur in the process of establishment of a symbiotic relationship between lactobacilli and a host. LacTetH-DG was also immunogenic to human, because of the presence of natural antibodies against it, and the antibody binding to it was comparable to that of blood group- and species-related glycosphingolipids.

  20. Comodules over semiperfect corings

    CERN Document Server

    Caenepeel, S

    2011-01-01

    We discuss when the Rat functor associated to a coring satisfying the left $\\alpha$-condition is exact. We study the category of comodules over a semiperfect coring. We characterize semiperfect corings over artinian rings and over qF-rings.

  1. Coring Sample Acquisition Tool

    Science.gov (United States)

    Haddad, Nicolas E.; Murray, Saben D.; Walkemeyer, Phillip E.; Badescu, Mircea; Sherrit, Stewart; Bao, Xiaoqi; Kriechbaum, Kristopher L.; Richardson, Megan; Klein, Kerry J.

    2012-01-01

    A sample acquisition tool (SAT) has been developed that can be used autonomously to sample drill and capture rock cores. The tool is designed to accommodate core transfer using a sample tube to the IMSAH (integrated Mars sample acquisition and handling) SHEC (sample handling, encapsulation, and containerization) without ever touching the pristine core sample in the transfer process.

  2. Banded transformer cores

    Science.gov (United States)

    Mclyman, C. W. T. (Inventor)

    1974-01-01

    A banded transformer core formed by positioning a pair of mated, similar core halves on a supporting pedestal. The core halves are encircled with a strap, selectively applying tension whereby a compressive force is applied to the core edge for reducing the innate air gap. A dc magnetic field is employed in supporting the core halves during initial phases of the banding operation, while an ac magnetic field subsequently is employed for detecting dimension changes occurring in the air gaps as tension is applied to the strap.

  3. The absence of core fucose up-regulates GnT-III and Wnt target genes: a possible mechanism for an adaptive response in terms of glycan function.

    Science.gov (United States)

    Kurimoto, Ayako; Kitazume, Shinobu; Kizuka, Yasuhiko; Nakajima, Kazuki; Oka, Ritsuko; Fujinawa, Reiko; Korekane, Hiroaki; Yamaguchi, Yoshiki; Wada, Yoshinao; Taniguchi, Naoyuki

    2014-04-25

    Glycans play key roles in a variety of protein functions under normal and pathological conditions, but several glycosyltransferase-deficient mice exhibit no or only mild phenotypes due to redundancy or compensation of glycan functions. However, we have only a limited understanding of the underlying mechanism for these observations. Our previous studies indicated that 70% of Fut8-deficient (Fut8(-/-)) mice that lack core fucose structure die within 3 days after birth, but the remainder survive for up to several weeks although they show growth retardation as well as emphysema. In this study, we show that, in mouse embryonic fibroblasts (MEFs) from Fut8(-/-) mice, another N-glycan branching structure, bisecting GlcNAc, is specifically up-regulated by enhanced gene expression of the responsible enzyme N-acetylglucosaminyltransferase III (GnT-III). As candidate target glycoproteins for bisecting GlcNAc modification, we confirmed that level of bisecting GlcNAc on β1-integrin and N-cadherin was increased in Fut8(-/-) MEFs. Moreover using mass spectrometry, glycan analysis of IgG1 in Fut8(-/-) mouse serum demonstrated that bisecting GlcNAc contents were also increased by Fut8 deficiency in vivo. As an underlying mechanism, we found that in Fut8(-/-) MEFs Wnt/β-catenin signaling is up-regulated, and an inhibitor against Wnt signaling was found to abrogate GnT-III expression, indicating that Wnt/β-catenin is involved in GnT-III up-regulation. Furthermore, various oxidative stress-related genes were also increased in Fut8(-/-) MEFs. These data suggest that Fut8(-/-) mice adapted to oxidative stress, both ex vivo and in vivo, by inducing various genes including GnT-III, which may compensate for the loss of core fucose functions.

  4. N-glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research

    DEFF Research Database (Denmark)

    Pedersen, Carina T; Loke, Ian; Lorentzen, Andrea

    2017-01-01

    Studies of protein N-glycosylation are important for answering fundamental questions on the diverse functions of glycoproteins in plant growth and development. Here we generated and characterised a comprehensive collection of Lotus japonicusLORE1 insertion mutants, each lacking the activity of one...

  5. IgG1 Fc N-glycan galactosylation as a biomarker for immune activation

    NARCIS (Netherlands)

    De Jong, Sanne E.; Selman, Maurice H J; Adegnika, Ayola A.; Adegnika, Ayola A.; Adegnika, Ayola A.; Amoah, Abena S.; Amoah, Abena S.; Van Riet, Elly; Kruize, Yvonne C M; Raynes, John G.; Rodriguez, Alejandro; Boakye, Daniel; Von Mutius, Erika; Von Mutius, Erika; Knulst, André C.; Genuneit, Jon; Cooper, Philip J.; Cooper, Philip J.; Hokke, Cornelis H.; Wuhrer, Manfred; Yazdanbakhsh, Maria

    2016-01-01

    Immunoglobulin G (IgG) Fc N-glycosylation affects antibody-mediated effector functions and varies with inflammation rooted in both communicable and non-communicable diseases. Worldwide, communicable and non-communicable diseases tend to segregate geographically. Therefore, we studied whether IgG Fc

  6. Glycoengineering of Chinese hamster ovary cells for enhanced erythropoietin N-glycan branching and sialylation

    DEFF Research Database (Denmark)

    Yin, Bojiao; Gao, Yuan; Chung, Cheng-yu

    2015-01-01

    -glycosylation of recombinant erythropoietin (rEPO), a human α2,6-sialyltransferase (ST6Gal1) was expressed in Chinese hamster ovary (CHO-K1) cells. Sialylation increased on both EPO and CHO cellular proteins as observed by SNA lectin analysis, and HPLC profiling revealed that the sialic acid content of total glycans on EPO......EPO from these engineered cells was increased ∼45% higher with tetra-sialylation accounting for ∼10% of total sugar chains compared to ∼3% for the wild-type parental CHO-K1. In this way, coordinated overexpression of these three glycosyltransferases for the first time in model CHO-K1 cell lines provides...

  7. The role of N-glycan modification of TNFR1 in inflammatory microglia activation.

    Science.gov (United States)

    Han, Lijian; Zhang, Dongmei; Tao, Tao; Sun, Xiaolei; Liu, Xiaojuan; Zhu, Guizhou; Xu, Zhiwei; Zhu, Liang; Zhang, Yu; Liu, Wangrui; Ke, Kaifu; Shen, Aiguo

    2015-12-01

    Accumulating evidences demonstrated that microglia activation and the autocrine loop of tumor necrosis factor-α (TNFα) greatly contribute to the pathogenesis of several CNS diseases. TNFα exerts its biological effects by interacting with two different receptors: TNF receptor 1 (TNFR1) and TNFR2. The classic proinflammatory activity of TNFα is mainly mediated by TNFR1. In the present study, we found that TNFR1 was modificated by N-glycosylation on Asn151 and Asn202 in microglia. The N-glycosylation of TNFR1 could facilitate its capability of binding to TNFα and further promote the formation of TNFα autocrine loop in microglia stimulated by TNFα, resulting in excessive microglia activation and CNS inflammation. All these processes were related to TNFR1-mediated NF-κB pathways. Elimination of N-glycosylation did not affect the subcellular transportation and cell surface localization of TNFR1, but suppressed ligand-binding affinity. These findings indicated that the N-glycosylation of TNFR1 played an important role during microglia activation in CNS inflammation. By this study, we aimed to provide some valuable experimental evidence for a better understanding of the significance of protein glycosylation in microglia inflammatory activation and CNS disease.

  8. The N-glycan glycoprotein deglycosylation complex (Gpd from Capnocytophaga canimorsus deglycosylates human IgG.

    Directory of Open Access Journals (Sweden)

    Francesco Renzi

    2011-06-01

    Full Text Available C. canimorsus 5 has the capacity to grow at the expenses of glycan moieties from host cells N-glycoproteins. Here, we show that C. canimorsus 5 also has the capacity to deglycosylate human IgG and we analyze the deglycosylation mechanism. We show that deglycosylation is achieved by a large complex spanning the outer membrane and consisting of the Gpd proteins and sialidase SiaC. GpdD, -G, -E and -F are surface-exposed outer membrane lipoproteins. GpdDEF could contribute to the binding of glycoproteins at the bacterial surface while GpdG is a endo-β-N-acetylglucosaminidase cleaving the N-linked oligosaccharide after the first N-linked GlcNAc residue. GpdC, resembling a TonB-dependent OM transporter is presumed to import the oligosaccharide into the periplasm after its cleavage from the glycoprotein. The terminal sialic acid residue of the oligosaccharide is then removed by SiaC, a periplasm-exposed lipoprotein in direct contact with GpdC. Finally, most likely degradation of the oligosaccharide proceeds sequentially from the desialylated non reducing end by the action of periplasmic exoglycosidases, including β-galactosidases, β-N-Acetylhexosaminidases and α-mannosidases.

  9. Recognition of TLR2 N-glycans: critical role in ArtinM immunomodulatory activity.

    Directory of Open Access Journals (Sweden)

    Vania Sammartino Mariano

    Full Text Available TLR2 plays a critical role in the protection against Paracoccidioides brasiliensis conferred by ArtinM administration. ArtinM, a D-mannose-binding lectin from Artocarpus heterophyllus, induces IL-12 production in macrophages and dendritic cells, which accounts for the T helper1 immunity that results from ArtinM administration. We examined the direct interaction of ArtinM with TLR2using HEK293A cells transfected with TLR2, alone or in combination with TLR1 or TLR6, together with accessory proteins. Stimulation with ArtinM induced NF-κB activation and interleukin (IL-8 production in cells transfected with TLR2, TLR2/1, or TLR2/6. Murine macrophages that were stimulated with ArtinM had augmented TLR2 mRNA expression. Furthermore, pre-incubation of unstimulated macrophages with an anti-TLR2 antibody reduced the cell labeling with ArtinM. In addition, a microplate assay revealed that ArtinM bound to TLR2 molecules that had been captured by specific antibodies from a macrophages lysate. Notably,ArtinM binding to TLR2 was selectively inhibited when the lectin was pre-incubated with mannotriose. The biological relevance of the direct interaction of ArtinM with TLR2 glycans was assessed using macrophages from TLR2-KOmice, which produced significantly lower levels of IL-12 and IL-10 in response to ArtinM than macrophages from wild-type mice. Pre-treatment of murine macrophages with pharmacological inhibitors of signaling molecules demonstrated the involvement of p38 MAPK and JNK in the IL-12 production induced by ArtinM and the involvement ofPI3K in IL-10 production. Thus, ArtinM interacts directly with TLR2 or TLR2 heterodimers in a carbohydrate recognition-dependent manner and functions as a TLR2 agonist with immunomodulatory properties.

  10. Stable Isotope Quantitative N-Glycan Analysis by Liquid Separation Techniques and Mass Spectrometry.

    Science.gov (United States)

    Mittermayr, Stefan; Albrecht, Simone; Váradi, Csaba; Millán-Martín, Silvia; Bones, Jonathan

    2017-01-01

    Liquid phase separation analysis and subsequent quantitation remains a challenging task for protein-derived oligosaccharides due to their inherent structural complexity and diversity. Incomplete resolution or co-detection of multiple glycan species complicates peak area-based quantitation and associated statistical analysis when optical detection methods are used. The approach outlined herein describes the utilization of stable isotope variants of commonly used fluorescent tags that allow for mass-based glycan identification and relative quantitation following separation by liquid chromatography (LC) or capillary electrophoresis (CE). Comparability assessment of glycoprotein-derived oligosaccharides is performed by derivatization with commercially available isotope variants of 2-aminobenzoic acid or aniline and analysis by LC- and CE-mass spectrometry. Quantitative information is attained from the extracted ion chromatogram/electropherogram ratios generated from the light and heavy isotope clusters.

  11. Different site-specific N-glycan types in wheat (Triticum aestivum L.) PAP phytase.

    Science.gov (United States)

    Dionisio, Giuseppe; Brinch-Pedersen, Henrik; Welinder, Karen Gjesing; Jørgensen, Malene

    2011-07-01

    Phytase activity in grain is essential to make phosphate available to cell metabolism, and in food and feed. Cereals contain the purple acid phosphatase type of phytases (PAPhy). Mature wheat grain is dominated by TaPAPhy_a which, in the present work, has been characterized by extensive peptide and glycopeptide sequencing by mass spectrometry. Seven N-linked glycosylation sites were found. Three of these sites were dominated by variant forms of the XylMan(3)FucGlcNAc(2), i.e. the HRP-type of glycan. Complex-type glycans with one or two additional GlcNAc were observed, however in trace amounts only. At four sites the glycan consisted of a single GlcNAc residue. The mature protein is ca. 500 residues in size and appears to be truncated at the N- and C-termini. Copyright © 2011. Published by Elsevier Ltd.

  12. The core paradox.

    Science.gov (United States)

    Kennedy, G. C.; Higgins, G. H.

    1973-01-01

    Rebuttal of suggestions from various critics attempting to provide an escape from the seeming paradox originated by Higgins and Kennedy's (1971) proposed possibility that the liquid in the outer core was thermally stably stratified and that this stratification might prove a powerful inhibitor to circulation of the outer core fluid of the kind postulated for the generation of the earth's magnetic field. These suggestions are examined and shown to provide no reasonable escape from the core paradox.

  13. K-core inflation

    OpenAIRE

    Alexander L. Wolman

    2011-01-01

    K-core inflation is a new class of underlying inflation measures. The two most popular measures of underlying inflation are core inflation and trimmed mean inflation. The former removes fixed categories of goods and services (food and energy) from the inflation calculation, and the latter removes fixed percentiles of the weighted distribution of price changes. In contrast, k-core inflation specifies a size of relative price change to be removed from the inflation calculation. Thus, the catego...

  14. Sediment Core Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — FUNCTION: Provides instrumentation and expertise for physical and geoacoustic characterization of marine sediments. DESCRIPTION: The multisensor core logger measures...

  15. Sediment Core Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — FUNCTION: Provides instrumentation and expertise for physical and geoacoustic characterization of marine sediments.DESCRIPTION: The multisensor core logger measures...

  16. Ice Core Investigations

    Science.gov (United States)

    Krim, Jessica; Brody, Michael

    2008-01-01

    What can glaciers tell us about volcanoes and atmospheric conditions? How does this information relate to our understanding of climate change? Ice Core Investigations is an original and innovative activity that explores these types of questions. It brings together popular science issues such as research, climate change, ice core drilling, and air…

  17. Making an Ice Core.

    Science.gov (United States)

    Kopaska-Merkel, David C.

    1995-01-01

    Explains an activity in which students construct a simulated ice core. Materials required include only a freezer, food coloring, a bottle, and water. This hands-on exercise demonstrates how a glacier is formed, how ice cores are studied, and the nature of precision and accuracy in measurement. Suitable for grades three through eight. (Author/PVD)

  18. Ice Core Investigations

    Science.gov (United States)

    Krim, Jessica; Brody, Michael

    2008-01-01

    What can glaciers tell us about volcanoes and atmospheric conditions? How does this information relate to our understanding of climate change? Ice Core Investigations is an original and innovative activity that explores these types of questions. It brings together popular science issues such as research, climate change, ice core drilling, and air…

  19. Iowa Core Annual Report

    Science.gov (United States)

    Iowa Department of Education, 2015

    2015-01-01

    One central component of a great school system is a clear set of expectations, or standards, that educators help all students reach. In Iowa, that effort is known as the Iowa Core. The Iowa Core represents the statewide academic standards, which describe what students should know and be able to do in math, science, English language arts, and…

  20. Mercury's core evolution

    Science.gov (United States)

    Deproost, Marie-Hélène; Rivoldini, Attilio; Van Hoolst, Tim

    2016-10-01

    Remote sensing data of Mercury's surface by MESSENGER indicate that Mercury formed under reducing conditions. As a consequence, silicon is likely the main light element in the core together with a possible small fraction of sulfur. Compared to sulfur, which does almost not partition into solid iron at Mercury's core conditions and strongly decreases the melting temperature, silicon partitions almost equally well between solid and liquid iron and is not very effective at reducing the melting temperature of iron. Silicon as the major light element constituent instead of sulfur therefore implies a significantly higher core liquidus temperature and a decrease in the vigor of compositional convection generated by the release of light elements upon inner core formation.Due to the immiscibility in liquid Fe-Si-S at low pressure (below 15 GPa), the core might also not be homogeneous and consist of an inner S-poor Fe-Si core below a thinner Si-poor Fe-S layer. Here, we study the consequences of a silicon-rich core and the effect of the blanketing Fe-S layer on the thermal evolution of Mercury's core and on the generation of a magnetic field.

  1. Mars' core and magnetism.

    Science.gov (United States)

    Stevenson, D J

    2001-07-12

    The detection of strongly magnetized ancient crust on Mars is one of the most surprising outcomes of recent Mars exploration, and provides important insight about the history and nature of the martian core. The iron-rich core probably formed during the hot accretion of Mars approximately 4.5 billion years ago and subsequently cooled at a rate dictated by the overlying mantle. A core dynamo operated much like Earth's current dynamo, but was probably limited in duration to several hundred million years. The early demise of the dynamo could have arisen through a change in the cooling rate of the mantle, or even a switch in convective style that led to mantle heating. Presently, Mars probably has a liquid, conductive outer core and might have a solid inner core like Earth.

  2. Importance of the Side Chain at Position 296 of Antibody Fc in Interactions with FcγRIIIa and Other Fcγ Receptors.

    Directory of Open Access Journals (Sweden)

    Yuya Isoda

    Full Text Available Antibody-dependent cellular cytotoxicity (ADCC is an important effector function determining the clinical efficacy of therapeutic antibodies. Core fucose removal from N-glycans on the Fc portion of immunoglobulin G (IgG improves the binding affinity for Fcγ receptor IIIa (FcγRIIIa and dramatically enhances ADCC. Our previous structural analyses revealed that Tyr-296 of IgG1-Fc plays a critical role in the interaction with FcγRIIIa, particularly in the enhanced FcγRIIIa binding of nonfucosylated IgG1. However, the importance of the Tyr-296 residue in the antibody in the interaction with various Fcγ receptors has not yet been elucidated. To further clarify the biological importance of this residue, we established comprehensive Tyr-296 mutants as fucosylated and nonfucosylated anti-CD20 IgG1s rituximab variants and examined their binding to recombinant soluble human Fcγ receptors: shFcγRI, shFcγRIIa, shFcγRIIIa, and shFcγRIIIb. Some of the mutations affected the binding of antibody to not only shFcγRIIIa but also shFcγRIIa and shFcγRIIIb, suggesting that the Tyr-296 residue in the antibody was also involved in interactions with FcγRIIa and FcγRIIIb. For FcγRIIIa binding, almost all Tyr-296 variants showed lower binding affinities than the wild-type antibody, irrespective of their core fucosylation, particularly in Y296K and Y296P. Notably, only the Y296W mutant showed improved binding to FcγRIIIa. The 3.00 Å-resolution crystal structure of the nonfucosylated Y296W mutant in complex with shFcγRIIIa harboring two N-glycans revealed that the Tyr-to-Trp substitution increased the number of potential contact atoms in the complex, thus improving the binding of the antibody to shFcγRIIIa. The nonfucosylated Y296W mutant retained high ADCC activity, relative to the nonfucosylated wild-type IgG1, and showed greater binding affinity for FcγRIIa. Our data may improve our understanding of the biological importance of human IgG1-Fc Tyr-296

  3. The t-core of an s-core

    OpenAIRE

    Fayers, Matthew

    2010-01-01

    We consider the $t$-core of an $s$-core partition, when $s$ and $t$ are coprime positive integers. Olsson has shown that the $t$-core of an $s$-core is again an $s$-core, and we examine certain actions of the affine symmetric group on $s$-cores which preserve the $t$-core of an $s$-core. Along the way, we give a new proof of Olsson's result. We also give a new proof of a result of Vandehey, showing that there is a simultaneous $s$- and $t$-core which contains all others.

  4. The t-core of an s-core

    OpenAIRE

    Fayers, Matthew

    2010-01-01

    We consider the $t$-core of an $s$-core partition, when $s$ and $t$ are coprime positive integers. Olsson has shown that the $t$-core of an $s$-core is again an $s$-core, and we examine certain actions of the affine symmetric group on $s$-cores which preserve the $t$-core of an $s$-core. Along the way, we give a new proof of Olsson's result. We also give a new proof of a result of Vandehey, showing that there is a simultaneous $s$- and $t$-core which contains all others.

  5. Korrelasjon mellom core styrke, core stabilitet og utholdende styrke i core

    OpenAIRE

    Berg-Olsen, Andrea Marie; Fugelsøy, Eivor; Maurstad, Ann-Louise

    2010-01-01

    Formålet med studien var å se hvilke korrelasjon det er mellom core styrke, core stabilitet og utholdende styrke i core. Testingen bestod av tre hoveddeler hvor vi testet core styrke, core stabilitet og utholdende styrke i core. Innenfor core styrke og utholdende styrke i core ble tre ulike tester utført. Ved måling av core stabilitet ble det gjennomført kun en test. I core styrke ble isometrisk abdominal fleksjon, isometrisk rygg ekstensjon og isometrisk lateral fleksjon testet. Sit-ups p...

  6. Korrelasjon mellom core styrke, core stabilitet og utholdende styrke i core

    OpenAIRE

    Berg-Olsen, Andrea Marie; Fugelsøy, Eivor; Maurstad, Ann-Louise

    2010-01-01

    Formålet med studien var å se hvilke korrelasjon det er mellom core styrke, core stabilitet og utholdende styrke i core. Testingen bestod av tre hoveddeler hvor vi testet core styrke, core stabilitet og utholdende styrke i core. Innenfor core styrke og utholdende styrke i core ble tre ulike tester utført. Ved måling av core stabilitet ble det gjennomført kun en test. I core styrke ble isometrisk abdominal fleksjon, isometrisk rygg ekstensjon og isometrisk lateral fleksjon testet. Sit-ups p...

  7. Earth's inner core: Innermost inner core or hemispherical variations?

    NARCIS (Netherlands)

    Lythgoe, K. H.; Deuss, A.; Rudge, J. F.; Neufeld, J. A.

    2014-01-01

    The structure of Earth's deep inner core has important implications for core evolution, since it is thought to be related to the early stages of core formation. Previous studies have suggested that there exists an innermost inner core with distinct anisotropy relative to the rest of the inner core.

  8. Earth's inner core: Innermost inner core or hemispherical variations?

    NARCIS (Netherlands)

    Lythgoe, K. H.; Deuss, A.; Rudge, J. F.; Neufeld, J. A.

    2014-01-01

    The structure of Earth's deep inner core has important implications for core evolution, since it is thought to be related to the early stages of core formation. Previous studies have suggested that there exists an innermost inner core with distinct anisotropy relative to the rest of the inner core.

  9. IGCSE core mathematics

    CERN Document Server

    Wall, Terry

    2013-01-01

    Give your core level students the support and framework they require to get their best grades with this book dedicated to the core level content of the revised syllabus and written specifically to ensure a more appropriate pace. This title has been written for Core content of the revised Cambridge IGCSE Mathematics (0580) syllabus for first teaching from 2013. ? Gives students the practice they require to deepen their understanding through plenty of practice questions. ? Consolidates learning with unique digital resources on the CD, included free with every book. We are working with Cambridge

  10. Core shroud corner joints

    Science.gov (United States)

    Gilmore, Charles B.; Forsyth, David R.

    2013-09-10

    A core shroud is provided, which includes a number of planar members, a number of unitary corners, and a number of subassemblies each comprising a combination of the planar members and the unitary corners. Each unitary corner comprises a unitary extrusion including a first planar portion and a second planar portion disposed perpendicularly with respect to the first planar portion. At least one of the subassemblies comprises a plurality of the unitary corners disposed side-by-side in an alternating opposing relationship. A plurality of the subassemblies can be combined to form a quarter perimeter segment of the core shroud. Four quarter perimeter segments join together to form the core shroud.

  11. Biospecimen Core Resource - TCGA

    Science.gov (United States)

    The Cancer Genome Atlas (TCGA) Biospecimen Core Resource centralized laboratory reviews and processes blood and tissue samples and their associated data using optimized standard operating procedures for the entire TCGA Research Network.

  12. NICHD Zebrafish Core

    Data.gov (United States)

    Federal Laboratory Consortium — The core[HTML_REMOVED]s goal is to help researchers of any expertise perform zebrafish experiments aimed at illuminating basic biology and human disease mechanisms,...

  13. iPSC Core

    Data.gov (United States)

    Federal Laboratory Consortium — The induced Pluripotent Stem Cells (iPSC) Core was created in 2011 to accelerate stem cell research in the NHLBI by providing investigators consultation, technical...

  14. Reference: -300CORE [PLACE

    Lifescience Database Archive (English)

    Full Text Available -300CORE Forde BG, Heyworth A, Pywell J, Kreis M Nucleotide sequence of a B1 hordein gene and the identifica...tion of possible upstream regulatory elements in endosperm storage protein genes fr

  15. INTEGRAL core programme

    Science.gov (United States)

    Gehrels, N.; Schoenfelder, V.; Ubertini, P.; Winkler, C.

    1997-01-01

    The International Gamma Ray Astrophysics Laboratory (INTEGRAL) mission is described with emphasis on the INTEGRAL core program. The progress made in the planning activities for the core program is reported on. The INTEGRAL mission has a nominal lifetime of two years with a five year extension option. The observing time will be divided between the core program (between 30 and 35 percent during the first two years) and general observations. The core program consists of three main elements: the deep survey of the Galactic plane in the central radian of the Galaxy; frequent scans of the Galactic plane in the search for transient sources, and pointed observations of several selected sources. The allocation of the observation time is detailed and the sensitivities of the observations are outlined.

  16. Focusing on Core Business

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    China is regulating state-owned enterprises that are investing outside of their core business realms, concerned that poor investment decisions could lead to loss of state-owned assets, but some doubt the effect of the new regulation

  17. Organizing Core Tasks

    DEFF Research Database (Denmark)

    Boll, Karen

    Civil servants conduct the work which makes welfare states functions on an everyday bases: Police men police, school teachers teach, and tax inspectors inspect. Focus in this paper is on the core tasks of tax inspectors. The paper argues that their core task of securing the collection of revenue...... has remained much the same within the last 10 years. However, how the core task has been organized has changed considerable under the influence of various “organizing devices”. The paper focusses on how organizing devices such as risk assessment, output-focus, effect orientation, and treatment...... projects influence the organization of core tasks within the tax administration. The paper shows that the organizational transformations based on the use of these devices have had consequences both for the overall collection of revenue and for the employees’ feeling of “making a difference”. All in all...

  18. Plant protein glycosylation

    Science.gov (United States)

    Strasser, Richard

    2016-01-01

    Protein glycosylation is an essential co- and post-translational modification of secretory and membrane proteins in all eukaryotes. The initial steps of N-glycosylation and N-glycan processing are highly conserved between plants, mammals and yeast. In contrast, late N-glycan maturation steps in the Golgi differ significantly in plants giving rise to complex N-glycans with β1,2-linked xylose, core α1,3-linked fucose and Lewis A-type structures. While the essential role of N-glycan modifications on distinct mammalian glycoproteins is already well documented, we have only begun to decipher the biological function of this ubiquitous protein modification in different plant species. In this review, I focus on the biosynthesis and function of different protein N-linked glycans in plants. Special emphasis is given on glycan-mediated quality control processes in the ER and on the biological role of characteristic complex N-glycan structures. PMID:26911286

  19. Packing in protein cores

    Science.gov (United States)

    Gaines, J. C.; Clark, A. H.; Regan, L.; O'Hern, C. S.

    2017-07-01

    Proteins are biological polymers that underlie all cellular functions. The first high-resolution protein structures were determined by x-ray crystallography in the 1960s. Since then, there has been continued interest in understanding and predicting protein structure and stability. It is well-established that a large contribution to protein stability originates from the sequestration from solvent of hydrophobic residues in the protein core. How are such hydrophobic residues arranged in the core; how can one best model the packing of these residues, and are residues loosely packed with multiple allowed side chain conformations or densely packed with a single allowed side chain conformation? Here we show that to properly model the packing of residues in protein cores it is essential that amino acids are represented by appropriately calibrated atom sizes, and that hydrogen atoms are explicitly included. We show that protein cores possess a packing fraction of φ ≈ 0.56 , which is significantly less than the typically quoted value of 0.74 obtained using the extended atom representation. We also compare the results for the packing of amino acids in protein cores to results obtained for jammed packings from discrete element simulations of spheres, elongated particles, and composite particles with bumpy surfaces. We show that amino acids in protein cores pack as densely as disordered jammed packings of particles with similar values for the aspect ratio and bumpiness as found for amino acids. Knowing the structural properties of protein cores is of both fundamental and practical importance. Practically, it enables the assessment of changes in the structure and stability of proteins arising from amino acid mutations (such as those identified as a result of the massive human genome sequencing efforts) and the design of new folded, stable proteins and protein-protein interactions with tunable specificity and affinity.

  20. Switching of the core structures of glycosphingolipids from globo- and lacto- to ganglio-series upon human embryonic stem cell differentiation

    Science.gov (United States)

    Liang, Yuh-Jin; Kuo, Huan-Hsien; Chen, Yen-Ying; Yang, Bei-Chia; Cheng, Yuan-Yuan; Yu, Alice L.; Khoo, Kay-Hooi; Yu, John

    2010-01-01

    A systematic survey of expression profiles of glycosphingolipids (GSLs) in two hESC lines and their differentiated embryoid body (EB) outgrowth with three germ layers was carried out using immunofluorescence, flow cytometry, and MALDI-MS and MS/MS analyses. In addition to the well-known hESC-specific markers stage-specific embryonic antigen 3 (SSEA-3) and SSEA-4, we identified several globosides and lacto-series GSLs, previously unrevealed in hESCs, including Gb4Cer, Lc4Cer, fucosyl Lc4Cer, Globo H, and disialyl Gb5Cer. During hESC differentiation into EBs, MS analysis revealed a clear-cut switch in the core structures of GSLs from globo- and lacto- to ganglio-series, which was not as evident by immunostaining with antibodies against SSEA-3 and SSEA-4, owing to their cross-reactivities with various glycosphingolipids. Such a switch was attributable to altered expression of key glycosyltransferases (GTs) in the biosynthetic pathways by the up-regulation of ganglio-series–related GTs with simultaneous down-regulation of globo- and lacto-series–related GTs. Thus, these results provide insights into the unique stage-specific transition and mechanism for alterations of GSL core structures during hESC differentiation. In addition, unique glycan structures uncovered by MS analyses may serve as surface markers for further delineation of hESCs and help identify of their functional roles not only in hESCs but also in cancers. PMID:21149694

  1. Switching of the core structures of glycosphingolipids from globo- and lacto- to ganglio-series upon human embryonic stem cell differentiation.

    Science.gov (United States)

    Liang, Yuh-Jin; Kuo, Huan-Hsien; Lin, Chi-Hung; Chen, Yen-Ying; Yang, Bei-Chia; Cheng, Yuan-Yuan; Yu, Alice L; Khoo, Kay-Hooi; Yu, John

    2010-12-28

    A systematic survey of expression profiles of glycosphingolipids (GSLs) in two hESC lines and their differentiated embryoid body (EB) outgrowth with three germ layers was carried out using immunofluorescence, flow cytometry, and MALDI-MS and MS/MS analyses. In addition to the well-known hESC-specific markers stage-specific embryonic antigen 3 (SSEA-3) and SSEA-4, we identified several globosides and lacto-series GSLs, previously unrevealed in hESCs, including Gb(4)Cer, Lc(4)Cer, fucosyl Lc(4)Cer, Globo H, and disialyl Gb(5)Cer. During hESC differentiation into EBs, MS analysis revealed a clear-cut switch in the core structures of GSLs from globo- and lacto- to ganglio-series, which was not as evident by immunostaining with antibodies against SSEA-3 and SSEA-4, owing to their cross-reactivities with various glycosphingolipids. Such a switch was attributable to altered expression of key glycosyltransferases (GTs) in the biosynthetic pathways by the up-regulation of ganglio-series-related GTs with simultaneous down-regulation of globo- and lacto-series-related GTs. Thus, these results provide insights into the unique stage-specific transition and mechanism for alterations of GSL core structures during hESC differentiation. In addition, unique glycan structures uncovered by MS analyses may serve as surface markers for further delineation of hESCs and help identify of their functional roles not only in hESCs but also in cancers.

  2. Inner core structure behind the PKP core phase triplication

    NARCIS (Netherlands)

    Blom, Nienke A.; Deuss, Arwen; Paulssen, Hanneke; Waszek, Lauren

    2015-01-01

    The structure of the Earth's inner core is not well known between depths of ∼100–200 km beneath the inner core boundary. This is a result of the PKP core phase triplication and the existence of strong precursors to PKP phases, which hinder the measurement of inner core compressional PKIKP waves at e

  3. Inner core structure behind the PKP core phase triplication

    NARCIS (Netherlands)

    Blom, Nienke A.; Deuss, Arwen; Paulssen, Hanneke; Waszek, Lauren

    The structure of the Earth's inner core is not well known between depths of ∼100–200 km beneath the inner core boundary. This is a result of the PKP core phase triplication and the existence of strong precursors to PKP phases, which hinder the measurement of inner core compressional PKIKP waves at

  4. Pressure Core Characterization

    Science.gov (United States)

    Santamarina, J. C.

    2014-12-01

    Natural gas hydrates form under high fluid pressure and low temperature, and are found in permafrost, deep lakes or ocean sediments. Hydrate dissociation by depressurization and/or heating is accompanied by a multifold hydrate volume expansion and host sediments with low permeability experience massive destructuration. Proper characterization requires coring, recovery, manipulation and testing under P-T conditions within the stability field. Pressure core technology allows for the reliable characterization of hydrate bearing sediments within the stability field in order to address scientific and engineering needs, including the measurement of parameters used in hydro-thermo-mechanical analyses, and the monitoring of hydrate dissociation under controlled pressure, temperature, effective stress and chemical conditions. Inherent sampling effects remain and need to be addressed in test protocols and data interpretation. Pressure core technology has been deployed to study hydrate bearing sediments at several locations around the world. In addition to pressure core testing, a comprehensive characterization program should include sediment analysis, testing of reconstituted specimens (with and without synthetic hydrate), and in situ testing. Pressure core characterization technology can be used to study other gas-charged formations such as deep sea sediments, coal bed methane and gas shales.

  5. GREEN CORE HOUSE

    Directory of Open Access Journals (Sweden)

    NECULAI Oana

    2017-05-01

    Full Text Available The Green Core House is a construction concept with low environmental impact, having as main central element a greenhouse. The greenhouse has the innovative role to use the biomass energy provided by plants to save energy. Although it is the central piece, the greenhouse is not the most innovative part of the Green Core House, but the whole building ensemble because it integrates many other sustainable systems as "waste purification systems", "transparent photovoltaic panels" or "double skin façades".

  6. Birefringent hollow core fibers

    DEFF Research Database (Denmark)

    Roberts, John

    2007-01-01

    Hollow core photonic crystal fiber (HC-PCF), fabricated according to a nominally non-birefringent design, shows a degree of un-controlled birefringence or polarization mode dispersion far in excess of conventional non polarization maintaining fibers. This can degrade the output pulse in many...... and an increased overlap between the polarization modes at the glass interfaces. The interplay between these effects leads to a wavelength for optimum polarization maintenance, lambda(PM), which is detuned from the wavelength of highest birefringence. By a suitable fiber design involving antiresonance of the core...

  7. Birefringent hollow core fibers

    DEFF Research Database (Denmark)

    Roberts, John

    2007-01-01

    Hollow core photonic crystal fiber (HC-PCF), fabricated according to a nominally non-birefringent design, shows a degree of un-controlled birefringence or polarization mode dispersion far in excess of conventional non polarization maintaining fibers. This can degrade the output pulse in many...... and an increased overlap between the polarization modes at the glass interfaces. The interplay between these effects leads to a wavelength for optimum polarization maintenance, lambda(PM), which is detuned from the wavelength of highest birefringence. By a suitable fiber design involving antiresonance of the core...

  8. Investigation of EAS cores

    Science.gov (United States)

    Shaulov, S. B.; Beyl, P. F.; Beysembaev, R. U.; Beysembaeva, E. A.; Bezshapov, S. P.; Borisov, A. S.; Cherdyntceva, K. V.; Chernyavsky, M. M.; Chubenko, A. P.; Dalkarov, O. D.; Denisova, V. G.; Erlykin, A. D.; Kabanova, N. V.; Kanevskaya, E. A.; Kotelnikov, K. A.; Morozov, A. E.; Mukhamedshin, R. A.; Nam, R. A.; Nesterova, N. M.; Nikolskaya, N. M.; Pavluchenko, V. P.; Piskal, V. V.; Puchkov, V. S.; Pyatovsky, S. E.; Ryabov, V. A.; Sadykov, T. Kh.; Schepetov, A. L.; Smirnova, M. D.; Stepanov, A. V.; Uryson, A. V.; Vavilov, Yu. N.; Vildanov, N. G.; Vildanova, L. I.; Zayarnaya, I. S.; Zhanceitova, J. K.; Zhukov, V. V.

    2017-06-01

    The development of nuclear-electromagnetic cascade models in air in the late forties have shown informational content of the study of cores of extensive air showers (EAS). These investigations were the main goal in different experiments which were carried out over many years by a variety of methods. Outcomes of such investigations obtained in the HADRON experiment using an X-ray emulsion chamber (XREC) as a core detector are considered. The Ne spectrum of EAS associated with γ-ray families, spectra of γ-rays (hadrons) in EAS cores and the Ne dependence of the muon number, ⟨Nμ⟩, in EAS with γ-ray families are obtained for the first time at energies of 1015-1017 eV with this method. A number of new effects were observed, namely, an abnormal scaling violation in hadron spectra which are fundamentally different from model predictions, an excess of muon number in EAS associated with γ-ray families, and the penetrating component in EAS cores. It is supposed that the abnormal behavior of γ-ray spectra and Ne dependence of the muon number are explained by the emergence of a penetrating component in the 1st PCR spectrum `knee' range. Nuclear and astrophysical explanations of the origin of the penetrating component are discussed. The necessity of considering the contribution of a single close cosmic-ray source to explain the PCR spectrum in the knee range is noted.

  9. Some Core Contested Concepts

    Science.gov (United States)

    Chomsky, Noam

    2015-01-01

    Core concepts of language are highly contested. In some cases this is legitimate: real empirical and conceptual issues arise. In other cases, it seems that controversies are based on misunderstanding. A number of crucial cases are reviewed, and an approach to language is outlined that appears to have strong conceptual and empirical motivation, and…

  10. Looking for Core Values

    Science.gov (United States)

    Carter, Margie

    2010-01-01

    People who view themselves as leaders, not just managers or teachers, are innovators who focus on clarifying core values and aligning all aspects of the organization with these values to grow their vision. A vision for an organization can't be just one person's idea. Visions grow by involving people in activities that help them name and create…

  11. Nucleosome Core Particle

    Science.gov (United States)

    1997-01-01

    Nucleosome Core Particle grown on STS-81. The fundamental structural unit of chromatin and is the basis for organization within the genome by compaction of DNA within the nucleus of the cell and by making selected regions of chromosomes available for transcription and replication. Principal Investigator's are Dr. Dan Carter and Dr. Gerard Bunick of New Century Pharmaceuticals.

  12. Investigation of EAS cores

    Directory of Open Access Journals (Sweden)

    Shaulov S.B.

    2017-01-01

    Full Text Available The development of nuclear-electromagnetic cascade models in air in the late forties have shown informational content of the study of cores of extensive air showers (EAS. These investigations were the main goal in different experiments which were carried out over many years by a variety of methods. Outcomes of such investigations obtained in the HADRON experiment using an X-ray emulsion chamber (XREC as a core detector are considered. The Ne spectrum of EAS associated with γ-ray families, spectra of γ-rays (hadrons in EAS cores and the Ne dependence of the muon number, ⟨Nμ⟩, in EAS with γ-ray families are obtained for the first time at energies of 1015–1017 eV with this method. A number of new effects were observed, namely, an abnormal scaling violation in hadron spectra which are fundamentally different from model predictions, an excess of muon number in EAS associated with γ-ray families, and the penetrating component in EAS cores. It is supposed that the abnormal behavior of γ-ray spectra and Ne dependence of the muon number are explained by the emergence of a penetrating component in the 1st PCR spectrum ‘knee’ range. Nuclear and astrophysical explanations of the origin of the penetrating component are discussed. The necessity of considering the contribution of a single close cosmic-ray source to explain the PCR spectrum in the knee range is noted.

  13. Modeling Core Collapse Supernovae

    Science.gov (United States)

    Mezzacappa, Anthony

    2017-01-01

    Core collapse supernovae, or the death throes of massive stars, are general relativistic, neutrino-magneto-hydrodynamic events. The core collapse supernova mechanism is still not in hand, though key components have been illuminated, and the potential for multiple mechanisms for different progenitors exists. Core collapse supernovae are the single most important source of elements in the Universe, and serve other critical roles in galactic chemical and thermal evolution, the birth of neutron stars, pulsars, and stellar mass black holes, the production of a subclass of gamma-ray bursts, and as potential cosmic laboratories for fundamental nuclear and particle physics. Given this, the so called ``supernova problem'' is one of the most important unsolved problems in astrophysics. It has been fifty years since the first numerical simulations of core collapse supernovae were performed. Progress in the past decade, and especially within the past five years, has been exponential, yet much work remains. Spherically symmetric simulations over nearly four decades laid the foundation for this progress. Two-dimensional modeling that assumes axial symmetry is maturing. And three-dimensional modeling, while in its infancy, has begun in earnest. I will present some of the recent work from the ``Oak Ridge'' group, and will discuss this work in the context of the broader work by other researchers in the field. I will then point to future requirements and challenges. Connections with other experimental, observational, and theoretical efforts will be discussed, as well.

  14. The Earth's Core.

    Science.gov (United States)

    Jeanloz, Raymond

    1983-01-01

    The nature of the earth's core is described. Indirect evidence (such as that determined from seismological data) indicates that it is an iron alloy, solid toward its center but otherwise liquid. Evidence also suggests that it is the turbulent flow of the liquid that generates the earth's magnetic field. (JN)

  15. Some Core Contested Concepts

    Science.gov (United States)

    Chomsky, Noam

    2015-01-01

    Core concepts of language are highly contested. In some cases this is legitimate: real empirical and conceptual issues arise. In other cases, it seems that controversies are based on misunderstanding. A number of crucial cases are reviewed, and an approach to language is outlined that appears to have strong conceptual and empirical motivation, and…

  16. Languages for Dublin Core.

    Science.gov (United States)

    Baker, Thomas

    1998-01-01

    Focusing on languages for the Dublin Core, examines the experience of some related ways to seek semantic interoperability through simplicity: planned languages, interlingua constructs, and pidgins. Also defines the conceptual and organizational problem of maintaining a metadata standard in multiple languages. (AEF)

  17. Core calculations of JMTR

    Energy Technology Data Exchange (ETDEWEB)

    Nagao, Yoshiharu [Japan Atomic Energy Research Inst., Oarai, Ibaraki (Japan). Oarai Research Establishment

    1998-03-01

    In material testing reactors like the JMTR (Japan Material Testing Reactor) of 50 MW in Japan Atomic Energy Research Institute, the neutron flux and neutron energy spectra of irradiated samples show complex distributions. It is necessary to assess the neutron flux and neutron energy spectra of an irradiation field by carrying out the nuclear calculation of the core for every operation cycle. In order to advance core calculation, in the JMTR, the application of MCNP to the assessment of core reactivity and neutron flux and spectra has been investigated. In this study, in order to reduce the time for calculation and variance, the comparison of the results of the calculations by the use of K code and fixed source and the use of Weight Window were investigated. As to the calculation method, the modeling of the total JMTR core, the conditions for calculation and the adopted variance reduction technique are explained. The results of calculation are shown. Significant difference was not observed in the results of neutron flux calculations according to the difference of the modeling of fuel region in the calculations by K code and fixed source. The method of assessing the results of neutron flux calculation is described. (K.I.)

  18. The core and cosmopolitans

    DEFF Research Database (Denmark)

    Dahlander, Linus; Frederiksen, Lars

    2012-01-01

    Users often interact and help each other solve problems in communities, but few scholars have explored how these relationships provide opportunities to innovate. We analyze the extent to which people positioned within the core of a community as well as people that are cosmopolitans positioned...

  19. Schumpeter's core works revisited

    DEFF Research Database (Denmark)

    Andersen, Esben Sloth

    2012-01-01

    This paper organises Schumpeter’s core books in three groups: the programmatic duology,the evolutionaryeconomic duology,and the socioeconomic synthesis. By analysing these groups and their interconnections from the viewpoint of modern evolutionaryeconomics,the paper summarises resolved problems...

  20. Adult educators' core competences

    DEFF Research Database (Denmark)

    Wahlgren, Bjarne

    2016-01-01

    environment as well as the kind of adult education concerned (e.g. basic education, work-related education etc.). However, it seems that it is possible to identify certain competence requirements which transcend national, cultural and functional boundaries. This research note summarises these common or “core...

  1. Adult educators' core competences

    DEFF Research Database (Denmark)

    Wahlgren, Bjarne

    2016-01-01

    environment as well as the kind of adult education concerned (e.g. basic education, work-related education etc.). However, it seems that it is possible to identify certain competence requirements which transcend national, cultural and functional boundaries. This research note summarises these common or “core...

  2. Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2 Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

    Directory of Open Access Journals (Sweden)

    Masaki Kurogochi

    Full Text Available Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain, and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC and complement dependent cytotoxicity (CDC. To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases, one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2, high-mannose type (Man4-9GlcNAc2, and complex type (Man3GlcNAc3-4 N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL, the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1 were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q, and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2 was performed using SKBR-3 and BT-474 as target

  3. Analysis of Asn-linked glycans from vegetable foodstuffs: widespread occurrence of Lewis a, core alpha1,3-linked fucose and xylose substitutions.

    Science.gov (United States)

    Wilson, I B; Zeleny, R; Kolarich, D; Staudacher, E; Stroop, C J; Kamerling, J P; Altmann, F

    2001-04-01

    The N-glycans from 27 "plant" foodstuffs, including one from a gymnospermic plant and one from a fungus, were prepared by a new procedure and examined by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For several samples, glycan structures were additionally investigated by size-fractionation and reverse-phase high-performance liquid chromatography in conjunction with exoglycosidase digests and finally also (1)H-nuclear magnetic resonance spectroscopy. The glycans found ranged from the typical vacuolar "horseradish peroxidase" type and oligomannose to complex Le(a)-carrying structures. Though the common mushroom exclusively contained N-glycans of the oligomannosidic type, all plant foods contained mixtures of the above-mentioned types. Apple, asparagus, avocado, banana, carrot, celery, hazelnut, kiwi, onion, orange, pear, pignoli, strawberry, and walnut were particularly rich in Le(a)-carrying N-glycans. Although traces of Le(a)-containing structures were also present in almond, pistachio, potato, and tomato, no such glycans could be found in cauliflower. Coconut exhibited almost exclusively N-glycans containing only xylose but no fucose. Oligomannosidic N-glycans dominated in buckwheat and especially in the legume seeds mung bean, pea, peanut, and soybean. Papaya presented a unique set of hybrid type structures partially containing the Le(a) determinant. These results are not only compatible with the hypothesis that the carbohydrate structures are another potential source of immunological cross-reaction between different plant allergens, but they also demonstrate that the Le(a)-type structure is very widespread among plants.

  4. Application of Core Dynamics Modeling to Core-Mantle Interactions

    Science.gov (United States)

    Kuang, Weijia

    2003-01-01

    Observations have demonstrated that length of day (LOD) variation on decadal time scales results from exchange of axial angular momentum between the solid mantle and the core. There are in general four core-mantle interaction mechanisms that couple the core and the mantle. Of which, three have been suggested likely the dominant coupling mechanism for the decadal core-mantle angular momentum exchange, namely, gravitational core-mantle coupling arising from density anomalies in the mantle and in the core (including the inner core), the electromagnetic coupling arising from Lorentz force in the electrically conducting lower mantle (e.g. D-layer), and the topographic coupling arising from non-hydrostatic pressure acting on the core-mantle boundary (CMB) topography. In the past decades, most effort has been on estimating the coupling torques from surface geomagnetic observations (kinematic approach), which has provided insights on the core dynamical processes. In the meantime, it also creates questions and concerns on approximations in the studies that may invalidate the corresponding conclusions. The most serious problem is perhaps the approximations that are inconsistent with dynamical processes in the core, such as inconsistencies between the core surface flow beneath the CMB and the CMB topography, and that between the D-layer electric conductivity and the approximations on toroidal field at the CMB. These inconsistencies can only be addressed with numerical core dynamics modeling. In the past few years, we applied our MoSST (Modular, Scalable, Self-consistent and Three-dimensional) core dynamics model to study core-mantle interactions together with geodynamo simulation, aiming at assessing the effect of the dynamical inconsistencies in the kinematic studies on core-mantle coupling torques. We focus on topographic and electromagnetic core-mantle couplings and find that, for the topographic coupling, the consistency between the core flow and the CMB topography is

  5. Characterization of the Ashbya gossypii secreted N-glycome and genomic insights into its N-glycosylation pathway.

    Science.gov (United States)

    Aguiar, Tatiana Q; Maaheimo, Hannu; Heiskanen, Annamari; Wiebe, Marilyn G; Penttilä, Merja; Domingues, Lucília

    2013-11-15

    The riboflavin producer Ashbya gossypii is a filamentous hemiascomycete, closely related to the yeast Saccharomyces cerevisiae, that has been used as a model organism to study fungal developmental biology. It has also been explored as a host for the expression of recombinant proteins. However, although N-glycosylation plays important roles in protein secretion, morphogenesis, and the development of multicellular organisms, the N-glycan structures synthesised by A. gossypii had not been elucidated. In this study, we report the first characterization of A. gossypii N-glycans and provide valuable insights into their biosynthetic pathway. By combined matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry profiling and nuclear magnetic resonance (NMR) spectroscopy we determined that the A. gossypii secreted N-glycome is characterized by high-mannose type structures in the range Man4-18GlcNAc2, mostly containing neutral core-type N-glycans with 8-10 mannoses. Cultivation in defined minimal media induced the production of acidic mannosylphosphorylated N-glycans, generally more elongated than the neutral N-glycans. Truncated neutral N-glycan structures similar to those found in other filamentous fungi (Man4-7GlcNAc2) were detected, suggesting the possible existence of trimming activity in A. gossypii. Homologs for all of the S. cerevisiae genes known to be involved in the endoplasmatic reticulum and Golgi N-glycan processing were found in the A. gossypii genome. However, processing of N-glycans by A. gossypii differs considerably from that by S. cerevisiae, allowing much shorter N-glycans. Genes for two putative N-glycan processing enzymes were identified, that did not have homologs in S. cerevisiae.

  6. USGS Core Research Center (CRC) Collection of Core

    Data.gov (United States)

    U.S. Geological Survey, Department of the Interior — The Core Research Center (CRC) was established in 1974 by the U.S. Geological Survey (USGS) to preserve valuable rock cores for use by scientists and educators from...

  7. Ice Cores of the National Ice Core Laboratory

    Data.gov (United States)

    U.S. Geological Survey, Department of the Interior — The U.S. National Ice Core Laboratory (NICL) is a facility for storing, curating, and studying ice cores recovered from the polar regions of the world. It provides...

  8. Glycomic analysis of tear and saliva in ocular rosacea patients: the search for a biomarker.

    Science.gov (United States)

    Vieira, Ana Carolina; An, Hyun Joo; Ozcan, Sureyya; Kim, Jae-Han; Lebrilla, Carlito B; Mannis, Mark J

    2012-07-01

    The purpose of this study was to study changes in glycosylation in tear and saliva obtained from control and ocular rosacea patients in order to identify potential biomarkers for rosacea. Tear fluid was collected from 51 subjects (28 healthy controls and 23 patients with ocular rosacea). Saliva was collected from 42 of the same subjects (25 controls and 17 patients). Pooled and individual samples were examined to determine overall glycan profiles and individual variations in glycosylation. O-and N- glycans were released from both patients and control subjects. Released glycans were purified and enriched by solid-phase extraction (SPE) with graphitized carbon. Glycans were eluted based on glycan size and polarity. SPE fractions were then analyzed by high-resolution mass spectrometry. Glycan compositions were assigned by accurate masses. Their structures were further elucidated by tandem mass spectrometric using collision-induced dissociation (CID), and specific linkage information was obtained by exoglycosidase digestion. N- and O-glycans were released from 20-μL samples without protein identification, separation, and purification. Approximately 50 N-glycans and 70 O-glycans were globally profiled by mass spectrometry. Most N-glycans were highly fucosylated, while O-glycans were sulfated. Normal tear fluid and saliva contain highly fucosylated glycans. The numbers of sulfated glycans were dramatically increased in tear and saliva of rosacea patients compared to controls. Glycans found in tear and saliva from roseatic patients present highly quantitative similarity. The abundance of highly fucosylated N-glycans in the control samples and sulfated O-glycans in ocular rosacea patient samples may lead to the discovery of an objective diagnostic marker for the disease.

  9. Core Outlet Temperature Study

    Energy Technology Data Exchange (ETDEWEB)

    Moisseytsev, A. [Argonne National Laboratory (ANL), Argonne, IL (United States); Hoffman, E. [Argonne National Laboratory (ANL), Argonne, IL (United States); Majumdar, S. [Argonne National Laboratory (ANL), Argonne, IL (United States)

    2008-07-28

    It is a known fact that the power conversion plant efficiency increases with elevation of the heat addition temperature. The higher efficiency means better utilization of the available resources such that higher output in terms of electricity production can be achieved for the same size and power of the reactor core or, alternatively, a lower power core could be used to produce the same electrical output. Since any nuclear power plant, such as the Advanced Burner Reactor, is ultimately built to produce electricity, a higher electrical output is always desirable. However, the benefits of the higher efficiency and electricity production usually come at a price. Both the benefits and the disadvantages of higher reactor outlet temperatures are analyzed in this work.

  10. Geomagnetism of earth's core

    Science.gov (United States)

    Benton, E. R.

    1983-01-01

    Instrumentation, analytical methods, and research goals for understanding the behavior and source of geophysical magnetism are reviewed. Magsat, launched in 1979, collected global magnetometer data and identified the main terrestrial magnetic fields. The data has been treated by representing the curl-free field in terms of a scalar potential which is decomposed into a truncated series of spherical harmonics. Solutions to the Laplace equation then extend the field upward or downward from the measurement level through intervening spaces with no source. Further research is necessary on the interaction between harmonics of various spatial scales. Attempts are also being made to analytically model the main field and its secular variation at the core-mantle boundary. Work is also being done on characterizing the core structure, composition, thermodynamics, energetics, and formation, as well as designing a new Magsat or a tethered satellite to be flown on the Shuttle.

  11. Geomagnetism of earth's core

    Science.gov (United States)

    Benton, E. R.

    1983-01-01

    Instrumentation, analytical methods, and research goals for understanding the behavior and source of geophysical magnetism are reviewed. Magsat, launched in 1979, collected global magnetometer data and identified the main terrestrial magnetic fields. The data has been treated by representing the curl-free field in terms of a scalar potential which is decomposed into a truncated series of spherical harmonics. Solutions to the Laplace equation then extend the field upward or downward from the measurement level through intervening spaces with no source. Further research is necessary on the interaction between harmonics of various spatial scales. Attempts are also being made to analytically model the main field and its secular variation at the core-mantle boundary. Work is also being done on characterizing the core structure, composition, thermodynamics, energetics, and formation, as well as designing a new Magsat or a tethered satellite to be flown on the Shuttle.

  12. Dynamics of core accretion

    Science.gov (United States)

    Nelson, Andrew F.; Ruffert, Maximilian

    2013-02-01

    We perform three-dimensional hydrodynamic simulations of gas flowing around a planetary core of mass Mpl = 10M⊕ embedded in a near Keplerian background flow, using a modified shearing box approximation. We assume an ideal gas behaviour following an equation of state with a fixed ratio of the specific heats, γ = 1.42, consistent with the conditions of a moderate-temperature background disc with solar composition. No radiative heating or cooling is included in the models. We employ a nested grid hydrodynamic code implementing the `Piecewise Parabolic Method' with as many as six fixed nested grids, providing spatial resolution on the finest grid comparable to the present-day diameters of Neptune and Uranus. We find that a strongly dynamically active flow develops such that no static envelope can form. The activity is not sensitive to plausible variations in the rotation curve of the underlying disc. It is sensitive to the thermodynamic treatment of the gas, as modelled by prescribed equations of state (either `locally isothermal' or `locally isentropic') and the temperature of the background disc material. The activity is also sensitive to the shape and depth of the core's gravitational potential, through its mass and gravitational softening coefficient. Each of these factors influences the magnitude and character of hydrodynamic feedback of the small-scale flow on the background, and we conclude that accurate modelling of such feedback is critical to a complete understanding of the core accretion process. The varying flow pattern gives rise to large, irregular eruptions of matter from the region around the core which return matter to the background flow: mass in the envelope at one time may not be found in the envelope at any later time. No net mass accretion into the envelope is observed over the course of the simulation and none is expected, due to our neglect of cooling. Except in cases of very rapid cooling however, as defined by locally isothermal or

  13. Some core contested concepts.

    Science.gov (United States)

    Chomsky, Noam

    2015-02-01

    Core concepts of language are highly contested. In some cases this is legitimate: real empirical and conceptual issues arise. In other cases, it seems that controversies are based on misunderstanding. A number of crucial cases are reviewed, and an approach to language is outlined that appears to have strong conceptual and empirical motivation, and to lead to conclusions about a number of significant issues that differ from some conventional beliefs.

  14. Identification of Antigenic Glycans from Schistosoma mansoni by Using a Shotgun Egg Glycan Microarray

    Science.gov (United States)

    Mickum, Megan L.; Prasanphanich, Nina Salinger; Song, Xuezheng; Dorabawila, Nelum; Mandalasi, Msano; Lasanajak, Yi; Luyai, Anthony; Secor, W. Evan; Wilkins, Patricia P.; Van Die, Irma; Smith, David F.; Nyame, A. Kwame

    2016-01-01

    Infection of mammals by the parasitic helminth Schistosoma mansoni induces antibodies to glycan antigens in worms and eggs, but the differential nature of the immune response among infected mammals is poorly understood. To better define these responses, we used a shotgun glycomics approach in which N-glycans from schistosome egg glycoproteins were prepared, derivatized, separated, and used to generate an egg shotgun glycan microarray. This array was interrogated with sera from infected mice, rhesus monkeys, and humans and with glycan-binding proteins and antibodies to gather information about the structures of antigenic glycans, which also were analyzed by mass spectrometry. A major glycan antigen targeted by IgG from different infected species is the FLDNF epitope [Fucα3GalNAcβ4(Fucα3)GlcNAc-R], which is also recognized by the IgG monoclonal antibody F2D2. The FLDNF antigen is expressed by all life stages of the parasite in mammalian hosts, and F2D2 can kill schistosomula in vitro in a complement-dependent manner. Different antisera also recognized other glycan determinants, including core β-xylose and highly fucosylated glycans. Thus, the natural shotgun glycan microarray of schistosome eggs is useful in identifying antigenic glycans and in developing new anti-glycan reagents that may have diagnostic applications and contribute to developing new vaccines against schistosomiasis. PMID:26883596

  15. Central core disease

    Directory of Open Access Journals (Sweden)

    Jungbluth Heinz

    2007-05-01

    Full Text Available Abstract Central core disease (CCD is an inherited neuromuscular disorder characterised by central cores on muscle biopsy and clinical features of a congenital myopathy. Prevalence is unknown but the condition is probably more common than other congenital myopathies. CCD typically presents in infancy with hypotonia and motor developmental delay and is characterized by predominantly proximal weakness pronounced in the hip girdle; orthopaedic complications are common and malignant hyperthermia susceptibility (MHS is a frequent complication. CCD and MHS are allelic conditions both due to (predominantly dominant mutations in the skeletal muscle ryanodine receptor (RYR1 gene, encoding the principal skeletal muscle sarcoplasmic reticulum calcium release channel (RyR1. Altered excitability and/or changes in calcium homeostasis within muscle cells due to mutation-induced conformational changes of the RyR protein are considered the main pathogenetic mechanism(s. The diagnosis of CCD is based on the presence of suggestive clinical features and central cores on muscle biopsy; muscle MRI may show a characteristic pattern of selective muscle involvement and aid the diagnosis in cases with equivocal histopathological findings. Mutational analysis of the RYR1 gene may provide genetic confirmation of the diagnosis. Management is mainly supportive and has to anticipate susceptibility to potentially life-threatening reactions to general anaesthesia. Further evaluation of the underlying molecular mechanisms may provide the basis for future rational pharmacological treatment. In the majority of patients, weakness is static or only slowly progressive, with a favourable long-term outcome.

  16. Core-seis: a code for LMFBR core seismic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Chellapandi, P.; Ravi, R.; Chetal, S.C.; Bhoje, S.B. [Indira Gandhi Centre for Atomic Research, Kalpakkam (India). Reactor Group

    1995-12-31

    This paper deals with a computer code CORE-SEIS specially developed for seismic analysis of LMFBR core configurations. For demonstrating the prediction capability of the code, results are presented for one of the MONJU reactor core mock ups which deals with a cluster of 37 subassemblies kept in water. (author). 3 refs., 7 figs., 2 tabs.

  17. Core Exercises: Why You Should Strengthen Your Core Muscles

    Science.gov (United States)

    Healthy Lifestyle Fitness You know core exercises are good for you — but do you include core exercises in your fitness routine? Here's why ... 18, 2014 Original article: http://www.mayoclinic.org/healthy-lifestyle/fitness/in-depth/core-exercises/art-20044751 . Mayo Clinic Footer Legal Conditions and ...

  18. ICF Core Sets for stroke

    National Research Council Canada - National Science Library

    Szilvia Geyh; Alarcos Cieza; Jan Schouten; Hugh Dickson; Peter Frommelt; Zaliha Omar; Nenad Kostanjsek; Haim Ring; Gerold Stucki

    2004-01-01

    Objective: To report on the results of the consensus process integrating evidence from preliminary studies to develop the first version of the Comprehensive ICF Core Set and the Brief ICF Core Set for stroke. Methods...

  19. Hollow-Core Fiber Lamp

    Science.gov (United States)

    Yi, Lin (Inventor); Tjoelker, Robert L. (Inventor); Burt, Eric A. (Inventor); Huang, Shouhua (Inventor)

    2016-01-01

    Hollow-core capillary discharge lamps on the millimeter or sub-millimeter scale are provided. The hollow-core capillary discharge lamps achieve an increased light intensity ratio between 194 millimeters (useful) and 254 millimeters (useless) light than conventional lamps. The capillary discharge lamps may include a cone to increase light output. Hollow-core photonic crystal fiber (HCPCF) may also be used.

  20. On core stability and extendability

    OpenAIRE

    Shellshear, Evan

    2007-01-01

    This paper investigates conditions under which the core of a TU cooperative game is stable. In particular the author extends the idea of extendability to find new conditions under which the core is stable. It is also shown that these new conditions are not necessary for core stability.

  1. Dual-core Itanium Processor

    CERN Multimedia

    2006-01-01

    Intel’s first dual-core Itanium processor, code-named "Montecito" is a major release of Intel's Itanium 2 Processor Family, which implements the Intel Itanium architecture on a dual-core processor with two cores per die (integrated circuit). Itanium 2 is much more powerful than its predecessor. It has lower power consumption and thermal dissipation.

  2. On core stability and extendability

    OpenAIRE

    Shellshear, Evan

    2007-01-01

    This paper investigates conditions under which the core of a TU cooperative game is stable. In particular the author extends the idea of extendability to find new conditions under which the core is stable. It is also shown that these new conditions are not necessary for core stability.

  3. Winning Cores in Parity Games

    DEFF Research Database (Denmark)

    Vester, Steen

    2016-01-01

    We introduce the novel notion of winning cores in parity games and develop a deterministic polynomial-time under-approximation algorithm for solving parity games based on winning core approximation. Underlying this algorithm are a number properties about winning cores which are interesting...... in their own right. In particular, we show that the winning core and the winning region for a player in a parity game are equivalently empty. Moreover, the winning core contains all fatal attractors but is not necessarily a dominion itself. Experimental results are very positive both with respect to quality...

  4. Loss of α1,6-fucosyltransferase suppressed liver regeneration: implication of core fucose in the regulation of growth factor receptor-mediated cellular signaling.

    Science.gov (United States)

    Wang, Yuqin; Fukuda, Tomohiko; Isaji, Tomoya; Lu, Jishun; Gu, Wei; Lee, Ho-Hsun; Ohkubo, Yasuhito; Kamada, Yoshihiro; Taniguchi, Naoyuki; Miyoshi, Eiji; Gu, Jianguo

    2015-02-05

    Core fucosylation is an important post-translational modification, which is catalyzed by α1,6-fucosyltransferase (Fut8). Increased expression of Fut8 has been shown in diverse carcinomas including hepatocarcinoma. In this study, we investigated the role of Fut8 expression in liver regeneration by using the 70% partial hepatectomy (PH) model, and found that Fut8 is also critical for the regeneration of liver. Interestingly, we show that the Fut8 activities were significantly increased in the beginning of PH (~4d), but returned to the basal level in the late stage of PH. Lacking Fut8 led to delayed liver recovery in mice. This retardation mainly resulted from suppressed hepatocyte proliferation, as supported not only by a decreased phosphorylation level of epidermal growth factor (EGF) receptor and hepatocyte growth factor (HGF) receptor in the liver of Fut8(-/-) mice in vivo, but by the reduced response to exogenous EGF and HGF of the primary hepatocytes isolated from the Fut8(-/-) mice. Furthermore, an administration of L-fucose, which can increase GDP-fucose synthesis through a salvage pathway, significantly rescued the delayed liver regeneration of Fut8(+/-) mice. Overall, our study provides the first direct evidence for the involvement of Fut8 in liver regeneration.

  5. Models of the earth's core

    Science.gov (United States)

    Stevenson, D. J.

    1981-01-01

    Combined inferences from seismology, high-pressure experiment and theory, geomagnetism, fluid dynamics, and current views of terrestrial planetary evolution lead to models of the earth's core with five basic properties. These are that core formation was contemporaneous with earth accretion; the core is not in chemical equilibrium with the mantle; the outer core is a fluid iron alloy containing significant quantities of lighter elements and is probably almost adiabatic and compositionally uniform; the more iron-rich inner solid core is a consequence of partial freezing of the outer core, and the energy release from this process sustains the earth's magnetic field; and the thermodynamic properties of the core are well constrained by the application of liquid-state theory to seismic and labroatory data.

  6. A pilot study of salivary N-glycome in HBV-induced chronic hepatitis, cirrhosis, and hepatocellular carcinoma.

    Science.gov (United States)

    Qin, Yannan; Zhong, Yaogang; Ma, Tianran; Zhang, Jiaxu; Yang, Ganglong; Guan, Feng; Li, Zheng; Li, Baozhen

    2017-08-01

    Hepatitis B is a potentially life-threatening liver infection caused by the hepatitis B virus (HBV), which can lead to chronic liver disease and put people at high risk of death from cirrhosis of the liver and liver cancer. However, little is known about the correlation of salivary N-linked glycans related to HBV-infected liver diseases. Here we investigated N-linked glycome in saliva from 200 subjects (50 healthy volunteers (HV), 40 HBV-infected patients (HB), 50 cirrhosis patients (HC), and 60 hepatocellular carcinoma patients (HCC) using MALDI-TOF/TOF-MS. Representative MS spectra of N-glycans with signal-to-noise ratios >6 were annotated using the GlycoWorkbench program. A total of 40, 47, 29, and 33 N-glycan peaks were identified and annotated from HV, HB, HC, and HCC groups, respectively. There were 15 N-glycan peaks (e.g., m/z 1647.587, 1688.613 and 2101.755) were present in all groups. Three N-glycan peaks (m/z 2596.925, 2756.962, and 2921.031) were unique in HV group, 2 N-glycan peaks (m/z 1898.676 and 1971.692) were unique in HB group, 5 N-glycan peaks (m/z 1954.677, 2507.914, 2580.930, 2637.952, and 3092.120) were unique in HC group, and 3 N-glycan peaks (m/z 2240.830, 2507.914, and 3931.338) were unique in HCC group. The proportion of fucosylated N-glycans was apparently increased in the HCC group (84.8%) than in any other group (73.1% ± 0.01), however, the proportion of sialylated N-glycans was decreased in HCC group (12.1%) than in any other group (17.23% ± 0.003). Our data provide pivotal information to distinguish between HBV-associated hepatitis, cirrhosis and HCC, and facilitate the discovery of biomarkers for HCC during its early stages based on precise alterations of N-linked glycans in saliva.

  7. Complex coacervate core micelles.

    Science.gov (United States)

    Voets, Ilja K; de Keizer, Arie; Cohen Stuart, Martien A

    2009-01-01

    In this review we present an overview of the literature on the co-assembly of neutral-ionic block, graft, and random copolymers with oppositely charged species in aqueous solution. Oppositely charged species include synthetic (co)polymers of various architectures, biopolymers - such as proteins, enzymes and DNA - multivalent ions, metallic nanoparticles, low molecular weight surfactants, polyelectrolyte block copolymer micelles, metallo-supramolecular polymers, equilibrium polymers, etcetera. The resultant structures are termed complex coacervate core/polyion complex/block ionomer complex/interpolyelectrolyte complex micelles (or vesicles); i.e., in short C3Ms (or C3Vs) and PIC, BIC or IPEC micelles (and vesicles). Formation, structure, dynamics, properties, and function will be discussed. We focus on experimental work; theory and modelling will not be discussed. Recent developments in applications and micelles with heterogeneous coronas are emphasized.

  8. Understanding core conductor fabrics

    Energy Technology Data Exchange (ETDEWEB)

    Swenson, D E, E-mail: deswenson@affinity-esd.com [Affinity Static Control Consulting, LLC 2609 Quanah Drive, Round Rock, Texas, 78681 (United States)

    2011-06-23

    ESD Association standard test method ANSI/ESD STM2.1 - Garments (STM2.1), provides electrical resistance test procedures that are applicable for materials and garments that have surface conductive or surface dissipative properties. As has been reported in other papers over the past several years{sup 1} fabrics are now used in many industries for electrostatic control purposes that do not have surface conductive properties and therefore cannot be evaluated using the procedures in STM2.1{sup 2}. A study was conducted to compare surface conductive fabrics with samples of core conductor fibre based fabrics in order to determine differences and similarities with regards to various electrostatic properties. This work will be used to establish a new work item proposal within WG-2, Garments, in the ESD Association Standards Committee in the USA.

  9. Growth outside the core.

    Science.gov (United States)

    Zook, Chris; Allen, James

    2003-12-01

    Growth in an adjacent market is tougher than it looks; three-quarters of the time, the effort fails. But companies can change those odds dramatically. Results from a five-year study of corporate growth conducted by Bain & Company reveal that adjacency expansion succeeds only when built around strong core businesses that have the potential to become market leaders. And the best place to look for adjacency opportunities is inside a company's strongest customers. The study also found that the most successful companies were able to consistently, profitably outgrow their rivals by developing a formula for pushing out the boundaries of their core businesses in predictable, repeatable ways. Companies use their repeatability formulas to expand into any number of adjacencies. Some companies make repeated geographic moves, as Vodafone has done in expanding from one geographic market to another over the past 13 years, building revenues from $1 billion in 1990 to $48 billion in 2003. Others apply a superior business model to new segments. Dell, for example, has repeatedly adapted its direct-to-customer model to new customer segments and new product categories. In other cases, companies develop hybrid approaches. Nike executed a series of different types of adjacency moves: it expanded into adjacent customer segments, introduced new products, developed new distribution channels, and then moved into adjacent geographic markets. The successful repeaters in the study had two common characteristics. First, they were extraordinarily disciplined, applying rigorous screens before they made an adjacency move. This discipline paid off in the form of learning curve benefits, increased speed, and lower complexity. And second, in almost all cases, they developed their repeatable formulas by studying their customers and their customers' economics very, very carefully.

  10. Advances in core drilling technology

    Science.gov (United States)

    Holdsworth, G.

    Some notable technical advances in drill design were reported at the meeting, held in Canada August 30-September 1, 1982, at the University of Calgary. Chief amongst these was a battery powered, computer assisted electromechanical core drill which has recently been used by the Danes in Greenland to continuously core to the base of the ice sheet at 2038 m. This is the deepest coring operation so far on the Greenland ice sheet. (The record for deep glacier drilling is held by the U.S. Army Cold Regions Research and Engineering Laboratory for the continuous coring through 2164 m of ice to bedrock at Byrd Station, Antarctica, in 1968). In early 1982, a current Soviet core drilling operation was reported to be at a depth of 2000 m at Vostok station, Antarctica, where the total ice thickness is about 4000 m; the goal of core drilling the entire ice thickness there could be achieved before the end of 1983.

  11. Core TuLiP

    OpenAIRE

    Czenko, M.R.; Etalle, Sandro

    2007-01-01

    We propose CoreTuLiP - the core of a trust management language based on Logic Programming. CoreTuLiP is based on a subset of moded logic programming, but enjoys the features of TM languages such as RT; in particular clauses are issued by different authorities and stored in a distributed manner. We present a lookup and inference algorithm which we prove to be correct and complete w.r.t. the declarative semantics. CoreTuLiP enjoys uniform syntax and the well-established semantics and is express...

  12. Biochemistry Instrumentation Core Technology Center

    Data.gov (United States)

    Federal Laboratory Consortium — The UCLA-DOE Biochemistry Instrumentation Core Facility provides the UCLA biochemistry community with easy access to sophisticated instrumentation for a wide variety...

  13. Waves in the core and mechanical core-mantle interactions

    DEFF Research Database (Denmark)

    Jault, D.; Finlay, Chris

    2015-01-01

    the motions in the direction parallel to the Earth'srotation axis. This property accounts for the signicance of the core-mantle topography.In addition, the stiening of the uid in the direction parallel to the rotation axis gives riseto a magnetic diusion layer attached to the core-mantle boundary, which would...

  14. Characterizing the Core via K-Core Covers

    NARCIS (Netherlands)

    Sanchez, S.M.; Borm, P.E.M.; Estevez, A.

    2013-01-01

    This paper extends the notion of individual minimal rights for a transferable utility game (TU-game) to coalitional minimal rights using minimal balanced families of a specific type, thus defining a corresponding minimal rights game. It is shown that the core of a TU-game coincides with the core of

  15. Characterizing the Core via K-Core Covers

    NARCIS (Netherlands)

    Sanchez, S.M.; Borm, P.E.M.; Estevez, A.

    2013-01-01

    This paper extends the notion of individual minimal rights for a transferable utility game (TU-game) to coalitional minimal rights using minimal balanced families of a specific type, thus defining a corresponding minimal rights game. It is shown that the core of a TU-game coincides with the core of

  16. Relativistic frozen core potential scheme with relaxation of core electrons

    Science.gov (United States)

    Nakajima, Yuya; Seino, Junji; Hayami, Masao; Nakai, Hiromi

    2016-10-01

    This letter proposes a relaxation scheme for core electrons based on the frozen core potential method at the infinite-order Douglas-Kroll-Hess level, called FCP-CR. The core electrons are self-consistently relaxed using frozen molecular valence potentials after the valence SCF calculation is performed. The efficiency of FCP-CR is confirmed by calculations of gold clusters. Furthermore, FCP-CR reproduces the results of the all-electron method for the energies of coinage metal dimers and the core ionization energies and core level shifts of vinyl acetate and three tungsten complexes at the Hartree-Fock and/or symmetry-adapted cluster configuration interaction levels.

  17. Adult educators' core competences

    Science.gov (United States)

    Wahlgren, Bjarne

    2016-06-01

    Which competences do professional adult educators need? This research note discusses the topic from a comparative perspective, finding that adult educators' required competences are wide-ranging, heterogeneous and complex. They are subject to context in terms of national and cultural environment as well as the kind of adult education concerned (e.g. basic education, work-related education etc.). However, it seems that it is possible to identify certain competence requirements which transcend national, cultural and functional boundaries. This research note summarises these common or "core" requirements, organising them into four thematic subcategories: (1) communicating subject knowledge; (2) taking students' prior learning into account; (3) supporting a learning environment; and (4) the adult educator's reflection on his or her own performance. At the end of his analysis of different competence profiles, the author notes that adult educators' ability to train adult learners in a way which then enables them to apply and use what they have learned in practice (thus performing knowledge transfer) still seems to be overlooked.

  18. Core of Cloud Computing

    Directory of Open Access Journals (Sweden)

    Prof. C.P.Chandgude

    2017-04-01

    Full Text Available Advancement in computing facilities marks back from 1960’s with introduction of mainframes. Each of the computing has one or the other issues, so keeping this in mind cloud computing was introduced. Cloud computing has its roots in older technologies such as hardware virtualization, distributed computing, internet technologies, and autonomic computing. Cloud computing can be described with two models, one is service model and second is deployment model. While providing several services, cloud management’s primary role is resource provisioning. While there are several such benefits of cloud computing, there are challenges in adopting public clouds because of dependency on infrastructure that is shared by many enterprises. In this paper, we present core knowledge of cloud computing, highlighting its key concepts, deployment models, service models, benefits as well as security issues related to cloud data. The aim of this paper is to provide a better understanding of the cloud computing and to identify important research directions in this field

  19. Maximum stellar iron core mass

    Indian Academy of Sciences (India)

    F W Giacobbe

    2003-03-01

    An analytical method of estimating the mass of a stellar iron core, just prior to core collapse, is described in this paper. The method employed depends, in part, upon an estimate of the true relativistic mass increase experienced by electrons within a highly compressed iron core, just prior to core collapse, and is significantly different from a more typical Chandrasekhar mass limit approach. This technique produced a maximum stellar iron core mass value of 2.69 × 1030 kg (1.35 solar masses). This mass value is very near to the typical mass values found for neutron stars in a recent survey of actual neutron star masses. Although slightly lower and higher neutron star masses may also be found, lower mass neutron stars are believed to be formed as a result of enhanced iron core compression due to the weight of non-ferrous matter overlying the iron cores within large stars. And, higher mass neutron stars are likely to be formed as a result of fallback or accretion of additional matter after an initial collapse event involving an iron core having a mass no greater than 2.69 × 1030 kg.

  20. The INTEGRAL Core Observing Programme

    OpenAIRE

    Winkler, C; Gehrels, N.; Lund, N.; Schoenfelder, V.; Ubertini, P.

    1999-01-01

    The Core Programme of the INTEGRAL mission is defined as the portion of the scientific programme covering the guaranteed time observations for the INTEGRAL Science Working Team. This paper describes the current status of the Core Programme preparations and summarizes the key elements of the observing programme.

  1. The INTEGRAL Core Observing Programme

    DEFF Research Database (Denmark)

    Winkler, C.; Gehrels, N.; Lund, Niels

    1999-01-01

    The Core Programme of the INTEGRAL mission is defined as the portion of the scientific programme covering the guaranteed time observations for the INTEGRAL Science Working Team. This paper describes the current status of the Core Programme preparations and summarizes the key elements...

  2. Complicated Politics to the Core

    Science.gov (United States)

    McGuinn, Patrick

    2015-01-01

    People dislike the Common Core for several different reasons, and so it is important to disaggregate the sources of opposition and to assess and then to dispel some of the myths that have built up around it. It also is important to understand the unusual political alliances that have emerged in opposition to Common Core implementation and how they…

  3. Common Core: Victory Is Yours!

    Science.gov (United States)

    Fink, Jennifer L. W.

    2012-01-01

    In this article, the author discusses how to implement the Common Core State Standards in the classroom. She presents examples and activities that will leave teachers feeling "rosy" about tackling the new standards. She breaks down important benchmarks and shows how other teachers are doing the Core--and loving it!

  4. The INTEGRAL Core Observing Programme

    DEFF Research Database (Denmark)

    Winkler, C.; Gehrels, N.; Lund, Niels

    1999-01-01

    The Core Programme of the INTEGRAL mission is defined as the portion of the scientific programme covering the guaranteed time observations for the INTEGRAL Science Working Team. This paper describes the current status of the Core Programme preparations and summarizes the key elements...

  5. Complicated Politics to the Core

    Science.gov (United States)

    McGuinn, Patrick

    2015-01-01

    People dislike the Common Core for several different reasons, and so it is important to disaggregate the sources of opposition and to assess and then to dispel some of the myths that have built up around it. It also is important to understand the unusual political alliances that have emerged in opposition to Common Core implementation and how they…

  6. Core body temperature in obesity.

    Science.gov (United States)

    Heikens, Marc J; Gorbach, Alexander M; Eden, Henry S; Savastano, David M; Chen, Kong Y; Skarulis, Monica C; Yanovski, Jack A

    2011-05-01

    A lower core body temperature set point has been suggested to be a factor that could potentially predispose humans to develop obesity. We tested the hypothesis that obese individuals have lower core temperatures than those in normal-weight individuals. In study 1, nonobese [body mass index (BMI; in kg/m(2)) temperature-sensing capsules, and we measured core temperatures continuously for 24 h. In study 2, normal-weight (BMI of 18-25) and obese subjects swallowed temperature-sensing capsules to measure core temperatures continuously for ≥48 h and kept activity logs. We constructed daily, 24-h core temperature profiles for analysis. Mean (±SE) daily core body temperature did not differ significantly between the 35 nonobese and 46 obese subjects (36.92 ± 0.03°C compared with 36.89 ± 0.03°C; P = 0.44). Core temperature 24-h profiles did not differ significantly between 11 normal-weight and 19 obese subjects (P = 0.274). Women had a mean core body temperature ≈0.23°C greater than that of men (36.99 ± 0.03°C compared with 36.76 ± 0.03°C; P body temperature. It may be necessary to study individuals with function-altering mutations in core temperature-regulating genes to determine whether differences in the core body temperature set point affect the regulation of human body weight. These trials were registered at clinicaltrials.gov as NCT00428987 and NCT00266500.

  7. COSIS: COre State Indication System

    Energy Technology Data Exchange (ETDEWEB)

    Cho, J. Y.; Lee, K. B.; Koo, B. S.; Lee, W. K.; Lee, C. C; Zee, S. Q

    2006-02-15

    COSIS (COre State Indication System) which implemented in the SMART research reactor plays a role to supply the core state parameters or graphs for the operator to recognize the core state effectively. The followings are the main functions of COSIS. (1) Validity Check for the Process Signals and Determination of the COSIS Inputs (SIGVAL) (2) Coolant Flow Rate Calculation (FLOW) (3) Core Thermal Power Calculation (COREPOW) (4) In-core 3-Dimensional Power Distribution Calculation and Peaking Parameters Generation (POWER3D) (5) Azimuthal Tilt Calculation (AZITILT). This report describes the methodology of COSIS which produces the core state parameters using the process and detector signals. In the SIGVAL module, COSIS checks most signals except for the CEA position and determines the input signals. In the FLOW module, the corelation coefficient between the RPM signal and coolant flow is updated from the energy balance at the steam generator, and the coolant flow rate is calculated using the RPM signal. In the COREPOW module, the secondary calorimetric power, the primary {delta}T power and the ex-core power are calculated, and the final core thermal power and biased core power are determined. In the POWER3D module, the 3-dimensional power distribution is calculated using the in-core detector signal, and the 3-D peaking factor, 2-D radial peaking factor, axial offset, maximum linear power density are produced. In the AZITILT module, the arithmetic averaged and vector averaged azimuthal tilts are calculated, and the final tilt is determined. The COSIS performance test of the COSIS is performed for the temperature compensation method, the COREPOW and the POWER3D modules. The test for the temperature compensation method is performed for the temperature variations of the linear, parabolic, exponential, sine function. The test shows that the implemented temperature compensation method works soundly. The COREPOW test is performed by varying the core power from the initial

  8. Epistemology and ontology in core ontologies: FOLaw and LRI-Core, two core ontologies for law

    NARCIS (Netherlands)

    Breukers, J.A.P.J.; Hoekstra, R.J.

    2004-01-01

    For more than a decade constructing ontologies for legal domains, we, at the Leibniz Center for Law, felt really the need to develop a core ontology for law that would enable us to re-use the common denominator of the various legal domains. In this paper we present two core ontologies for law. The

  9. Identification of Haloferax volcanii Pilin N-Glycans with Diverse Roles in Pilus Biosynthesis, Adhesion, and Microcolony Formation.

    Science.gov (United States)

    Esquivel, Rianne N; Schulze, Stefan; Xu, Rachel; Hippler, Michael; Pohlschroder, Mechthild

    2016-05-13

    N-Glycosylation is a post-translational modification common to all three domains of life. In many archaea, the oligosacharyltransferase (AglB)-dependent N-glycosylation of flagellins is required for flagella assembly. However, whether N-glycosylation is required for the assembly and/or function of the structurally related archaeal type IV pili is unknown. Here, we show that of six Haloferax volcanii adhesion pilins, PilA1 and PilA2, the most abundant pilins in pili of wild-type and ΔaglB strains, are modified under planktonic conditions in an AglB-dependent manner by the same pentasaccharide detected on H. volcanii flagellins. However, unlike wild-type cells, which have surfaces decorated with discrete pili and form a dispersed layer of cells on a plastic surface, ΔaglB cells have thick pili bundles and form microcolonies. Moreover, expressing PilA1, PilA2, or PilA6 in ΔpilA[1-6]ΔaglB stimulates microcolony formation compared with their expression in ΔpilA[1-6]. Conversely, expressing PilA3 or PilA4 in ΔpilA[1-6] cells results in strong surface adhesion, but not microcolony formation, and neither pilin stimulates surface adhesion in ΔpilA[1-6]ΔaglB cells. Although PilA4 assembles into pili in the ΔpilA[1-6]ΔaglB cells, these pili are, unlike wild-type pili, curled, perhaps rendering them non-functional. To our knowledge, this is the first demonstration of a differential effect of glycosylation on pilus assembly and function of paralogous pilins. The growth of wild-type cells in low salt media, a condition that decreases AglB glycosylation, also stimulates microcolony formation and inhibits motility, supporting our hypothesis that N-glycosylation plays an important role in regulating the transition between planktonic to sessile cell states as a response to stress.

  10. Characterization of N-Glycan Structures on the Surface of Mature Dengue 2 Virus Derived from Insect Cells: e0132122

    National Research Council Canada - National Science Library

    Y Lei; H Yu; Y Dong; J Yang; W Ye; Y Wang; W Chen; Z Jia; Z Xu; Z Li; F Zhang

    2015-01-01

      DENV envelope glycoprotein (E) is responsible for interacting with host cell receptors and is the main target for the development of a dengue vaccine based on an induction of neutralizing antibodies...

  11. Characterization of N-Glycan Structures on the Surface of Mature Dengue 2 Virus Derived from Insect Cells

    National Research Council Canada - National Science Library

    Lei, Y; Yu, H; Dong, Y; Yang, J; Ye, W; Wang, Y; Chen, W; Jia, Z; Xu, Z; Li, Z; Zhang, F

    2015-01-01

    DENV envelope glycoprotein (E) is responsible for interacting with host cell receptors and is the main target for the development of a dengue vaccine based on an induction of neutralizing antibodies...

  12. Proton nuclear magnetic resonance spectroscopic detection of oligomannosidic n glycans in alpha-mannosidosis: a method of monitoring treatment.

    Science.gov (United States)

    Avenarius, Derk Frederik Matthaus; Svendsen, John-Sigurd; Malm, Dag

    2011-10-01

    In Alpha-mannosidosis (MIM 248500) the patients accumulate mainly unbranched oligosaccharide chains in the lysosomes in all body tissues, including the brain. With ensuing therapeutic modalities in man (BMT and ERT) non-invasive methods of monitoring the effect of treatment are needed. Paramount is the possible effect of the treatment on the brain, since this organ is regarded as difficult to reach because of the blood-brain barrier. We therefore performed proton nuclear magnetic resonance spectroscopy (MRS) of the brain in two untreated patients, and a 16-year-old patient treated with BMT at the age of 10 to assess whether this non-invasive method could be applied in the monitoring of the accumulation of abnormal chemicals in the brain of patients. We found an abnormal peak that was not present in the treated patient. A similar pattern was also found in MRS of urine from patients, reflecting the concentration of oligosaccharides in serum and tissues. We therefore conclude that MRS can be a useful method to monitor the effect of treatment for Alpha-Mannosidosis.

  13. Influence of N-glycans on Expression of Cell Wall Remodeling Related Genes in Paracoccidioides brasiliensis Yeast Cells

    Science.gov (United States)

    Almeida, Fausto; Antoniêto, Amanda Cristina Campos; Pessoni, André Moreira; Monteiro, Valdirene Neves; Alegre-Maller, Ana Claudia Paiva; Pigosso, Laurine Lacerda; Pereira, Maristela; Soares, Célia Maria de Almeida; Roque-Barreira, Maria Cristina

    2016-01-01

    Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America. It is caused by the temperature-dependent dimorphic fungus Paracoccidioides brasiliensis. The P. brasiliensis cell wall is a dynamic outer structure, composed of a network of glycoproteins and polysaccharides, such as chitin, glucan and N-glycosylated proteins. These glycoproteins can interact with the host to affect infection rates, and are known to perform other functions. We inhibited N-linked glycosylation using tunicamycin (TM), and then evaluated the expression of P. brasiliensis genes related to cell wall remodeling. Our results suggest that cell wall synthesis related genes, such as β-1,3-glucanosyltransferase (PbGEL3), 1,3-β-D-glucan synthase (PbFKS1), and α-1,4-amylase (PbAMY), as well as cell wall degrading related genes, such as N-acetyl-β-D-glucosaminidase (PbNAG1), α-1,3-glucanase (PbAGN), and β-1,3-glucanase (PbBGN1 and PbBGN2), have their expression increased by the N-glycosylation inhibition, as detected by qRT-PCR. The observed increases in gene expression levels reveal possible compensatory mechanisms for diminished enzyme activity due to the lack of glycosylation caused by TM. PMID:27226767

  14. Comprehensive N-Glycan Profiling of Cetuximab Biosimilar Candidate by NP-HPLC and MALDI-MS

    Science.gov (United States)

    He, Zhenyu; Feng, Xiaojun; Liu, Bi-Feng; Liu, Xin

    2017-01-01

    Monitoring glycosylation of the mAbs have been emphasized and routinely characterized in biopharmaceutical industries because the carbohydrate components are closely related to the safety, efficacy, and consistency of the antibodies. In this study, the comprehensive glycan profiling of a biosimilar candidate of cetuximab was successfully characterized using Normal phase high-performance liquid chromatography (NP-HPLC) in combination with Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The presence of minor N-linked glycans containing sialic acid lactone residues (NeuAcLac) was observed in the biosimilar for the first time, which could influence the quantitative analysis of sialylated glycans and interfere with quantification of neutral glycans when it was analyzed by high performance liquid chromatography fluorescence (HPLC-FL). To overcome this issue, mild alkali treatment was used to hydrolyze lactone of the sialic acid to their neutral formation, which had no impact on the analysis of other glycans before and after the treatment. As a result, the mild alkali treatment might be helpful to obtain quantitative glycan profiling of the mAbs drugs with enhanced accuracy and robustness. PMID:28072827

  15. Different site-specific N-glycan types in wheat (Triticum aestivum L.) PAP phytase

    DEFF Research Database (Denmark)

    Dionisio, Giuseppe; Brinch-Pedersen, Henrik; Welinder, Karen Gjesing

    2011-01-01

    Phytase activity in grain is essential to make phosphate available to cell metabolism, and in food and feed. Cereals contain the purple acid phosphatase type of phytases (PAPhy). Mature wheat grain is dominated by TaPAPhy_a which, in the present work, has been characterized by extensive peptide a...

  16. Multi-Core Cache Hierarchies

    CERN Document Server

    Balasubramonian, Rajeev

    2011-01-01

    A key determinant of overall system performance and power dissipation is the cache hierarchy since access to off-chip memory consumes many more cycles and energy than on-chip accesses. In addition, multi-core processors are expected to place ever higher bandwidth demands on the memory system. All these issues make it important to avoid off-chip memory access by improving the efficiency of the on-chip cache. Future multi-core processors will have many large cache banks connected by a network and shared by many cores. Hence, many important problems must be solved: cache resources must be allocat

  17. Characterizing Facesheet/Core Disbonding in Honeycomb Core Sandwich Structure

    Science.gov (United States)

    Rinker, Martin; Ratcliffe, James G.; Adams, Daniel O.; Krueger, Ronald

    2013-01-01

    Results are presented from an experimental investigation into facesheet core disbonding in carbon fiber reinforced plastic/Nomex honeycomb sandwich structures using a Single Cantilever Beam test. Specimens with three, six and twelve-ply facesheets were tested. Specimens with different honeycomb cores consisting of four different cell sizes were also tested, in addition to specimens with three different widths. Three different data reduction methods were employed for computing apparent fracture toughness values from the test data, namely an area method, a compliance calibration technique and a modified beam theory method. The compliance calibration and modified beam theory approaches yielded comparable apparent fracture toughness values, which were generally lower than those computed using the area method. Disbonding in the three-ply facesheet specimens took place at the facesheet/core interface and yielded the lowest apparent fracture toughness values. Disbonding in the six and twelve-ply facesheet specimens took place within the core, near to the facesheet/core interface. Specimen width was not found to have a significant effect on apparent fracture toughness. The amount of scatter in the apparent fracture toughness data was found to increase with honeycomb core cell size.

  18. Introduction to the Core Curriculum in GIS

    OpenAIRE

    2015-01-01

    This short narrative introduces the Core Curriculum in GIS and provides a historical overview of the Core Curriculum Project, including the later Core Curriculum in GIScience and Core Curriculum for Technical Programs. Appended to this description is an original pamphlet advertising the Core Curriculum in GIS.

  19. Assessing Core Competencies

    Science.gov (United States)

    Narayanan, M.

    2004-12-01

    Catherine Palomba and Trudy Banta offer the following definition of assessment, adapted from one provided by Marches in 1987. Assessment in the systematic collection, review, and use of information about educational programs undertaken for the purpose of improving student learning and development. (Palomba and Banta 1999). It is widely recognized that sophisticated computing technologies are becoming a key element in today's classroom instructional techniques. Regardless, the Professor must be held responsible for creating an instructional environment in which the technology actually supplements learning outcomes of the students. Almost all academic disciplines have found a niche for computer-based instruction in their respective professional domain. In many cases, it is viewed as an essential and integral part of the educational process. Educational institutions are committing substantial resources to the establishment of dedicated technology-based laboratories, so that they will be able to accommodate and fulfill students' desire to master certain of these specific skills. This type of technology-based instruction may raise some fundamental questions about the core competencies of the student learner. Some of the most important questions are : 1. Is the utilization of these fast high-powered computers and user-friendly software programs creating a totally non-challenging instructional environment for the student learner ? 2. Can technology itself all too easily overshadow the learning outcomes intended ? 3. Are the educational institutions simply training students how to use technology rather than educating them in the appropriate field ? 4. Are we still teaching content-driven courses and analysis oriented subject matter ? 5. Are these sophisticated modern era technologies contributing to a decline in the Critical Thinking Capabilities of the 21st century technology-savvy students ? The author tries to focus on technology as a tool and not on the technology

  20. Viral Evolution Core | FNLCR Staging

    Science.gov (United States)

    Brandon F. Keele, Ph.D. PI/Senior Principal Investigator, Retroviral Evolution Section Head, Viral Evolution Core Leidos Biomedical Research, Inc. Frederick National Laboratory for Cancer Research Frederick, MD 21702-1201 Tel: 301-846-173

  1. Lunar Core Drive Tubes Summary

    Data.gov (United States)

    National Aeronautics and Space Administration — Contains a brief summary and high resolution imagery from various lunar rock and core drive tubes collected from the Apollo and Luna missions to the moon.

  2. Viscosity of the earth's core.

    Science.gov (United States)

    Gans, R. F.

    1972-01-01

    Calculation of the viscosity of the core at the boundary of the inner and outer core. It is assumed that this boundary is a melting transition and the viscosity limits of the Andrade (1934,1952) hypothesis (3.7 to 18.5 cp) are adopted. The corresponding kinematic viscosities are such that the precessional system explored by Malkus (1968) would be unstable. Whether it would be sufficiently unstable to overcome a severely subadiabatic temperature gradient cannot be determined.

  3. Nanoporous polymer liquid core waveguides

    DEFF Research Database (Denmark)

    Gopalakrishnan, Nimi; Christiansen, Mads Brøkner; Ndoni, Sokol

    2010-01-01

    We demonstrate liquid core waveguides defined by UV to enable selective water infiltration in nanoporous polymers, creating an effective refractive index shift Δn=0.13. The mode confinement and propagation loss in these waveguides are presented.......We demonstrate liquid core waveguides defined by UV to enable selective water infiltration in nanoporous polymers, creating an effective refractive index shift Δn=0.13. The mode confinement and propagation loss in these waveguides are presented....

  4. Crystallization in Earth's Core after High-Temperature Core Formation

    Science.gov (United States)

    Hirose, K.; Morard, G.; Hernlund, J. W.; Helffrich, G. R.; Ozawa, H.

    2015-12-01

    Recent core formation models based on the metal-silicate partitioning of siderophile elements suggest that the Earth's core was formed by metal segregation at high pressure and high temperature in a deep magma ocean. It is also thought that the simultaneous solubility of silicon and oxygen in liquid iron are strongly enhanced at high pressure and high temperature, such that at the end of accretion the core was rich in both silicon and oxygen. Here we performed crystallization experiments on the Fe-Si binary and Fe-Si-O ternary systems up to core pressure in a laser-heated diamond-anvil cell. The starting material for the latter was a homogeneous mixture of fine-grain Fe-Si and SiO2 (sustain without extreme degrees of secular cooling. However, even for modest degrees of joint Si-O incorporation into the early core, the buoyancy released by crystallization of SiO2 is sufficient to overcome thermal stratification and sustain the geodynamo.

  5. Core formation in silicate bodies

    Science.gov (United States)

    Nimmo, F.; O'Brien, D. P.; Kleine, T.

    2008-12-01

    Differentiation of a body into a metallic core and silicate mantle occurs most efficiently if temperatures are high enough to allow at least the metal to melt [1], and is enhanced if matrix deformation occurs [2]. Elevated temperatures may occur due to either decay of short-lived radio-isotopes, or gravitational energy release during accretion [3]. For bodies smaller than the Moon, core formation happens primarily due to radioactive decay. The Hf-W isotopic system may be used to date core formation; cores in some iron meteorites and the eucrite parent body (probably Vesta) formed within 1 My and 1-4~My of solar system formation, respectively [4]. These formation times are early enough to ensure widespread melting and differentiation by 26Al decay. Incorporation of Fe60 into the core, together with rapid early mantle solidification and cooling, may have driven early dynamo activity on some bodies [5]. Iron meteorites are typically depleted in sulphur relative to chondrites, for unknown reasons [6]. This depletion contrasts with the apparently higher sulphur contents of cores in larger planetary bodies, such as Mars [7], and also has a significant effect on the timing of core solidification. For bodies of Moon-size and larger, gravitational energy released during accretion is probably the primary cause of core formation [3]. The final stages of accretion involve large, stochastic collisions [8] between objects which are already differentiated. During each collision, the metallic cores of the colliding objects merge on timescales of a few hours [9]. Each collision will reset the Hf-W isotopic signature of both mantle and core, depending on the degree to which the impactor core re-equilibrates with the mantle of the target [10]. The re-equilibration efficiency depends mainly on the degree to which the impactor emulsifies [11], which is very uncertain. Results from N-body simulations [8,12] suggest that significant degrees of re- equilibration are required [4,10]. Re

  6. Viscosity of Earth's Outer Core

    CERN Document Server

    Smylie, D E

    2007-01-01

    A viscosity profile across the entire fluid outer core is found by interpolating between measured boundary values, using a differential form of the Arrhenius law governing pressure and temperature dependence. The discovery that both the retrograde and prograde free core nutations are in free decay (Palmer and Smylie, 2005) allows direct measures of viscosity at the top of the outer core, while the reduction in the rotational splitting of the two equatorial translational modes of the inner core allows it to be measured at the bottom. We find 2,371 plus/minus 1,530 Pa.s at the top and 1.247 plus/minus 0.035 x 10^11 Pa.s at the bottom. Following Brazhkin (1998) and Brazhkin and Lyapin (2000) who get 10^2 Pa.s at the top, 10^11 Pa.s at the bottom, by an Arrhenius extrapolation of laboratory experiments, we use a differential form of the Arrhenius law to interpolate along the melting temperature curve to find a viscosity profile across the outer core. We find the variation to be closely log-linear between the meas...

  7. SMART core protection system design

    Energy Technology Data Exchange (ETDEWEB)

    Lee, J. K.; Park, H. Y.; Koo, I. S. [KAERI, Taejon (Korea, Republic of); Park, H. S.; Kim, J. S.; Son, C. H. [Samchang Enterprise Co., Ltd., Taejon (Korea, Republic of)

    2003-10-01

    SMART COre Protection System(SCOPS) is designed with real-tims Digital Signal Processor(DSP) board and Network Interface Card(NIC) board. SCOPS has a Control Rod POSition (CRPOS) software module while Core Protection Calculator System(CPCS) consists of Core Protection Calculators(CPCs) and Control Element Assembly(CEA) Calculators(CEACs) in the commercial nuclear plant. It's not necessary to have a independent cabinets for SCOPS because SCOPS is physically very small. Then SCOPS is designed to share the cabinets with Plant Protection System(PPS) of SMART. Therefor it's very easy to maintain the system because CRPOS module is used instead of the computer with operating system.

  8. Atmospheric Methane in Ice Cores

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    The reconstruction of air trapped in ice cores provides us the most direct information about atmospheric CH4 variations in the past history. Ice core records from the "Three Poles (Antarctica, Greenland and Tibetan Plateau)" reveal the detailed fluctuations of atmospheric CH4 concentration with time and are allowed to quantify the CH4 differences among latitudes. These data are indispensably in the farther study of the relationship between greenhouse gases and climatic change, and of the past changes in terrestrial CH4 emissions. Ice cores reconstruction indicates that atmospheric CH4 concentration has increased quickly since industrialization, and the present day's level of atmospheric CH4 (1800 ppbv) is unprecedented during the past Glacial-Interglacial climate cycles.

  9. Distributed k-Core Decomposition

    CERN Document Server

    Montresor, Alberto; Miorandi, Daniele

    2011-01-01

    Among the novel metrics used to study the relative importance of nodes in complex networks, k-core decomposition has found a number of applications in areas as diverse as sociology, proteinomics, graph visualization, and distributed system analysis and design. This paper proposes new distributed algorithms for the computation of the k-core decomposition of a network, with the purpose of (i) enabling the run-time computation of k-cores in "live" distributed systems and (ii) allowing the decomposition, over a set of connected machines, of very large graphs, that cannot be hosted in a single machine. Lower bounds on the algorithms complexity are given, and an exhaustive experimental analysis on real-world graphs is provided.

  10. Identifying ELIXIR Core Data Resources.

    Science.gov (United States)

    Durinx, Christine; McEntyre, Jo; Appel, Ron; Apweiler, Rolf; Barlow, Mary; Blomberg, Niklas; Cook, Chuck; Gasteiger, Elisabeth; Kim, Jee-Hyub; Lopez, Rodrigo; Redaschi, Nicole; Stockinger, Heinz; Teixeira, Daniel; Valencia, Alfonso

    2016-01-01

    The core mission of ELIXIR is to build a stable and sustainable infrastructure for biological information across Europe. At the heart of this are the data resources, tools and services that ELIXIR offers to the life-sciences community, providing stable and sustainable access to biological data. ELIXIR aims to ensure that these resources are available long-term and that the life-cycles of these resources are managed such that they support the scientific needs of the life-sciences, including biological research. ELIXIR Core Data Resources are defined as a set of European data resources that are of fundamental importance to the wider life-science community and the long-term preservation of biological data. They are complete collections of generic value to life-science, are considered an authority in their field with respect to one or more characteristics, and show high levels of scientific quality and service. Thus, ELIXIR Core Data Resources are of wide applicability and usage. This paper describes the structures, governance and processes that support the identification and evaluation of ELIXIR Core Data Resources. It identifies key indicators which reflect the essence of the definition of an ELIXIR Core Data Resource and support the promotion of excellence in resource development and operation. It describes the specific indicators in more detail and explains their application within ELIXIR's sustainability strategy and science policy actions, and in capacity building, life-cycle management and technical actions. The identification process is currently being implemented and tested for the first time. The findings and outcome will be evaluated by the ELIXIR Scientific Advisory Board in March 2017. Establishing the portfolio of ELIXIR Core Data Resources and ELIXIR Services is a key priority for ELIXIR and publicly marks the transition towards a cohesive infrastructure.

  11. Earth's core and the geodynamo

    Science.gov (United States)

    Buffett

    2000-06-16

    Earth's magnetic field is generated by fluid motion in the liquid iron core. Details of how this occurs are now emerging from numerical simulations that achieve a self-sustaining magnetic field. Early results predict a dominant dipole field outside the core, and some models even reproduce magnetic reversals. The simulations also show how different patterns of flow can produce similar external fields. Efforts to distinguish between the various possibilities appeal to observations of the time-dependent behavior of the field. Important constraints will come from geological records of the magnetic field in the past.

  12. Magnetic Probing of Core Geodynamics

    Science.gov (United States)

    Voorhies, Coerte V.

    2004-01-01

    To better understand geomagnetic theory and observation, we can use spatial magnetic spectra for the main field and secular variation to test core dynmcal hypotheses against seismology. The hypotheses lead to theoretical spectra which are fitted to observational spectra. Each fit yields an estimate of the radius of Earth's core and uncertainty. If this agrees with the seismologic value, then the hypothes pass the test. A new way to obtain theoretical spectra extends the hydromagnetic scale analysis of Benton to scale-variant field and flow. For narrow scale flow and a dynamically weak field by the top of Earth's core, this yields a generalized Stevenson-McLeod spectrum for the core-source field, and a secular variation spectrum modulated by a cubic polynomial in spherical harmonic degree n. The former passes the tests. The latter passes many tests, but does not describe rapid dipole decline and quadrupole rebound; some tests suggest it is a bit hard, or rich in narrow scale change. In a core geodynamo, motion of the fluid conductor does work against the Lorentz force. This converts kinetic into magnetic energy which, in turn, is lost to heat via Ohmic dissipation. In the analysis at lentgh-scale l/k, if one presumes kinetic energy is converted in either eddy- overturning or magnetic free-decay time-scales, then Kolmogorov or other spectra in conflict with observational spectra can result. Instead, the rate work is done roughly balances the dissipation rate, which is consistent with small scale flow. The conversion time-scale depends on dynamical constraints. These are summarized by the magneto-geostrophic vertical vorticity balance by the top of the core, which includes anisotropic effects of rotation, the magnetic field, and the core- mantle boundary. The resulting theoretical spectra for the core-source field and its SV are far more compatible with observation. The conversion time-scale of order l20 years is pseudo-scale-invarient. Magnetic spectra of other

  13. Core competencies in internal medicine.

    Science.gov (United States)

    Porcel, José Manuel; Casademont, Jordi; Conthe, Pedro; Pinilla, Blanca; Pujol, Ramón; García-Alegría, Javier

    2012-06-01

    The working group on Competencies of Internal Medicine from the Spanish Society of Internal Medicine (SEMI) proposes a series of core competencies that we consider should be common to all European internal medicine specialists. The competencies include aspects related to patient care, clinical knowledge, technical skills, communication skills, professionalism, cost-awareness in medical care and academic activities. The proposal could be used as a working document for the Internal Medicine core curriculum in the context of the educational framework of medical specialties in Europe.

  14. Core Task and Organizational Reality

    DEFF Research Database (Denmark)

    Vikkelsø, Signe

    2015-01-01

    of core objects such as ‘task’ and ‘coordination,’ contemporary organization studies emphasize, much like other social science disciplines, broader topics such as ‘network,’ ‘identity,’ and ‘change.’ The paper argues that this altered focus and vocabulary is accompanied by a diminished ability to specify...... and intervene into the practical reality of organizations. It further argues that a discipline's core objects are not anachronisms to be discarded with, but crucial for specifying reality in ways that have proven practically relevant and still are....

  15. Producing gapped-ferrite transformer cores

    Science.gov (United States)

    Mclyman, W. T.

    1980-01-01

    Improved manufacturing techniques make reproducible gaps and minimize cracking. Molded, unfired transformer cores are cut with thin saw and then fired. Hardened semicircular core sections are bonded together, placed in aluminum core box, and fluidized-coated. After winding is run over box, core is potted. Economical method significantly reduces number of rejects.

  16. Multi-core Architectures and Streaming Applications

    NARCIS (Netherlands)

    Smit, Gerard J.M.; Kokkeler, André B.J.; Wolkotte, Pascal T.; Burgwal, van de Marcel D.; Mandoiu, I.; Kennings, A.

    2008-01-01

    In this paper we focus on algorithms and reconfigurable multi-core architectures for streaming digital signal processing (DSP) applications. The multi-core concept has a number of advantages: (1) depending on the requirements more or fewer cores can be switched on/off, (2) the multi-core structure f

  17. Core Stability Training for Injury Prevention

    OpenAIRE

    2013-01-01

    Context: Enhancing core stability through exercise is common to musculoskeletal injury prevention programs. Definitive evidence demonstrating an association between core instability and injury is lacking; however, multifaceted prevention programs including core stabilization exercises appear to be effective at reducing lower extremity injury rates. Evidence Acquisition: PubMed was searched for epidemiologic, biomechanic, and clinical studies of core stability for injury prevention (keywords: ...

  18. Visual Feedback for Rover-based Coring

    Science.gov (United States)

    Backes, Paul; Helmick, Daniel; Bajracharya, Max

    2008-01-01

    Technology for coring from a low-mass rover has been developed to enable core sample acquisition where a planetary rover experiences moderate slip during the coring operation. A new stereo vision technique, Absolute Motion Visual Odometry, is used to measure rover slip during coring and the slip is accommodated through corresponding arm pose updating. Coring rate is controlled by feedback of themeasured force of the coring tool against the environment. Test results in the JPL Marsyard show for the first time that coring from a low-mass rover with slip is feasible.

  19. Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure.

    Science.gov (United States)

    Nagai-Okatani, Chiaki; Minamino, Naoto

    2016-01-01

    Targeted proteomics focusing on post-translational modifications, including glycosylation, is a useful strategy for discovering novel biomarkers. To apply this strategy effectively to cardiac hypertrophy and resultant heart failure, we aimed to characterize glycosylation profiles in the left ventricle and plasma of rats with cardiac hypertrophy. Dahl salt-sensitive hypertensive rats, a model of hypertension-induced cardiac hypertrophy, were fed a high-salt (8% NaCl) diet starting at 6 weeks. As a result, they exhibited cardiac hypertrophy at 12 weeks and partially impaired cardiac function at 16 weeks compared with control rats fed a low-salt (0.3% NaCl) diet. Gene expression analysis revealed significant changes in the expression of genes encoding glycosyltransferases and glycosidases. Glycoproteome profiling using lectin microarrays indicated upregulation of mucin-type O-glycosylation, especially disialyl-T, and downregulation of core fucosylation on N-glycans, detected by specific interactions with Amaranthus caudatus and Aspergillus oryzae lectins, respectively. Upregulation of plasma α-l-fucosidase activity was identified as a biomarker candidate for cardiac hypertrophy, which is expected to support the existing marker, atrial natriuretic peptide and its related peptides. Proteomic analysis identified cysteine and glycine-rich protein 3, a master regulator of cardiac muscle function, as an O-glycosylated protein with altered glycosylation in the rats with cardiac hypertrophy, suggesting that alternations in O-glycosylation affect its oligomerization and function. In conclusion, our data provide evidence of significant changes in glycosylation pattern, specifically mucin-type O-glycosylation and core defucosylation, in the pathogenesis of cardiac hypertrophy and heart failure, suggesting that they are potential biomarkers for these diseases.

  20. Core Competence Development : paradigm and practical implementations

    OpenAIRE

    Koay, Ze Wei; E.Markov, Denis

    2011-01-01

    The theory of core competence has drawn a large amount of attention in the academic field as well as of practitioners in the corporate world. Theory asserts that long-term value creation and competitiveness of the corporation relies on full-scale exploitation and timely development of company Core Competences; business strategies should be built around the core competencies of a firm. Identification and exploitation of Core Competences as well as essential elements comprising Core Competences...

  1. Fuzzy Cores and Fuzzy Balancedness

    NARCIS (Netherlands)

    van Gulick, G.; Norde, H.W.

    2011-01-01

    We study the relation between the fuzzy core and balancedness for fuzzy games. For regular games, this relation has been studied by Bondareva (1963) and Shapley (1967). First, we gain insight in this relation when we analyse situations where the fuzzy game is continuous. Our main result shows that a

  2. Competition for cores in remanufacturing

    NARCIS (Netherlands)

    Bulmus, Serra Caner; Zhu, Stuart X.; Teunter, Ruud

    2014-01-01

    We study competition between an original equipment manufacturer (OEM) and an independently operating remanufacturer (IO). Different from the existing literature, the OEM and IO compete not only for selling their products but also for collecting returned products (cores) through their acquisition pri

  3. One Health Core Competency Domains.

    Science.gov (United States)

    Frankson, Rebekah; Hueston, William; Christian, Kira; Olson, Debra; Lee, Mary; Valeri, Linda; Hyatt, Raymond; Annelli, Joseph; Rubin, Carol

    2016-01-01

    The emergence of complex global challenges at the convergence of human, animal, and environmental health has catalyzed a movement supporting "One Health" approaches. Despite recognition of the importance of One Health approaches to address these complex challenges, little effort has been directed at identifying the seminal knowledge, skills, and attitudes necessary for individuals to successfully contribute to One Health efforts. Between 2008 and 2011, three groups independently embarked on separate initiatives to identify core competencies for professionals involved with One Health approaches. Core competencies were considered critically important for guiding curriculum development and continuing professional education, as they describe the knowledge, skills, and attitudes required to be effective. A workshop was convened in 2012 to synthesize the various strands of work on One Health competencies. Despite having different mandates, participants, and approaches, all of these initiatives identified similar core competency domains: management; communication and informatics; values and ethics; leadership; teams and collaboration; roles and responsibilities; and systems thinking. These core competency domains have been used to develop new continuing professional education programs for One Health professionals and help university curricula prepare new graduates to be able to contribute more effectively to One Health approaches.

  4. Fuzzy Cores and Fuzzy Balancedness

    NARCIS (Netherlands)

    van Gulick, G.; Norde, H.W.

    2011-01-01

    We study the relation between the fuzzy core and balancedness for fuzzy games. For regular games, this relation has been studied by Bondareva (1963) and Shapley (1967). First, we gain insight in this relation when we analyse situations where the fuzzy game is continuous. Our main result shows that

  5. Common Core: Solve Math Problems

    Science.gov (United States)

    Strom, Erich

    2012-01-01

    The new common core standards for mathematics demand that students (and teachers!) exhibit deeper conceptual understanding. That's music to the ears of education professor John Tapper, who says teachers have overemphasized teaching procedures--and getting right answers. In his new book, "Solving for Why," he makes a powerful case for moving beyond…

  6. Reference: GT1CORE [PLACE

    Lifescience Database Archive (English)

    Full Text Available GT1CORE Green PJ, Yong M-H, Cuozzo M, Kano-Murakami Y, Silverstein P, Chua N-H Binding site require...ments for pea nuclear protein factor GT-1 correlate with sequences required for light-depend

  7. One Health Core Competency Domains

    Science.gov (United States)

    Frankson, Rebekah; Hueston, William; Christian, Kira; Olson, Debra; Lee, Mary; Valeri, Linda; Hyatt, Raymond; Annelli, Joseph; Rubin, Carol

    2016-01-01

    The emergence of complex global challenges at the convergence of human, animal, and environmental health has catalyzed a movement supporting “One Health” approaches. Despite recognition of the importance of One Health approaches to address these complex challenges, little effort has been directed at identifying the seminal knowledge, skills, and attitudes necessary for individuals to successfully contribute to One Health efforts. Between 2008 and 2011, three groups independently embarked on separate initiatives to identify core competencies for professionals involved with One Health approaches. Core competencies were considered critically important for guiding curriculum development and continuing professional education, as they describe the knowledge, skills, and attitudes required to be effective. A workshop was convened in 2012 to synthesize the various strands of work on One Health competencies. Despite having different mandates, participants, and approaches, all of these initiatives identified similar core competency domains: management; communication and informatics; values and ethics; leadership; teams and collaboration; roles and responsibilities; and systems thinking. These core competency domains have been used to develop new continuing professional education programs for One Health professionals and help university curricula prepare new graduates to be able to contribute more effectively to One Health approaches. PMID:27679794

  8. Stability of Molten Core Materials

    Energy Technology Data Exchange (ETDEWEB)

    Layne Pincock; Wendell Hintze

    2013-01-01

    The purpose of this report is to document a literature and data search for data and information pertaining to the stability of nuclear reactor molten core materials. This includes data and analysis from TMI-2 fuel and INL’s LOFT (Loss of Fluid Test) reactor project and other sources.

  9. One Health Core Competency Domains

    Directory of Open Access Journals (Sweden)

    Rebekah Frankson

    2016-09-01

    Full Text Available The emergence of complex global challenges at the convergence of human, animal, and environmental health has catalyzed a movement supporting ‘One Health’ approaches. Despite recognition of the importance of One Health approaches to address these complex challenges, little effort has been directed at identifying the seminal knowledge, skills and attitudes necessary for individuals to successfully contribute to One Health efforts. Between 2008 and 2011, three groups independently embarked on separate initiatives to identify core competencies for professionals involved with One Health approaches. Core competencies were considered critically important for guiding curriculum development and continuing professional education as they describe the knowledge, skills and attitudes required to be effective. A workshop was convened in 2012 to synthesize the various strands of work on One Health competencies. Despite having different mandates, participants, and approaches, all of these initiatives identified similar core competency domains: management; communication and informatics; values and ethics; leadership; teams and collaboration; roles and responsibilities; and systems thinking. These core competency domains have been used to develop new continuing professional education programs for One Health professionals and help university curricula prepare new graduates to be able to contribute more effectively to One Health approaches.

  10. Earth rotation and core topography

    Science.gov (United States)

    Hager, Bradford H.; Clayton, Robert W.; Spieth, Mary Ann

    1988-01-01

    The NASA Geodynamics program has as one of its missions highly accurate monitoring of polar motion, including changes in length of day (LOD). These observations place fundamental constraints on processes occurring in the atmosphere, in the mantle, and in the core of the planet. Short-timescale (t less than or approx 1 yr) variations in LOD are mainly the result of interaction between the atmosphere and the solid earth, while variations in LOD on decade timescales result from the exchange of angular momentum between the mantle and the fluid core. One mechanism for this exchange of angular momentum is through topographic coupling between pressure variations associated with flow in the core interacting with topography at the core-mantel boundary (CMB). Work done under another NASA grant addressing the origin of long-wavelength geoid anomalies as well as evidence from seismology, resulted in several models of CMB topography. The purpose of work supported by NAG5-819 was to study further the problem of CMB topography, using geodesy, fluid mechanics, geomagnetics, and seismology. This is a final report.

  11. CopperCore Service Integration

    Science.gov (United States)

    Vogten, Hubert; Martens, Harrie; Nadolski, Rob; Tattersall, Colin; van Rosmalen, Peter; Koper, Rob

    2007-01-01

    In an e-learning environment there is a need to integrate various e-learning services like assessment services, collaboration services, learning design services and communication services. In this article we present the design and implementation of a generic integrative service framework, called CopperCore Service Integration (CCSI). We will…

  12. Core shift effect in blazars

    Science.gov (United States)

    Agarwal, A.; Mohan, P.; Gupta, Alok C.; Mangalam, A.; Volvach, A. E.; Aller, M. F.; Aller, H. D.; Gu, M. F.; Lähteenmäki, A.; Tornikoski, M.; Volvach, L. N.

    2017-07-01

    We studied the pc-scale core shift effect using radio light curves for three blazars, S5 0716+714, 3C 279 and BL Lacertae, which were monitored at five frequencies (ν) between 4.8 and 36.8 GHz using the University of Michigan Radio Astronomical Observatory (UMRAO), the Crimean Astrophysical Observatory (CrAO) and Metsähovi Radio Observatory for over 40 yr. Flares were Gaussian fitted to derive time delays between observed frequencies for each flare (Δt), peak amplitude (A) and their half width. Using A ∝ να, we infer α in the range of -16.67-2.41 and using Δ t ∝ ν ^{1/k_r}, we infer kr ∼ 1, employed in the context of equipartition between magnetic and kinetic energy density for parameter estimation. From the estimated core position offset (Ωrν) and the core radius (rcore), we infer that opacity model may not be valid in all cases. The mean magnetic field strengths at 1 pc (B1) and at the core (Bcore) are in agreement with previous estimates. We apply the magnetically arrested disc model to estimate black hole spins in the range of 0.15-0.9 for these blazars, indicating that the model is consistent with expected accretion mode in such sources. The power-law-shaped power spectral density has slopes -1.3 to -2.3 and is interpreted in terms of multiple shocks or magnetic instabilities.

  13. Magnectic Probing of Core Geodynamics

    Science.gov (United States)

    Voorhies, Coerte

    2004-01-01

    To better understand geomagnetic theory and observation, we can use spatial magnetic spectra for the main field and secular variation to test core dynamical hypotheses against seismology. The hypotheses lead to theoretical spectra which are fitted to observational spectra. Each fit yields an estimate of the radius of Earth s core and uncertainty. If this agrees with the seismologic value, then the hypotheses pass the test. A new way to obtain theoretical spectra extends the hydromagnetic scale analysis of Benton to scale-variant field and flow. For narrow scale flow and a dynamically weak field by the top of Earth s core, this yields a JGR-PI, and a secular variation spectrum modulated by a cubic polynomial in spherical harmonic degree n. The former passes the tests. The latter passes many tests, but does not describe rapid dipole decline and quadrupole rebound; some tests suggest it is a bit hard, or rich in narrow scale change.In a core geodynamo, motion of the fluid conductor does work against the Lorentz force. This converts kinetic into magnetic energy which, in turn, is lost to heat via Ohmic dissipation. In the analysis at length- scale l/k, if one presumes kinetic energy is converted in either eddy- overturning or magnetic free-decay time-scales, then Kolmogorov or other spectra in conflict with observational spectra can result. Instead, the rate work is done roughly balances the dissipation rate, which is consistent with small scale flow. The conversion time-scale depends on dynamical constraints. These are summarized by the magneto- geostrophic vertical vorticity balance by the top of the core, which includes anisotropic effects of rotation, the magnetic field, and the core-mantle boundary. The resulting theoretical spectra for the core- source field and its SV are far more compatible with observation. The conversion time-scale of order 120 years is pseudo-scale-invariant. Magnetic spectra of other planets may differ; however, if a transition to non

  14. Interleaved Core Assignment for Bidirectional Transmission in Multi-Core Fibers

    DEFF Research Database (Denmark)

    Ye, Feihong; Morioka, Toshio

    2013-01-01

    We study interleaved core assignment for bidirectional transmission in multi-core fibers. By combining it with heterogeneous core structure in an 18-core fiber, the transmission distance is extended by 10 times compared to homogeneous core structure with unidirectional transmission, achieving...

  15. Interleaved Core Assignment for Bidirectional Transmission in Multi-Core Fibers

    OpenAIRE

    2013-01-01

    We study interleaved core assignment for bidirectional transmission in multi-core fibers. By combining it with heterogeneous core structure in an 18-core fiber, the transmission distance is extended by 10 times compared to homogeneous core structure with unidirectional transmission, achieving a total capacity of 1 Pb/s per direction.

  16. Interleaved Core Assignment for Bidirectional Transmission in Multi-Core Fibers

    DEFF Research Database (Denmark)

    Ye, Feihong; Morioka, Toshio

    2013-01-01

    We study interleaved core assignment for bidirectional transmission in multi-core fibers. By combining it with heterogeneous core structure in an 18-core fiber, the transmission distance is extended by 10 times compared to homogeneous core structure with unidirectional transmission, achieving a t...

  17. Cores to the rescue: how old cores enable new science

    Science.gov (United States)

    Ito, E.; Noren, A. J.; Brady, K.

    2016-12-01

    The value of archiving scientific specimens and collections for the purpose of enabling further research using new analytical techniques, resolving conflicting results, or repurposing them for entirely new research, is often discussed in abstract terms. We all agree that samples with adequate metadata ought to be archived systematically for easy access, for a long time and stored under optimal conditions. And yet, as storage space fills, there is a temptation to cull the collection, or when a researcher retires, to discard the collection unless the researcher manages to make his or her own arrangement for the collection to be accessioned elsewhere. Nobody has done anything with these samples in over 20 years! Who would want them? It turns out that plenty of us do want them, if we know how to find them and if they have sufficient metadata to assess past work and suitability for new analyses. The LacCore collection holds over 33 km of core from >6700 sites in diverse geographic locations worldwide with samples collected as early as 1950s. From these materials, there are many examples to illustrate the scientific value of archiving geologic samples. One example that benefitted Ito personally were cores from Lakes Mirabad and Zeribar, Iran, acquired in 1963 by Herb Wright and his associates. Several doctoral and postdoctoral students generated and published paleoecological reconstructions based on cladocerans, diatoms, pollen or plant macrofossils, mostly between 1963 and 1967. The cores were resampled in 1990s by a student being jointly advised by Wright and Ito for oxygen isotope analysis of endogenic calcite. The results were profitably compared with pollen and the results published in 2001 and 2006. From 1979 until very recently, visiting Iran for fieldwork was not pallowed for US scientists. Other examples will be given to further illustrate the power of archived samples to advance science.

  18. Hydrologic characterization of four cores from the Geysers Coring Project

    Energy Technology Data Exchange (ETDEWEB)

    Persoff, Peter; Hulen, Jeffrey B.

    1996-01-24

    Results of hydrologic tests conducted on four representative core plugs from Geysers Coring Project drill hole SB-15-D have been related to detailed mineralogic and textural characterization of the plugs to yield new information about permeability, porosity, and capillary-pressure characteristics of the uppermost Geysers steam reservoir and its immediately overlying caprock. The core plugs are all fine- to medium-grained, Franciscan-assemblage (late Mesozoic) metagraywacke with sparse Franciscan metamorphic quartz-calcite veins and late Cenozoic, hydrothermal quartz-calcite-pyrite veins. The matrices of three plugs from the caprock are rich in metamorphic mixed-layer illite/smectite and disseminated hydrothermal pyrite; the reservoir plug instead contains abundant illite and only minor pyrite. The reservoir plug and one caprock plug are sparsely disrupted by latest-stage, unmineralized microfractures which both follow and crosscut veinlets but which could be artifacts. Porosities of the plugs, measured by Boyles-law gas expansion, range between 1.9 and 2.5%. Gas permeability and Klinkenberg slip factor were calculated from gas-pressure-pulse-decay measurements using a specially designed permeameter with small (2 mL) reservoirs. Matrix permeabilities in the range 10-21 m² ( = 1 nanodarcy) were measured for two plugs that included mineral-filled veins but no unfilled microfractures. Greater permeabilities were measured on plugs that contained microfractures; at 500 psi net confining pressure, an effective aperture of 1.6 µm was estimated for one plug. Capillary pressure curves were determined for three cores by measuring saturation as weight gain of plugs equilibrated with atmospheres in which the relative humidity was controlled by saturated brines.

  19. Hydrologic characterization of four cores from the Geysers Coring Project

    Energy Technology Data Exchange (ETDEWEB)

    Persoff, P. [Lawrence Berkeley National Lab., CA (United States); Hulen, J.B. [Univ. of Utah, Salt Lake City, UT (United States). Earth Sciences and Resources Institute

    1996-01-01

    Results of hydrologic tests on 4 representative core plugs from Geysers Coring Project drill hole SB-15-D were related to mineralogy and texture. Permeability measurements were made on 3 plugs from caprock and one plug from the steam reservoir. Late-stage microfractures present in 2 of the plugs contributed to greater permeability, but the values for the 2 other plugs indicate a typical matrix permeability of 1 to 2 {times} 10{sup {minus}21}m{sup 2}. Klinkenberg slip factor b for these plugs is generally consistent with the inverse relation between slip factor and permeability observed by Jones (1972) for plugs of much more permeable material. The caprock and reservoir samples are nearly identical metagraywackes with slight mineralogical differences which appear to have little effect on hydrology. The late stage microfractures are suspected of being artifacts. The capillary pressure curves for 3 cores are fit by power-law relations which can be used to estimate relative permeability curves for the matrix rocks.

  20. Logging-while-coring method and apparatus

    Science.gov (United States)

    Goldberg, David S.; Myers, Gregory J.

    2007-11-13

    A method and apparatus for downhole coring while receiving logging-while-drilling tool data. The apparatus includes core collar and a retrievable core barrel. The retrievable core barrel receives core from a borehole which is sent to the surface for analysis via wireline and latching tool The core collar includes logging-while-drilling tools for the simultaneous measurement of formation properties during the core excavation process. Examples of logging-while-drilling tools include nuclear sensors, resistivity sensors, gamma ray sensors, and bit resistivity sensors. The disclosed method allows for precise core-log depth calibration and core orientation within a single borehole, and without at pipe trip, providing both time saving and unique scientific advantages.

  1. Conceptual study of advanced PWR core design

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young Jin; Chang, Moon Hee; Kim, Keung Ku; Joo, Hyung Kuk; Kim, Young Il; Noh, Jae Man; Hwang, Dae Hyun; Kim, Taek Kyum; Yoo, Yon Jong

    1997-09-01

    The purpose of this project is for developing and verifying the core design concepts with enhanced safety and economy, and associated methodologies for core analyses. From the study of the sate-of-art of foreign advanced reactor cores, we developed core concepts such as soluble boron free, high convertible and enhanced safety core loaded semi-tight lattice hexagonal fuel assemblies. To analyze this hexagonal core, we have developed and verified some neutronic and T/H analysis methodologies. HELIOS code was adopted as the assembly code and HEXFEM code was developed for hexagonal core analysis. Based on experimental data in hexagonal lattices and the COBRA-IV-I code, we developed a thermal-hydraulic analysis code for hexagonal lattices. Using the core analysis code systems developed in this project, we designed a 600 MWe core and studied the feasibility of the core concepts. Two additional scopes were performed in this project : study on the operational strategies of soluble boron free core and conceptual design of large scale passive core. By using the axial BP zoning concept and suitable design of control rods, this project showed that it was possible to design a soluble boron free core in 600 MWe PWR. The results of large scale core design showed that passive concepts and daily load follow operation could be practiced. (author). 15 refs., 52 tabs., 101 figs.

  2. Whole Core Transport Calculation Methodology for a Hexagonal Core

    Energy Technology Data Exchange (ETDEWEB)

    Cho, J. Y.; Kim, K. S.; Lee, C. C.; Zee, S. Q.; Joo, H. G

    2007-07-15

    This report discusses the hexagonal module implemented to the DeCART code and the performance of them. The implemented hexagonal module includes the hexagonal ray tracing and the CMFD acceleration modules. The performance of the implemented hexagonal module is examined for 4 tests of: (1) CMFD acceleration test, (2) the accuracy test of the hexagonal module, (3) the performance test for 2-D NGNP problem and (4) the applicability test for 3-D NGNP problem. The features of the implemented hexagonal modules are: (1) The Modular ray tracing scheme based on a hexagonal assembly and a path linking scheme between the modular rays. (2) Segment generation based on the structure unit. (3) Cell ray approximation: This feature is developed to reduce the memory required to store the segment information. (4) Modified cycle ray scheme that begins the ray tracing at a given surface and finishes if the reflected ray meets the starting surface. This feature is developed to reduce the memory required for the angular flux at the core boundary. (5) Fixed assembly geometry. The pin geometry of the single pin per assembly problem is different from that of the multi-pin problem. The core geometry of a single assembly problem is also different from that of the multi-assembly problem. (6) CMFD module based on unstructured cell. This feature is to deal with the irregular gap cells that are positioned at the assembly boundaries. The examination results of the 4 tests can be summarized as: (1) The CMFD acceleration test shows that the CMFD module speedups about greater than 200 for the core problem. (2) The accuracy test shows that the hexagonal MOC module produces an accurate solution of less than 60 pcm of eigenvalue and less than 2 % of local pin power errors. (3) The performance test for 2-D NGNP problem shows that the implemented hexagonal module works soundly and produces a reasonable solution by cooperating with the existing DeCART library and the other modules. (4) The applicability

  3. Sediments at the top of Earth's core.

    Science.gov (United States)

    Buffett, B A; Garnero, E J; Jeanloz, R

    2000-11-17

    Unusual physical properties at the core-mantle boundary have been inferred from seismic and geodetic observations in recent years. We show how both types of observations can be explained by a layer of silicate sediments, which accumulate at the top of the core as Earth cools. Compaction of the sediments expels most of the liquid iron but leaves behind a small amount of core material, which is entrained in mantle convection and may account for the isotopic signatures of core material in some hot spot plumes. Extraction of light elements from the liquid core also enhances the vigor of convection in the core and may increase the power available to the geodynamo.

  4. Core and Lumbopelvic Stabilization in Runners.

    Science.gov (United States)

    Rivera, Carlos E

    2016-02-01

    Core muscles provide stability that allows generation of force and motion in the lower extremities, as well as distributing impact forces and allowing controlled and efficient body movements. Imbalances or deficiencies in the core muscles can result in increased fatigue, decreased endurance, and injury in runners. Core strengthening should incorporate the intrinsic needs of the core for flexibility, strength, balance, and endurance, and the function of the core in relation to its role in extremity function and dysfunction. Specific exercises are effective in strengthening the core muscles.

  5. Accelerator driven sub-critical core

    Science.gov (United States)

    McIntyre, Peter M; Sattarov, Akhdiyor

    2015-03-17

    Systems and methods for operating an accelerator driven sub-critical core. In one embodiment, a fission power generator includes a sub-critical core and a plurality of proton beam generators. Each of the proton beam generators is configured to concurrently provide a proton beam into a different area of the sub-critical core. Each proton beam scatters neutrons within the sub-critical core. The plurality of proton beam generators provides aggregate power to the sub-critical core, via the proton beams, to scatter neutrons sufficient to initiate fission in the sub-critical core.

  6. L183, a Quiescent Core?

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Some observed results of NH3 (1, 1) and (2, 2) line emission in the starless dark cloud L183 are reported. Our observation suggests that the dense core of L183 has a size of ~ 0.16pc ×0.1pc with a mass of ~ 12M⊙. A velocity gradient of 4km s-1pc-1 from the north to the south was detected. The velocity shift corresponds to a central mass of ~ 5M⊙. If it is caused by rotation, the mass would be much less than the value above. This suggests that there may be more mass in the envelope of L183 than in the central region. The analysis of our data and the evidence in the literature about L183 indicate that it may be undergoing a process of collapsing to form a low-mass binary dense core.

  7. CORE CULTURE IN LANGUAGE LEARNING

    Institute of Scientific and Technical Information of China (English)

    Jane Orton

    2002-01-01

    This paper presents the essential role of culture in the creation of meaning of language-inuse, showing it to be the source of both the contextual shaping of a particular text and the fundamental core of beliefs and values from which contemporary contextual features derive. As a result of this essential relationship, it is argued, modern language learners need to master the cultural system as well as the linguistic system of their new language if they are to be able to use it competently in real life, as mo st intend today. Samples of texts are examined to show how the meanings of both context and core culture are naturally embedded in ordinary language and suggestions are provided for how these might be successfully brought to students' attention and mastery in a classroom.

  8. Beyond core knowledge: Natural geometry

    Science.gov (United States)

    Spelke, Elizabeth; Lee, Sang Ah; Izard, Véronique

    2010-01-01

    For many centuries, philosophers and scientists have pondered the origins and nature of human intuitions about the properties of points, lines, and figures on the Euclidean plane, with most hypothesizing that a system of Euclidean concepts either is innate or is assembled by general learning processes. Recent research from cognitive and developmental psychology, cognitive anthropology, animal cognition, and cognitive neuroscience suggests a different view. Knowledge of geometry may be founded on at least two distinct, evolutionarily ancient, core cognitive systems for representing the shapes of large-scale, navigable surface layouts and of small-scale, movable forms and objects. Each of these systems applies to some but not all perceptible arrays and captures some but not all of the three fundamental Euclidean relationships of distance (or length), angle, and direction (or sense). Like natural number (Carey, 2009), Euclidean geometry may be constructed through the productive combination of representations from these core systems, through the use of uniquely human symbolic systems. PMID:20625445

  9. Continuous Chemistry in Ice Cores

    DEFF Research Database (Denmark)

    Kjær, Helle Astrid

    originating from volcanic eruptions, crucial for cross-dating ice cores and relevant for climate interpretations. The method includes a heat bath to minimize the acidifying effect of CO2 both from the laboratory and from the ice itself. While for acidic ice the method finds similar concentrations of H......Ice cores provide high resolution records of past climate and environment. In recent years the use of continuous flow analysis (CFA) systems has increased the measurement throughput, while simultaneously decreasing the risk of contaminating the ice samples. CFA measurements of high temporal...... resolution increase our knowledge on fast climate variations and cover a wide range of proxies informing on a variety of components such as atmospheric transport, volcanic eruptions, forest fires and many more. New CFA methods for the determination of dissolved reactive phosphorus (DRP) and pH are presented...

  10. Composite Structure with Origami Core

    Science.gov (United States)

    2016-07-19

    preparation). Hence, we are able to produce foldcores in any given shape. Mechanical behaviour of sandwich shells with foldcores Equipped with the...being pressed in order to invert the tube inside out. For a tube with circular section, this particular mode of failure has been proven to consume the...to design the most suitable folded core structure for given applications. Gattas J M and You Z, The Behaviour of Curved-Crease Foldcores under

  11. Hollow-core grating fiber

    Science.gov (United States)

    Barillé, R.; Tajalli, P.; Roy, P.; Ahmadi-kandjani, S.; Kucharski, S.; Ortyl, E.

    2012-02-01

    We propose a new type of hollow-core fiber where the propagation is ensured by a photoinduced self-pattern acting as a surface relief grating (SRG). The SRG is written by launching a suitable laser beam with proper polarization in a capillary glass fiber with the inner surface previously coated with an azopolymer thin film. Such a grating acts as a wavelength/angle dependant reflective mirror and enhances the confinement and the propagation of the light.

  12. Understanding Core-Collapse Supernovae

    CERN Document Server

    Burrows, A

    2004-01-01

    I summarize, in the form of an extended abstract, the ongoing efforts at the University of Arizona (and in collaboration) to understand core-collapse supernovae theoretically. Included are short discussions of 1D (SESAME) and 2D (VULCAN/2D) codes and results, as well as discussions of the possible role of rotation. Highlighted are recent developments in multi-dimensional radiation hydrodynamics and the essential physics of the neutrino-driven mechanism.

  13. Finding your next core business.

    Science.gov (United States)

    Zook, Chris

    2007-04-01

    How do you know when your core needs to change? And how do you determine what should replace it? From an in-depth study of 25 companies, the author, a strategy consultant, has discovered that it's possible to measure the vitality of a business's core. If it needs reinvention, he says, the best course is to mine hidden assets. Some of the 25 companies were in deep crisis when they began the process of redefining themselves. But, says Zook, management teams can learn to recognize early signs of erosion. He offers five diagnostic questions with which to evaluate the customers, key sources of differentiation, profit pools, capabilities, and organizational culture of your core business. The next step is strategic regeneration. In four-fifths of the companies Zook examined, a hidden asset was the centerpiece of the new strategy. He provides a map for identifying the hidden assets in your midst, which tend to fall into three categories: undervalued business platforms, untapped insights into customers, and underexploited capabilities. The Swedish company Dometic, for example, was manufacturing small absorption refrigerators for boats and RVs when it discovered a hidden asset: its understanding of, and access to, customers in the RV market. The company took advantage of a boom in that market to refocus on complete systems for live-in vehicles. The Danish company Novozymes, which produced relatively low-tech commodity enzymes such as those used in detergents, realized that its underutilized biochemical capability in genetic and protein engineering was a hidden asset and successfully refocused on creating bioengineered specialty enzymes. Your next core business is not likely to announce itself with fanfare. Use the author's tools to conduct an internal audit of possibilities and pinpoint your new focus.

  14. Rich-cores in networks

    CERN Document Server

    Ma, Athen

    2014-01-01

    A core is said to be a group of central and densely connected nodes which governs the overall behavior of a network. Profiling this meso--scale structure currently relies on a limited number of methods which are often complex, and have scalability issues when dealing with very large networks. As a result, we are yet to fully understand its impact on network properties and dynamics. Here we introduce a simple method to profile this structure by combining the concepts of core/periphery and rich-club. The key challenge in addressing such association of the two concepts is to establish a way to define the membership of the core. The notion of a "rich-club" describes nodes which are essentially the hub of a network, as they play a dominating role in structural and functional properties. Interestingly, the definition of a rich-club naturally emphasizes high degree nodes and divides a network into two subgroups. Our approach theoretically couples the underlying principle of a rich-club with the escape time of a rand...

  15. Grain alignment in starless cores

    Energy Technology Data Exchange (ETDEWEB)

    Jones, T. J.; Bagley, M. [Minnesota Institute for Astrophysics, University of Minnesota, Minneapolis, MN 55455 (United States); Krejny, M. [Cree Inc., 4600 Silicon Dr., Durham, NC (United States); Andersson, B.-G. [SOFIA Science Center, USRA, Moffett Field, CA (United States); Bastien, P., E-mail: tjj@astro.umn.edu [Centre de recherche en astrophysique du Québec and Départment de Physique, Université de Montréal, Montréal (Canada)

    2015-01-01

    We present near-IR polarimetry data of background stars shining through a selection of starless cores taken in the K band, probing visual extinctions up to A{sub V}∼48. We find that P{sub K}/τ{sub K} continues to decline with increasing A{sub V} with a power law slope of roughly −0.5. Examination of published submillimeter (submm) polarimetry of starless cores suggests that by A{sub V}≳20 the slope for P versus τ becomes ∼−1, indicating no grain alignment at greater optical depths. Combining these two data sets, we find good evidence that, in the absence of a central illuminating source, the dust grains in dense molecular cloud cores with no internal radiation source cease to become aligned with the local magnetic field at optical depths greater than A{sub V}∼20. A simple model relating the alignment efficiency to the optical depth into the cloud reproduces the observations well.

  16. Helium in Earth's early core

    Science.gov (United States)

    Bouhifd, M. A.; Jephcoat, Andrew P.; Heber, Veronika S.; Kelley, Simon P.

    2013-11-01

    The observed escape of the primordial helium isotope, 3He, from the Earth's interior indicates that primordial helium survived the energetic process of planetary accretion and has been trapped within the Earth to the present day. Two distinct reservoirs in the Earth's interior have been invoked to account for variations in the 3He/4He ratio observed at the surface in ocean basalts: a conventional depleted mantle source and a deep, still enigmatic, source that must have been isolated from processing throughout Earth history. The Earth's iron-based core has not been considered a potential helium source because partitioning of helium into metal liquid has been assumed to be negligible. Here we determine helium partitioning in experiments between molten silicates and iron-rich metal liquids at conditions up to 16GPa and 3,000K. Analyses of the samples by ultraviolet laser ablation mass spectrometry yield metal-silicate helium partition coefficients that range between 4.7×10-3 and 1.7×10-2 and suggest that significant quantities of helium may reside in the core. Based on estimated concentrations of primordial helium, we conclude that the early core could have incorporated enough helium to supply deep-rooted plumes enriched in 3He throughout the age of the Earth.

  17. Core shifts in blazar jets

    CERN Document Server

    Zdziarski, Andrzej A; Pjanka, Patryk; Tchekhovskoy, Alexander

    2014-01-01

    We study the effect of core shift in jets, which is the dependence of the position of the jet radio core on the frequency. We derive a new method to measure the jet magnetic field based on both the value of the shift and the observed flux, which compliments the standard method assuming equipartition. Using both methods, we re-analyse the blazar sample of Zamaninasab et al. We find that equipartition is satisfied only if the jet opening angle in the radio core region is close to the values found observationally, $\\simeq$0.1--0.2 divided by the bulk Lorentz factor, $\\Gamma_{\\rm j}$. Larger values, e.g., $1/\\Gamma_{\\rm j}$, would imply very strong departures from equipartition. A small jet opening angle implies in turn the magnetization parameter of $\\ll 1$. We determine the jet magnetic flux taking this effect into account. We find that the average jet magnetic flux is compatible with the model of jet formation due to black-hole spin energy extraction and accretion being magnetically arrested. We calculate the ...

  18. Core stability training for injury prevention.

    Science.gov (United States)

    Huxel Bliven, Kellie C; Anderson, Barton E

    2013-11-01

    Enhancing core stability through exercise is common to musculoskeletal injury prevention programs. Definitive evidence demonstrating an association between core instability and injury is lacking; however, multifaceted prevention programs including core stabilization exercises appear to be effective at reducing lower extremity injury rates. PUBMED WAS SEARCHED FOR EPIDEMIOLOGIC, BIOMECHANIC, AND CLINICAL STUDIES OF CORE STABILITY FOR INJURY PREVENTION (KEYWORDS: "core OR trunk" AND "training OR prevention OR exercise OR rehabilitation" AND "risk OR prevalence") published between January 1980 and October 2012. Articles with relevance to core stability risk factors, assessment, and training were reviewed. Relevant sources from articles were also retrieved and reviewed. Stabilizer, mobilizer, and load transfer core muscles assist in understanding injury risk, assessing core muscle function, and developing injury prevention programs. Moderate evidence of alterations in core muscle recruitment and injury risk exists. Assessment tools to identify deficits in volitional muscle contraction, isometric muscle endurance, stabilization, and movement patterns are available. Exercise programs to improve core stability should focus on muscle activation, neuromuscular control, static stabilization, and dynamic stability. Core stabilization relies on instantaneous integration among passive, active, and neural control subsystems. Core muscles are often categorized functionally on the basis of stabilizing or mobilizing roles. Neuromuscular control is critical in coordinating this complex system for dynamic stabilization. Comprehensive assessment and training require a multifaceted approach to address core muscle strength, endurance, and recruitment requirements for functional demands associated with daily activities, exercise, and sport.

  19. CORE SHAPES AND ORIENTATIONS OF CORE-SÉRSIC GALAXIES

    Energy Technology Data Exchange (ETDEWEB)

    Dullo, Bililign T.; Graham, Alister W., E-mail: Bdullo@astro.swin.edu.au [Centre for Astrophysics and Supercomputing, Swinburne University of Technology, Hawthorn, Victoria 3122 (Australia)

    2015-01-01

    The inner and outer shapes and orientations of core-Sérsic galaxies may hold important clues to their formation and evolution. We have therefore measured the central and outer ellipticities and position angles for a sample of 24 core-Sérsic galaxies using archival Hubble Space Telescope (HST) images and data. By selecting galaxies with core-Sérsic break radii R{sub b} —a measure of the size of their partially depleted core—that are ≳ 0.''2, we find that the ellipticities and position angles are quite robust against HST seeing. For the bulk of the galaxies, there is a good agreement between the ellipticities and position angles at the break radii and the average outer ellipticities and position angles determined over R {sub e}/2 < R < R {sub e}, where R {sub e} is the spheroids' effective half light radius. However there are some interesting differences. We find a median ''inner'' ellipticity at R{sub b} of ε{sub med} = 0.13 ± 0.01, rounder than the median ellipticity of the ''outer'' regions ε{sub med} = 0.20 ± 0.01, which is thought to reflect the influence of the central supermassive black hole at small radii. In addition, for the first time we find a trend, albeit weak (2σ significance), such that galaxies with larger (stellar deficit-to-supermassive black hole) mass ratios—thought to be a measure of the number of major dry merger events—tend to have rounder inner and outer isophotes, suggesting a connection between the galaxy shapes and their merger histories. We show that this finding is not simply reflecting the well known result that more luminous galaxies are rounder, but it is no doubt related.

  20. Optimized Cellular Core for Rotorcraft Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Patz Materials and Technologies has developed, produced and tested, as part of the Phase-I SBIR, a new form of composite cellular core material, named Interply Core,...

  1. Scaling Turbo Boost to a 1000 cores

    CERN Document Server

    S, Ananth Narayan; Fedorova, Alexandra

    2010-01-01

    The Intel Core i7 processor code named Nehalem provides a feature named Turbo Boost which opportunistically varies the frequencies of the processor's cores. The frequency of a core is determined by core temperature, the number of active cores, the estimated power consumption, the estimated current consumption, and operating system frequency scaling requests. For a chip multi-processor(CMP) that has a small number of physical cores and a small set of performance states, deciding the Turbo Boost frequency to use on a given core might not be difficult. However, we do not know the complexity of this decision making process in the context of a large number of cores, scaling to the 100s, as predicted by researchers in the field.

  2. Investigating the translation of Earth's inner core

    DEFF Research Database (Denmark)

    Day, Elizabeth A; Cormier, Vernon F; Geballe, Zachary M;

    2012-01-01

    The Earth’s inner core provides unique insights into processes that are occurring deep within our Earth today, as well as processes that occurred in the past. The seismic structure of the inner core is complex, and is dominated by anisotropic and isotropic differences between the Eastern...... and Western ‘hemispheres’ of the inner core. Recent geodynamical models suggest that this hemispherical dichotomy can be explained by a fast translation of the inner core. In these models one side of the inner core is freezing, while the other side is melting, leading to the development of different seismic...... properties on either side of the inner core. A simple translating model of the inner core, however, does not seem to easily explain all of the seismically observed features, including the innermost inner core; the observed sharp lateral gradient in seismic properties between the two hemispheres...

  3. High Efficiency Solar Furnace Core Project

    Data.gov (United States)

    National Aeronautics and Space Administration — It is proposed to develop a high efficiency solar furnace core that greatly lessens the heat losses from the furnace core, either greatly reducing the amount of...

  4. Characterizing core stability with fuzzy games

    OpenAIRE

    Shellshear, Evan

    2011-01-01

    This paper investigates core stability of cooperative, TU games via a fuzzy extension of the totally balanced cover of a TU game. The stability of the core of the fuzzy extension of a game, the concave extension, is shown to reflect the core stability of the original game and vice versa. Stability of the core is then shown to be equivalent to the existence of an equilibrium of a certain correspondence.

  5. Characterizing core stability with fuzzy games

    OpenAIRE

    Shellshear, Evan

    2009-01-01

    This paper investigates core stability of cooperative, TU games via a fuzzy extension of the totally balanced cover of a TU game. The stability of the core of the fuzzy extension of a game, the concave extension, is shown to reflect the core stability of the original game and vice versa. Stability of the core is then shown to be equivalent to the existence of an equilibrium of a certain correspondence.

  6. Core Processes: Earth's eccentric magnetic field

    DEFF Research Database (Denmark)

    Finlay, Chris

    2012-01-01

    Earth’s magnetic field is characterized by a puzzling hemispheric asymmetry. Calculations of core dynamo processes suggest that lopsided growth of the planet’s inner core may be part of the cause.......Earth’s magnetic field is characterized by a puzzling hemispheric asymmetry. Calculations of core dynamo processes suggest that lopsided growth of the planet’s inner core may be part of the cause....

  7. Rotation and Magnetism of Earth's Inner Core

    Science.gov (United States)

    Glatzmaier; Roberts

    1996-12-13

    Three-dimensional numerical simulations of the geodynamo suggest that a super- rotation of Earth's solid inner core relative to the mantle is maintained by magnetic coupling between the inner core and an eastward thermal wind in the fluid outer core. This mechanism, which is analogous to a synchronous motor, also plays a fundamental role in the generation of Earth's magnetic field.

  8. Core Competencies in Information Management Education.

    Science.gov (United States)

    Gorman, G. E.; Corbitt, B. J.

    2002-01-01

    Discusses core competencies in library and information science and in information systems to use as a background for an examination of core competencies in information management. Suggests a set of core competencies and educational outcomes that might be applied to curricula in both developed and developing countries. (Author/LRW)

  9. Simplifying the ELA Common Core; Demystifying Curriculum

    Science.gov (United States)

    Schmoker, Mike; Jago, Carol

    2013-01-01

    The English Language Arts (ELA) Common Core State Standards ([CCSS], 2010) could have a transformational effect on American education. Though the process seems daunting, one can begin immediately integrating the essence of the ELA Common Core in every subject area. This article shows how one could implement the Common Core and create coherent,…

  10. Heterogeneity and anisotropy of Earth's inner core

    NARCIS (Netherlands)

    Deuss, Arwen

    2014-01-01

    Seismic observations provide strong evidence that Earth's inner core is anisotropic, with larger velocity in the polar than in the equatorial direction. The top 60-80 km of the inner core is isotropic; evidence for an innermost inner core is less compelling. The anisotropy is most likely due to alig

  11. Honeycomb Core Permeability Under Mechanical Loads

    Science.gov (United States)

    Glass, David E.; Raman, V. V.; Venkat, Venki S.; Sankaran, Sankara N.

    1997-01-01

    A method for characterizing the air permeability of sandwich core materials as a function of applied shear stress was developed. The core material for the test specimens was either Hexcel HRP-3/16-8.0 and or DuPont Korex-1/8-4.5 and was nominally one-half inch thick and six inches square. The facesheets where made of Hercules' AS4/8552 graphite/epoxy (Gr/Ep) composites and were nominally 0.059-in. thick. Cytec's Metalbond 1515-3M epoxy film adhesive was used for co-curing the facesheets to the core. The permeability of the specimens during both static (tension) and dynamic (reversed and non-reversed) shear loads were measured. The permeability was measured as the rate of air flow through the core from a circular 1-in2 area of the core exposed to an air pressure of 10.0 psig. In both the static and dynamic testing, the Korex core experienced sudden increases in core permeability corresponding to a core catastrophic failure, while the URP core experienced a gradual increase in the permeability prior to core failure. The Korex core failed at lower loads than the HRP core both in the transverse and ribbon directions.

  12. Heterogeneity and anisotropy of Earth's inner core

    NARCIS (Netherlands)

    Deuss, Arwen

    2014-01-01

    Seismic observations provide strong evidence that Earth's inner core is anisotropic, with larger velocity in the polar than in the equatorial direction. The top 60-80 km of the inner core is isotropic; evidence for an innermost inner core is less compelling. The anisotropy is most likely due to

  13. Improving Core Strength to Prevent Injury

    Science.gov (United States)

    Oliver, Gretchen D.; Adams-Blair, Heather R.

    2010-01-01

    Regardless of the sport or skill, it is essential to have correct biomechanical positioning, or postural control, in order to maximize energy transfer. Correct postural control requires a strong, stable core. A strong and stable core allows one to transfer energy effectively as well as reduce undue stress. An unstable or weak core, on the other…

  14. The core health science library in Canada.

    Science.gov (United States)

    Huntley, J L

    1974-04-01

    Core lists in Canada are characterized by regional differences. The lists of current importance are: (1) the British Columbia acquisitions guide for hospital libraries, (2) three Saskatchewan lists for hospitals of different sizes, (3) a core list recommended for Ontario hospitals, (4) Quebec core lists, including French language lists.

  15. The Core Health Science Library in Canada *

    Science.gov (United States)

    Huntley, June Leath

    1974-01-01

    Core lists in Canada are characterized by regional differences. The lists of current importance are: (1) the British Columbia acquisitions guide for hospital libraries, (2) three Saskatchewan lists for hospitals of different sizes, (3) a core list recommended for Ontario hospitals, (4) Quebec core lists, including French language lists. PMID:4826482

  16. Common Core: Teaching Optimum Topic Exploration (TOTE)

    Science.gov (United States)

    Karge, Belinda Dunnick; Moore, Roxane Kushner

    2015-01-01

    The Common Core has become a household term and yet many educators do not understand what it means. This article explains the historical perspectives of the Common Core and gives guidance to teachers in application of Teaching Optimum Topic Exploration (TOTE) necessary for full implementation of the Common Core State Standards. An effective…

  17. Core Journal Lists: Classic Tool, New Relevance

    Science.gov (United States)

    Paynter, Robin A.; Jackson, Rose M.; Mullen, Laura Bowering

    2010-01-01

    Reviews the historical context of core journal lists, current uses in collection assessment, and existing methodologies for creating lists. Outlines two next generation core list projects developing new methodologies and integrating novel information/data sources to improve precision: a national-level core psychology list and the other a local…

  18. Structure of Hot Molecular Cores

    OpenAIRE

    Rolffs, Rainer

    2011-01-01

    High-mass stars form deeply embedded in dense molecular gas, which they heat up and ionize due to their high energy output. During an early phase, the ionization is confined to small regions, and the stellar radiation is absorbed by dust. The high temperatures lead to the evaporation of ice mantles around dust grains, and many highly excited and complex molecules can be observed in these Hot Molecular Cores. At later stages, the whole molecular cloud is ionized and disrupted, and a...

  19. Antiferromagnetic hedgehogs with superconducting cores

    Energy Technology Data Exchange (ETDEWEB)

    Goldbart, P.M.; Sheehy, D.E. [Department of Physics and Materials Research Laboratory, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States)

    1998-09-01

    Excitations of the antiferromagnetic state that resemble antiferromagnetic hedgehogs at large distances but are predominantly superconducting inside a core region are discussed within the context of Zhang{close_quote}s SO(5)-symmetry-based approach to the physics of high-temperature superconducting materials. Nonsingular, in contrast with their hedgehog cousins in pure antiferromagnetism, these texture excitations are what hedgehogs become when the antiferromagnetic order parameter is permitted to {open_quotes}escape{close_quotes} into superconducting directions. The structure of such excitations is determined in a simple setting, and a number of their experimental implications are examined. {copyright} {ital 1998} {ital The American Physical Society}

  20. Evaluation of Fugen core management code (POLESTAR)

    Energy Technology Data Exchange (ETDEWEB)

    Shiratori, Yoshitake (Power Reactor and Nuclear Fuel Development Corp., Tsuruga, Fukui (Japan). Fugen Nuclear Power Station); Matsumoto, Mitsuo; Deshimaru, Takehide; Saito, Kuniyoshi

    1991-06-01

    Core management code POLESTAR has been developed by PNC and it has enough functions for core management. The code has been successfully used to carry out core management of Fugen such as making a long term or next cycle fuel loading plan, predicting detailed characteristics of a next cycle core, planning of a control rod pattern and evaluating the core life time after reactor start-up and so on. This code has been contributed to the reliable and economical operation of Fugen, since its accuracy has been checked and the code has been tailored by comparing its calculation results with various measured data. (author).

  1. Double U-Core Switched Reluctance Machine

    DEFF Research Database (Denmark)

    2016-01-01

    The present invention relates to an electrical machine stator comprising a plurality of stator segments (131,132,133), each segment comprises a first U-core and a second U-core wound with a winding, where the winding being arranged with at least one coil turn, each coil turn comprises a first axial......(s), wherein the first U-core and the second U-core are located adjacent to each other, whereby the winding spans the first and second U-cores. The invention also relates to a SRM machine with a stator mentioned above and a rotor....

  2. Hydrophobic-Core Microcapsules and Their Formation

    Science.gov (United States)

    Calle, Luz M. (Inventor); Li, Wenyan (Inventor); Buhrow, Jerry W. (Inventor); Jolley, Scott T. (Inventor)

    2016-01-01

    Hydrophobic-core microcapsules and methods of their formation are provided. A hydrophobic-core microcapsule may include a shell that encapsulates a hydrophobic substance with a core substance, such as dye, corrosion indicator, corrosion inhibitor, and/or healing agent, dissolved or dispersed therein. The hydrophobic-core microcapsules may be formed from an emulsion having hydrophobic-phase droplets, e.g., containing the core substance and shell-forming compound, dispersed in a hydrophilic phase. The shells of the microcapsules may be capable of being broken down in response to being contacted by an alkali, e.g., produced during corrosion, contacting the shell.

  3. Hydrophilic-Core Microcapsules and Their Formation

    Science.gov (United States)

    Calle, Luz M. (Inventor); Li, Wenyan (Inventor); Buhrow, Jerry W. (Inventor); Jolley, Scott T. (Inventor)

    2016-01-01

    Hydrophilic-core microcapsules and methods of their formation are provided. A hydrophilic-core microcapsule may include a shell that encapsulates water with the core substance dissolved or dispersed therein. The hydrophilic-core microcapsules may be formed from an emulsion having hydrophilic-phase droplets dispersed in a hydrophobic phase, with shell-forming compound contained in the hydrophilic phase or the hydrophobic phase and the core substance contained in the hydrophilic phase. The shells of the microcapsules may be capable of being broken down in response to being contacted by an alkali, e.g., produced during corrosion, contacting the shell.

  4. 77 FR 30435 - In-core Thermocouples at Different Elevations and Radial Positions in Reactor Core

    Science.gov (United States)

    2012-05-23

    ... COMMISSION 10 CFR Part 50 In-core Thermocouples at Different Elevations and Radial Positions in Reactor Core... ``require all holders of operating licenses for nuclear power plants (``NPP'') to operate NPPs with in-core thermocouples at different elevations and radial positions throughout the reactor core.'' DATES: Submit...

  5. Impaired N-linked glycosylation of uptake and efflux transporters in human non-alcoholic fatty liver disease.

    Science.gov (United States)

    Clarke, John D; Novak, Petr; Lake, April D; Hardwick, Rhiannon N; Cherrington, Nathan J

    2017-07-01

    N-linked glycosylation of proteins is critical for proper protein folding and trafficking to the plasma membrane. Drug transporters are one class of proteins that have reduced function when glycosylation is impaired. N-linked glycosylation of plasma proteins has also been investigated as a biomarker for several liver diseases, including non-alcoholic fatty liver disease (NAFLD). The purpose of this study was to assess the transcriptomic expression of genes involved in protein processing and glycosylation, and to determine the glycosylation status of key drug transporters during human NAFLD progression. Human liver samples diagnosed as healthy, steatosis, and non-alcoholic steatohepatitis (NASH) were analysed for gene expression of glycosylation-related genes and for protein glycosylation using immunoblot. Genes involved in protein processing in the ER and biosynthesis of N-glycans were significantly enriched for down-regulation in NAFLD progression. Included in the down regulated N-glycan biosynthesis category were genes involved in the oligosaccharyltransferase complex, N-glycan quality control, N-glycan precursor biosynthesis, N-glycan trimming to the core, and N-glycan extension from the core. N-glycan degradation genes were unaltered in the progression to NASH. Immunoblot analysis of the uptake transporters organic anion transporting polypeptide-1B1 (OATP1B1), OATP1B3, OATP2B1, and Sodium/Taurocholate Co-transporting Polypeptide (NTCP) and the efflux transporter multidrug resistance-associated protein 2 (MRP2) demonstrated a significant loss of glycosylation following the progression to NASH. These data suggest that the loss of glycosylation of key uptake and efflux transporters in humans NASH may influence transporter function and contribute to altered drug disposition observed in NASH. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Dependence of Core and Extended Flux on Core Dominance Parameter for Radio Sources

    Indian Academy of Sciences (India)

    J. J. Nie; J. H. Yang

    2014-09-01

    Based on two extragalactic radio source samples, the core dominance parameter is calculated, and the correlations between the core/extended flux density and core dominance parameter are investigated. When the core dominance parameter is lower than unity, it is linearly correlated with the core flux density, but it is not correlated with the extended flux density. When the core dominance parameter is higher than unity, it is not correlated with the core flux density, but it is linearly correlated with the extended flux density. Therefore, there are different results from different samples. The results can be explained using a relativistic beaming model.

  7. Drilling history core hole DC-8

    Energy Technology Data Exchange (ETDEWEB)

    1978-10-01

    Core hole DC-8 was completed in August, 1978 by Boyles Brothers Drilling Company, Spokane, Washington, under subcontract to Fenix and Scission, Inc. The hole was cored for the US Department of Energy and the Rockwell Hanford Operations' Basalt Waste Isolation Program. Fenix and Scisson, Inc. furnished the engineering, daily supervision of the core drilling activities, and geologic core logging for hole DC-8. Core hole DC-8 is located on the Hanford Site near the Wye Barricade and 50 feet northwest of rotary hole DC-7. The Hanford Site vation coordinates for DC-8 are North 14,955.94 feet and West 14,861.92 coordinates for DC-8 are North 14,955.94 feet and West 14,861.92 mean sea level. The purpose of core hole DC-8 was to core drill vertically through the basalt and interbed units for stratigraphic depth determination and core collection, and to provide a borehole for hydrologic testing and cross-hole seismic shear and pressure wave velocity studies with rotary hole DC-7. The total depth of core hole DC-8 was 4100.5 feet. Core recovery exceeded 97 percent of the total footage cored.

  8. Functional Screening of Core Promoter Activity.

    Science.gov (United States)

    Even, Dan Y; Kedmi, Adi; Ideses, Diana; Juven-Gershon, Tamar

    2017-01-01

    The core promoter is the DNA sequence that recruits the basal transcription machinery and directs accurate initiation of transcription. It is an active contributor to gene expression that can be rationally designed to manipulate the levels of expression. Core promoter function can be analyzed using different experimental approaches. Here, we describe the qualitative and quantitative analysis of engineered core promoter functions using the EGFP reporter gene that is driven by distinct core promoters. Expression plasmids are transfected into different mammalian cell lines, and the resulting fluorescence is monitored by live cell imaging , as well as by flow cytometry. In order to verify that the transcriptional activity of the examined core promoters is indeed a function of their activity, as opposed to differences in DNA uptake, real-time quantitative PCR analysis is performed. Importantly, the described methodology for functional screening of core promoter activity has enabled the analysis of engineered potent core promoters for extended time periods.

  9. Research on plasma core reactors

    Energy Technology Data Exchange (ETDEWEB)

    Jarvis, G.A.; Barton, D.M.; Helmick, H.H.; Bernard, W.; White, R.H.

    1977-01-01

    Experiments and theoretical studies are being conducted for NASA on critical assemblies with 1-m-diam by 1-m-long low-density cores surrounded by a thick beryllium reflector. These assemblies make extensive use of existing nuclear propulsion reactor components, facilities, and instrumentation. Due to excessive porosity in the reflector, the initial critical mass was 19 kg U(93.2). Addition of a 17-cm-thick by 89-cm-diam beryllium flux trap in the cavity reduced the critical mass to 7 kg when all the uranium was in the zone just outside the flux trap. A mockup aluminum UF/sub 6/ container was placed inside the flux trap and fueled with uranium-graphite elements. Fission distributions and reactivity worths of fuel and structural materials were measured. Finally, an 85,000-cm/sup 3/ aluminum canister in the central region was fueled with UF/sub 6/ gas and fission density distributions determined. These results will be used to guide the design of a prototype plasma core reactor which will test energy removal by optical radiation.

  10. Electrostatically enhanced core separator system

    Energy Technology Data Exchange (ETDEWEB)

    Easom, B.H.; Smolensky, L.A.; Altman, R.F. [LSR Technologies, Inc., Acton, MA (United States)

    1997-12-31

    The Electrostatically Enhanced Core Separator (EECS) system employs the same design principles as the mechanical Core Separator system plus an electrostatic separation enhancing technique. The EECS system contains a special type of separator, the EECS element, a conventional solids collector and means for flow recirculation. In the EECS system solids separation and collection are accomplished in two different components. The EECS element acts as a separator, not as a collector so particles are not collected on its walls. This eliminates or at least mitigates the problems associated with reentrainment (due to high or low dust resistivity), seepage (due to gas flow below the precipitator plates and over the hoppers), sneakage (due to gas flow both above and below the precipitator plates), and rapping reentrainment. If the EECS separation efficiency is high enough, particles cannot leave the system with the process stream. They recirculate until they are extracted by the collector. As a result, the separation efficiency of the EECS element determines the efficiency of the system, even if the collector efficiency is relatively low. 8 refs., 3 figs.

  11. PHYLOMETABOLIC CORE OF INTESTINAL MICROBIOTA

    Directory of Open Access Journals (Sweden)

    S. I. Sitkin

    2015-01-01

    Full Text Available The authors  discuss the  theory  of human  superorganism and its microbiota (microbiome, whose mutualistic  interactions  is realized within the  microbiota – gut – brain axis that includes endocrine, immune and neurohumoral pathways. The newest concepts  of microbiome enterotypes and core microbiota  are  presented, which are  important  for understanding of the  role of symbiotic  microorganisms  in human  vital activities, for explanation of pathophysiology of many  chronic  human  diseases  (beyond  gastrointestinal disorders, as well as for the  search of effective therapeutic targets. As highly promising are considered  the functional approaches to studies  of microbiota  that  allowed to formulate the concept  of phylometabolic (phylofunctional core. This is a series of evolutionally stable microorganisms  responsible  for majority of the  main microbiome  functions, such as fermentation  of polysaccharides  (glycans, production of short-chain  fatty acids (butyrate, propionate, acetate, hydrogen  utilization, production of lactate, metabolism of aminoacids, bile acids, choline, production  of vitamins and  some  biologically active substances – anti-inflammatory, anti-microbial, immunostimulatory. The authors are first to describe the main functional groups  of microorganisms  of   gut microbiota phylometabolic core, providing key metabolic functions, as well as the leading characteristics of the  phylometabolic core as such. The perspectives  of modification  of composition  and functions  of phylometabolic microbiota  core are discussed based on metabiotics  as a virtually new class of therapeutic agents. A hypothesis has been proposed that  the  ratios  between main  components of the key gut microbiota may reflect fundamental  processed  related  to a mutualistic interactions between microbiota and human body, as well as they may serve as effective biological markers of

  12. Melting of the Earth's inner core.

    Science.gov (United States)

    Gubbins, David; Sreenivasan, Binod; Mound, Jon; Rost, Sebastian

    2011-05-19

    The Earth's magnetic field is generated by a dynamo in the liquid iron core, which convects in response to cooling of the overlying rocky mantle. The core freezes from the innermost surface outward, growing the solid inner core and releasing light elements that drive compositional convection. Mantle convection extracts heat from the core at a rate that has enormous lateral variations. Here we use geodynamo simulations to show that these variations are transferred to the inner-core boundary and can be large enough to cause heat to flow into the inner core. If this were to occur in the Earth, it would cause localized melting. Melting releases heavy liquid that could form the variable-composition layer suggested by an anomaly in seismic velocity in the 150 kilometres immediately above the inner-core boundary. This provides a very simple explanation of the existence of this layer, which otherwise requires additional assumptions such as locking of the inner core to the mantle, translation from its geopotential centre or convection with temperature equal to the solidus but with composition varying from the outer to the inner core. The predominantly narrow downwellings associated with freezing and broad upwellings associated with melting mean that the area of melting could be quite large despite the average dominance of freezing necessary to keep the dynamo going. Localized melting and freezing also provides a strong mechanism for creating seismic anomalies in the inner core itself, much stronger than the effects of variations in heat flow so far considered.

  13. Comparative N-linked glycan analysis of wild-type and α1,3-galactosyltransferase gene knock-out pig fibroblasts using mass spectrometry approaches.

    Science.gov (United States)

    Park, Hae-Min; Kim, Yoon-Woo; Kim, Kyoung-Jin; Kim, Young June; Yang, Yung-Hun; Jin, Jang Mi; Kim, Young Hwan; Kim, Byung-Gee; Shim, Hosup; Kim, Yun-Gon

    2015-01-31

    Carbohydrate antigens expressed on pig cells are considered to be major barriers in pig-to-human xenotransplantation. Even after α1,3-galactosyltransferase gene knock-out (GalT-KO) pigs are generated, potential non-Gal antigens are still existed. However, to the best of our knowledge there is no extensive study analyzing N-glycans expressed on the GalT-KO pig tissues or cells. Here, we identified and quantified totally 47 N-glycans from wild-type (WT) and GalT-KO pig fibroblasts using mass spectrometry. First, our results confirmed the absence of galactose-alpha-1,3-galactose (α-Gal) residue in the GalT-KO pig cells. Interestingly, we showed that the level of overall fucosylated N-glycans from GalT-KO pig fibroblasts is much higher than from WT pig fibroblasts. Moreover, the relative quantity of the N-glycolylneuraminic acid (NeuGc) antigen is slightly higher in the GalT-KO pigs. Thus, this study will contribute to a better understanding of cellular glycan alterations on GalT-KO pigs for successful xenotransplantation.

  14. The Transcendental Core of Correlationism

    Directory of Open Access Journals (Sweden)

    Paul J. Ennis

    2011-06-01

    Full Text Available In this paper I read Quentin Meillassoux’s critique of correlationism as truly a critique of transcendentalism and the transcendental method. I do so by considering the two correlationist rejoinders that occur in the English edition of Meillassoux’s After Finitude. The first rejoinder is from an idealist and relies on adumbrations for its defence. This reliance on adumbrations will be shown to be itself transcendentally implicated through Edmund Husserl’s Crisis of the European Sciences and Transcendental Phenomenology. I then turn to the explicit engagement with the transcendental method that arises from the transcendentalist’s rejoinder. Considered together I hope to convince the reader that the core of correlationism is transcendentalism.

  15. Thermohydraulics of LMFBR core catchers

    Science.gov (United States)

    Turland, B. D.

    Characterization of the likely form of fuel debris after an accident, following interaction with sodium in the primary vessel and mechanisms controlling the location of the debris in the primary system is discussed. Heat transfer from particulate to liquid sodium and the development of models predicting the amount of debris that may be retained in a coolable form on a structure are considered. The evaluation of the coolability of the structure itself in post accident conditions, particularly the cooling provided by natural convection alone is treated. The response of structures at elevated temperatures and under high thermal loads is considered. The potential for vessel failure if significant quantities of debris accumulate at the bottom of the vessel is shown. The performance of a flat plate core catcher, or similar structure with good cooling from underneath is evaluated.

  16. Final Report - BRER Core Support

    Energy Technology Data Exchange (ETDEWEB)

    Evan B. Douple

    2007-01-09

    This contract provided core support for activities of the advisory committee of experts comprising the Board on Radiation Effects Research (BRER), in The National Academies' Division on Earth and Life Studies. That committee met two times during the funding period. The committee members provided oversight and advice regarding ongoing BRER projects and also assisted in the identification of potential committee members for new studies and the development of proposals for projects in the radiation sciences worthy for future study. In addition, funding provided support for the planning, advertisement, and invited speakers' travel-expense reimbursement for the Third and Fourth Gilbert W. Beebe Symposia held at The National Academies on December 1, 2004 and on November 30, 2005, respectively.

  17. On-line core axial power distribution synthesis method from in-core and ex-core neutron detectors

    Energy Technology Data Exchange (ETDEWEB)

    In, Wang Kee; Cho, Byung Oh [Korea Atomic Energy Research Institute, Taejon (Korea)

    1999-10-01

    This document describes the methodology in detail and the synthesis coefficients of the Fourier series expansion and the cubic spline synthesis techniques. A computer program was developed to generate the synthesis coefficients and the core power distribution. For the illustration, various axial power shapes for YGN 3 Cycle 1 and SMART were synthesized using the simulated in-core and/or ex-core detector signals. The results of this study will be useful to select the best synthesis method for the SMART core monitoring and protection systems and to evaluate the accuracy of the synthesized power shape. 4 refs., 13 figs., 5 tabs. (Author)

  18. 75 FR 52596 - Financial Education Core Competencies; Comment Request

    Science.gov (United States)

    2010-08-26

    ... Financial Education Core Competencies; Comment Request AGENCY: Department of the Treasury. ACTION: Notice... proposed set of financial education core competencies (``Core Competencies''). Comments are requested specifically on whether the list of Core Competencies referenced in the Supplementary Section is complete...

  19. Inner Core Rotation from Geomagnetic Westward Drift and a Stationary Spherical Vortex in Earth's Core

    Science.gov (United States)

    Voorhies, C. V.

    1999-01-01

    The idea that geomagnetic westward drift indicates convective leveling of the planetary momentum gradient within Earth's core is pursued in search of a differentially rotating mean state, upon which various oscillations and secular effects might be superimposed. The desired state conforms to roughly spherical boundary conditions, minimizes dissipative interference with convective cooling in the bulk of the core, yet may aide core cooling by depositing heat in the uppermost core and lower mantle. The variational calculus of stationary dissipation applied to a spherical vortex within the core yields an interesting differential rotation profile akin to spherical Couette flow bounded by thin Hartmann layers. Four boundary conditions are required. To concentrate shear induced dissipation near the core-mantle boundary, these are taken to be: (i) no-slip at the core-mantle interface; (ii) geomagnetically estimated bulk westward flow at the base of the core-mantle boundary layer; (iii) no-slip at the inner-outer core interface; and, to describe magnetic locking of the inner core to the deep outer core, (iv) hydrodynamically stress-free at the inner-outer core boundary. By boldly assuming the axial core angular momentum anomaly to be zero, the super-rotation of the inner core is calculated to be at most 1.5 degrees per year.

  20. Advantages of iron core in a tokamak

    Energy Technology Data Exchange (ETDEWEB)

    Bettis, E.S.; Ballou, J.K.; Becraft, W.R.; Peng, Y.K.M.; Watts, H.L.

    1977-01-01

    A quantitative comparison of the iron core vs air core concepts was carried out on a preliminary basis by using a representative tokamak reactor design with the following self-consistent reference parameters. In the area of plasma engineering, poloidal field and MHD equilibrium considerations with an unsaturated iron core is discussed. The question of proper poloidal field coils to maintain D-shaped plasmas of relatively high anti ..beta.. (7%) with a saturated iron core is also discussed. Estimates of the required iron core size, volt seconds, magnetic flux and its influence on force loading on the superconducting toroidal field coils are shown. Conceptual designs of the mechanical structure of an iron core device are presented. Favorable impacts on the OH power supply cost and complexity are indicated.

  1. Cool Core Clusters from Cosmological Simulations

    CERN Document Server

    Rasia, E; Murante, G; Planelles, S; Beck, A M; Biffi, V; Ragone-Figueroa, C; Granato, G L; Steinborn, L K; Dolag, K

    2015-01-01

    We present results obtained from a set of cosmological hydrodynamic simulations of galaxy clusters, aimed at comparing predictions with observational data on the diversity between cool-core and non-cool-core clusters. Our simulations include the effects of stellar and AGN feedback and are based on an improved version of the Smoothed-Particle-Hydrodynamics code GADGET-3, which ameliorates gas mixing and better captures gas-dynamical instabilities by including a suitable artificial thermal diffusion. In this Letter, we focus our analysis on the entropy profiles, our primary diagnostic to classify the degree of cool-coreness of clusters, and on the iron profiles. In keeping with observations, our simulated clusters display a variety of behaviors in entropy profiles: they range from steadily decreasing profiles at small radii, characteristic of cool-core systems, to nearly flat core isentropic profiles, characteristic of non cool-core systems. Using observational criteria to distinguish between the two classes of...

  2. Modeling of Pulsed Transformer with Nanocrystalline Cores

    Directory of Open Access Journals (Sweden)

    Amir Baktash

    2014-07-01

    Full Text Available Recently tape wound cores, due to their excellent properties, are widely used in transformers for pulsed or high frequency applications. The spiral structure of these cores affects the flux distribution inside the core and causes complication of the magnetic analysis and consequently the circuit analysis. In this paper, a model based on reluctance networks method is used to analyze the magnetic flux in toroidal wound cores and losses calculation. A Preisach based hysteresis model is included in the model to consider the nonlinear characteristic of the core. Magnetic losses are calculated by having the flux density in different points of the core and using the hysteresis model. A transformer for using in a series resonant converter is modeled and implemented. The modeling results are compared with experimental measurements and FEM results to evaluate the validity of the model. Comparisons show the accuracy of the model besides its simplicity and fast convergence.

  3. Reinventing the Platform Core Through Acquisition

    DEFF Research Database (Denmark)

    Toppenberg, Gustav; Henningsson, Stefan; Eaton, Ben

    2016-01-01

    Digital platform leaders need to continuously innovate the platform core to drive the technological trajectory of the overall architecture and business system, of which the platform is a core element. This papers analyses the potential of and challenges to completing this task through...... the acquisition and integration of companies presenting innovative technologies of relevance to the platform core. Using a revelatory case study of Cisco Systems, we develop the explanatory notion of ‘coring acquisition’. In this type of acquisition value is created through the acquisition of companies...... that provide products external to the acquirer that can be assimilated into the platform core. This creates value through the transformational process that we term ‘coring’. We also analyze how the benefits of coring acquisitions are contingent on challenges concerning the integration of acquisitions offering...

  4. Optical fiber sensor having an active core

    Science.gov (United States)

    Egalon, Claudio Oliveira (Inventor); Rogowski, Robert S. (Inventor)

    1993-01-01

    An optical fiber is provided. The fiber is comprised of an active fiber core which produces waves of light upon excitation. A factor ka is identified and increased until a desired improvement in power efficiency is obtained. The variable a is the radius of the active fiber core and k is defined as 2 pi/lambda wherein lambda is the wavelength of the light produced by the active fiber core. In one embodiment, the factor ka is increased until the power efficiency stabilizes. In addition to a bare fiber core embodiment, a two-stage fluorescent fiber is provided wherein an active cladding surrounds a portion of the active fiber core having an improved ka factor. The power efficiency of the embodiment is further improved by increasing a difference between the respective indices of refraction of the active cladding and the active fiber core.

  5. Design Principles for Synthesizable Processor Cores

    DEFF Research Database (Denmark)

    Schleuniger, Pascal; McKee, Sally A.; Karlsson, Sven

    2012-01-01

    As FPGAs get more competitive, synthesizable processor cores become an attractive choice for embedded computing. Currently popular commercial processor cores do not fully exploit current FPGA architectures. In this paper, we propose general design principles to increase instruction throughput...... on FPGA-based processor cores: first, superpipelining enables higher-frequency system clocks, and second, predicated instructions circumvent costly pipeline stalls due to branches. To evaluate their effects, we develop Tinuso, a processor architecture optimized for FPGA implementation. We demonstrate...

  6. Design Principles for Synthesizable Processor Cores

    DEFF Research Database (Denmark)

    Schleuniger, Pascal; McKee, Sally A.; Karlsson, Sven

    2012-01-01

    As FPGAs get more competitive, synthesizable processor cores become an attractive choice for embedded computing. Currently popular commercial processor cores do not fully exploit current FPGA architectures. In this paper, we propose general design principles to increase instruction throughput...... through the use of micro-benchmarks that our principles guide the design of a processor core that improves performance by an average of 38% over a similar Xilinx MicroBlaze configuration....

  7. The Fluxgate Ring-Core Internal Field

    DEFF Research Database (Denmark)

    Primdahl, Fritz; Brauer, Peter; Merayo, José M.G.

    2002-01-01

    A large number of measured demagnetizing factors for fluxgate ring cores of a wide range of cross section shapes have been compiled from the literature and plotted against the core cross-sectional area over the squared mean core diameter. The points group close to a straight line through the origin...... that the demagnetizing factor for thin rings is proportional to the ring cross-sectional area divided by the diameter squared....

  8. Briefing paper for universities on Core Maths

    OpenAIRE

    Glaister, Paul

    2015-01-01

    This briefing paper outlines the rationale for and development of the new Core Maths qualifications, the characteristics of Core Maths, and why Core Maths is important for higher education. It is part of a communication to university vice-chancellors from the Department for Business, Innovation and Skills (BIS) comprising this paper and a joint Ministerial letter from Jo Johnson, Minister of State for Universities and Science in BIS, and Nick Gibb, Minister of State for Schools in the Departm...

  9. Impact of core-cladding boundary shape on the waveguide properties of hollow core microstructured fibers

    Science.gov (United States)

    Pryamikov, A. D.; Alagashev, G. K.; Kosolapov, A. F.; Biriukov, A. S.

    2016-12-01

    In this paper we consider an interaction between the air core modes (ACMs) of hollow core waveguide microstructures and core-cladding boundary walls in various shapes. The analysis is based on well-established models such as the ARROW (anti-resonant reflecting optical waveguide) model and on the models proposed for the first time. In particular, we consider the dynamics of light localization in the polygonal core cladding boundary wall as dependant on the type of its discrete rotational symmetry. Based on our findings we analyze the mechanisms of light localization in the core-cladding boundary walls of negative curvature hollow core microstructured fibers (NC HCMFs).

  10. Fucosylation and protein glycosylation create functional receptors for cholera toxin

    DEFF Research Database (Denmark)

    Wands, Amberlyn M; Fujita, Akiko; McCombs, Janet E

    2015-01-01

    Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors using its B subunit (CTB). The ganglioside (glycolipid) GM1 is thought to be the sole CT receptor; however, the mechanism by which CTB binding to GM1 mediates internalization of CT remains enigmatic. Here we...

  11. Material with core-shell structure

    Science.gov (United States)

    Luhrs, Claudia; Richard, Monique N.; Dehne, Aaron; Phillips, Jonathan; Stamm, Kimber L.; Fanson, Paul T.

    2011-11-15

    Disclosed is a material having a composite particle, the composite particle including an outer shell and a core. The core is made from a lithium alloying material and the outer shell has an inner volume that is greater in size than the core of the lithium alloying material. In some instances, the outer mean diameter of the outer shell is less than 500 nanometers and the core occupies between 5 and 99% of the inner volume. In addition, the outer shell can have an average wall thickness of less than 100 nanometers.

  12. The Cores of Elliptical Galaxies in Coma

    Science.gov (United States)

    Lucey, John

    1995-07-01

    The cores of galaxies are astrophysically unique. They canhost high energy nuclei, star formation and perhaps even blackholes. HST observations have established that the cores ofellipticals are related to their global properties, and so canbe used as diagnostics of the physical processes occurring atthe time of formation. HST images of galaxy cores havedistinguished two different types of core luminosity profiles:`soft' and `hard' types. It is suggested that luminous, slowlyrotating galaxies have `soft' cores and the less luminousdisky galaxies have `hard' cores. This can be interpreted interms of a formation scenario based on a merger hierarchy inwhich the low luminosity systems experience highly dissipativemergers, but as the luminous systems are assembled the mergersbecome increasingly stellar. In this picture, the type of corea galaxy generates is intimately related to its evolutionaryhistory, i.e. the degree of interaction/merging experiencedand the availability of cold gas. In turn, this should notonly depend on luminosity but also on the galaxy's localenvironment. Here we propose to test the gaseous/stellarmerger picture by imaging a set of Coma cluster ellipticalsfrom a wide range of cluster radii. In the gas poorenvironment of the cluster core there may be insufficent coldgas for the low luminosity galaxies to form `hard' cores.Similarly, at the cluster turnround radius even luminousgalaxies may have experienced a dissipative core formation andpossess

  13. The nature of the earth's core

    Science.gov (United States)

    Jeanloz, Raymond

    1990-01-01

    The properties of the earth's core are overviewed with emphasis on seismologically determined regions and pressures and seismologically measured density, elastic wave velocities, and gravitational acceleration. Attention is given to solid-state convection of the inner core, and it is noted that though seismological results do not conclusively prove that the inner core is convective, the occurrence and magnitude of seismic anisotropy are explained by the effects of solid-state convection. Igneous petrology and geochemistry of the inner core, a layer at the base of the mantle and contact metasomatism at the core-mantle boundary, and evolution of the core-mantle system are discussed. It is pointed out that high-pressure melting experiments indicate that the temperature of the core is ranging from 4500 to 6500 K, and a major implication of such high temperature is that the tectonics and convection of the mantle, as well as the resulting geological processes observed at the surface, are powered by heat from the core. As a result of the high temperatures, along with the compositional contrast between silicates and iron alloy, the core-mantle boundary is considered to be most chemically active region of the earth.

  14. Optimized Cellular Core for Rotorcraft Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Patz Materials and Technologies proposes to develop a unique structural cellular core material to improve mechanical performance, reduce platform weight and lower...

  15. Recent Problems of Transformer Core Design

    Science.gov (United States)

    Valkovic, Z.

    1988-01-01

    The paper describes the result of the investigations of the efficiency of power loss reduction in transformer cores made with high-permeability (HGO) and laser scribed (LS) grain-oriented electrical steels, and also the phenomena in three-limb three-phase cores with the so-called staggered T-joint design. The efficiency of the HGO material depends on core form and core induction. The efficiency is better for single-phase than for three-phase cores and also for higher induction. The localised efficiency of HGO material is not uniform and it is significantly lower in the yoke than in other parts. The efficiency of LS material (grade ZDKH) is better than that of the HGO material and also somewhat higher for single-phase than for three-phase cores. The localised flux distribution in the central limb of the core with staggered T-joint is more uniform and the content of higher harmonics is smaller than in the core with conventional V-45° T-joint. This results in a 13% loss reduction in the central limb and in a 4-5% reduction of total core loss.

  16. Challenges Regarding IP Core Functional Reliability

    Science.gov (United States)

    Berg, Melanie D.; LaBel, Kenneth A.

    2017-01-01

    For many years, intellectual property (IP) cores have been incorporated into field programmable gate array (FPGA) and application specific integrated circuit (ASIC) design flows. However, the usage of large complex IP cores were limited within products that required a high level of reliability. This is no longer the case. IP core insertion has become mainstream including their use in highly reliable products. Due to limited visibility and control, challenges exist when using IP cores and subsequently compromise product reliability. We discuss challenges and suggest potential solutions to critical application IP insertion.

  17. More on core instabilities of magnetic monopoles

    CERN Document Server

    Striet, J

    2003-01-01

    In this paper we present new results on the core instability of the 't Hooft Polyakov monopoles we reported on before. This instability, where the spherical core decays in a toroidal one, typically occurs in models in which charge conjugation is gauged. In this paper we also discuss a third conceivable configuration denoted as ``split core'', which brings us to some details of the numerical methods we employed. We argue that a core instability of 't Hooft Polyakov type monopoles is quite a generic feature of models with charged Higgs particles.

  18. Nuclear reactor core modelling in multifunctional simulators

    Energy Technology Data Exchange (ETDEWEB)

    Puska, E.K. [VTT Energy, Nuclear Energy, Espoo (Finland)

    1999-06-01

    The thesis concentrates on the development of nuclear reactor core models for the APROS multifunctional simulation environment and the use of the core models in various kinds of applications. The work was started in 1986 as a part of the development of the entire APROS simulation system. The aim was to create core models that would serve in a reliable manner in an interactive, modular and multifunctional simulator/plant analyser environment. One-dimensional and three-dimensional core neutronics models have been developed. Both models have two energy groups and six delayed neutron groups. The three-dimensional finite difference type core model is able to describe both BWR- and PWR-type cores with quadratic fuel assemblies and VVER-type cores with hexagonal fuel assemblies. The one- and three-dimensional core neutronics models can be connected with the homogeneous, the five-equation or the six-equation thermal hydraulic models of APROS. The key feature of APROS is that the same physical models can be used in various applications. The nuclear reactor core models of APROS have been built in such a manner that the same models can be used in simulator and plant analyser applications, as well as in safety analysis. In the APROS environment the user can select the number of flow channels in the three-dimensional reactor core and either the homogeneous, the five- or the six-equation thermal hydraulic model for these channels. The thermal hydraulic model and the number of flow channels have a decisive effect on the calculation time of the three-dimensional core model and thus, at present, these particular selections make the major difference between a safety analysis core model and a training simulator core model. The emphasis on this thesis is on the three-dimensional core model and its capability to analyse symmetric and asymmetric events in the core. The factors affecting the calculation times of various three-dimensional BWR, PWR and WWER-type APROS core models have been

  19. Core-size-dependent catalytic properties of bimetallic Au/Ag core-shell nanoparticles.

    Science.gov (United States)

    Haldar, Krishna Kanta; Kundu, Simanta; Patra, Amitava

    2014-12-24

    Bimetallic core-shell nanoparticles have recently emerged as a new class of functional materials because of their potential applications in catalysis, surface enhanced Raman scattering (SERS) substrate and photonics etc. Here, we have synthesized Au/Ag bimetallic core-shell nanoparticles with varying the core diameter. The red-shifting of the both plasmonic peaks of Ag and Au confirms the core-shell structure of the nanoparticles. Transmission electron microscopy (TEM) analysis, line scan EDS measurement and UV-vis study confirm the formation of core-shell nanoparticles. We have examined the catalytic activity of these core-shell nanostructures in the reaction between 4-nitrophenol (4-NP) and NaBH4 to form 4-aminophenol (4-AP) and the efficiency of the catalytic reaction is found to be increased with increasing the core size of Au/Ag core-shell nanocrystals. The catalytic efficiency varies from 41.8 to 96.5% with varying core size from 10 to 100 nm of Au/Ag core-shell nanoparticles, and the Au100/Ag bimetallic core-shell nanoparticle is found to be 12-fold more active than that of the pure Au nanoparticles with 100 nm diameter. Thus, the catalytic properties of the metal nanoparticles are significantly enhanced because of the Au/Ag core-shell structure, and the rate is dependent on the size of the core of the nanoparticles.

  20. Core Forensics: Earth's Accretion and Differentiation

    Science.gov (United States)

    Badro, J.; Brodholt, J. P.; Siebert, J.; Piet, H.; Ryerson, F. J.

    2013-12-01

    Earth's accretion and its primitive differentiation are intimately interlinked processes. One way to constrain accretionary processes is by looking at the major differentiation event that took place during accretion: core formation. Understanding core formation and core composition can certainly shed a new light on early and late accretionary processes. On the other hand, testing certain accretionary models and hypothesis (fluxes, chemistries, timing) allows -short of validating them- at the very least to unambiguously refute them, through the 'filter'' of core formation and composition. Earth's core formed during accretion as a result of melting, phase-separation, and segregation of accretionary building blocks (from meteorites to planetesimals). The bulk composition of the core and mantle depends on the evolution (pressure, temperature, composition) of core extraction during accretion. The entire process left a compositional imprint on both reservoirs: (1) in the silicate Earth, in terms of siderophile trace-element (Ni, Co, V, Cr, among others) concentrations and isotopic fractionation (Si, Cu, among others), a record that is observed in present-day mantle rocks; and (2) on the core, in terms of major element composition and light elements dissolved in the metal, a record that is observed by seismology through the core density-deficit. This imprint constitutes actually a fairly impressive set of evidence (siderophile element concentration and fractionation, volatile and siderophile element isotopic fractionation), can be used today to trace back the primordial processes that occurred 4.5 billion years ago. We are seeking to provide an overhaul of the standard core formation/composition models, by using a new rationale that bridges geophysics and geochemistry. The new ingredients are (1) new laser-heated diamond anvil cell partitioning data, dramatically extending the previous P-T conditions for experimental work, (2) ab initio molecular dynamics calculations to