text:
Get notified about updates to this publication
Page 1
Drosophila neurally altered carbohydrate mutant 1 The Drosophila Neurally Altered Carbohydrate Mutant Has a Defective Golgi GDP-Fucose Transporter* Christoph Geisler‡, Varshika Kotu§, Mary Sharrow§, Dubravko Rendić¶, Gerald Pöltl¶, Michael Tiemeyer§, Iain B.H. Wilson¶, and Donald L. Jarvis‡,1 From ‡the Department of Molecular Biology, University of Wyoming, Laramie, WY 82071; §the Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602; and ¶the Department für Chemie, Universität für Bodenkultur, A-1190 Wien, Austria Running title: Drosophila neurally altered carbohydrate mutant 1To whom correspondence should be addressed: Donald L. Jarvis, Department of Molecular Biology, University of Wyoming, Laramie, WY, USA, Tel.: (307)-766-4282; Fax: (307)-766-5098; E-mail: email@example.com Keywords: Congenital disorders of glycosylation, Drosophila, Fucosylation, GDP-fucose transporter, Glycobiology, Glycoprotein biosynthesis, Mutant, N-glycosylation, HRP epitope, Sugar transport Background: The defect underlying reduced HRP epitope expression in Drosophila nac1 mutants has not been identified. Results: nac1 flies have a defective GDP-fucose transporter. Conclusion: The defective nac1 transporter cannot support normal N-glycan core fucosylation, leading to reduced HRP epitope expression. Significance: nac1 flies are a valid model for the human congenital disorder of glycosylation, CDG-IIc, which is caused by a similar molecular defect. SUMMARY Studying genetic disorders in model organisms can provide insights into heritable human diseases. The Drosophila neurally altered carbohydrate (nac) mutant is deficient for neural expression of the HRP epitope, which consists of N-glycans with core α1,3-linked fucose residues. Here, we show that a conserved serine residue in the Golgi GDP-fucose transporter (GFR) is substituted by leucine in nac1 flies, which abolishes GDP-fucose transport in vivo and in vitro. This loss of function is due to a biochemical defect, not to destabilization or mis-targeting of the mutant GFR protein. Mass spectrometry and HPLC analysis showed that nac1 mutants lack not only core α1,3-linked, but also core α1,6-linked fucose residues on their N-glycans. Thus, the nac1 Gfr mutation produces a previously unrecognized general defect in N-glycan core fucosylation. Transgenic expression of a wild-type Gfr gene restored the HRP epitope in neural tissues, directly demonstrating that the Gfr mutation is solely responsible for the neural HRP epitope deficiency in the nac1 mutant. These results validate the Drosophila nac1 mutant as a model for the human congenital disorder of glycosylation, CDG-IIc (also known as LAD-II), which is also the result of a GFR deficiency. Congenital disorders of glycosylation (CDG's) are a phenotypically diverse group of heritable diseases caused by mutations in genes functioning in glycosylation (recently reviewed by 1-3). The first congenital disorders that were recognized to have altered glycosylation patterns were identified in humans (4-7). These disorders were later found to involve deficiencies in N-glycan biosynthesis and processing (8-11) that were caused by mutations in genes encoding those functions (12-15). http://www.jbc.org/cgi/doi/10.1074/jbc.M112.379313The latest version is at JBC Papers in Press. Published on June 28, 2012 as Manuscript M112.379313 Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc. at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 2
Drosophila neurally altered carbohydrate mutant 2 The first CDG reported in a non-human organism was nac (neurally altered carbohydrate), which was identified in Drosophila. nac mutants have reduced levels of a neural carbohydrate epitope that is produced by α1,3-linkage of a fucose residue to the N-glycan core (16-18). Due to its identification as a dominant epitope in the plant glycoprotein horseradish peroxidase, this core α1,3-fucosylated N-glycan is also known as the horseradish peroxidase (HRP) epitope (19,20). In Drosophila, the HRP epitope is expressed mainly in the central nervous system (CNS, 21-23), where it is produced by a fucosyltransferase designated FucTA (24-26). FucTA is a Golgi resident enzyme that transfers fucose from the donor substrate GDP-fucose to the proximal N-acetylglucosamine residue of N-glycans (Fig. 1A). GDP-fucose is produced in the cytoplasm (27) and transported into the Golgi apparatus by GFR, a specific GDP-fucose transporter, in exchange for GMP (Fig. 1A, 28,29). The Drosophila Gfr gene is homologous to the human Gfr gene, which is defective in a congenital disorder of glycosylation known as CDG-IIc and also known as Type II leukocyte adhesion deficiency (LAD-II, 30,31) or SLC35C1-CDG, (32). The original nac mutant, which was later re-designated nac1 to distinguish it from other alleles, has a temperature-insensitive loss of the neural HRP epitope associated with other cold-sensitive phenotypes expressed at 18°C, but not 25°C (33,34). Katz and coworkers cytogenetically mapped the nac1 mutation to the region between 84F4 and 84F11-12 (33), which includes about 32 genes. Subsequent work showed that the Gfr gene, which encodes a Golgi GDP-fucose transporter, is located in this region (28). This finding hinted that a Gfr mutation might be responsible for nac1 because a defect in the ability to transport GDP-fucose into the Golgi apparatus could account for the reduced neural HRP epitope in the nac1 fly. This speculation was strengthened by data showing that homozygous Gfr knockout flies have temperature-sensitive Notch-like wing phenotypes (35), which are similar to the temperature-sensitive scalloped wing phenotype observed in the nac1 mutant (34). However, neither the gene(s) mutated in nac1 flies nor the precise nature of the mutation have been elucidated. Thus, we examined the Gfr gene in the Drosophila nac1 mutant and found that nac1 flies have a mutant Gfr gene, which encodes a defective Golgi GDP-fucose transporter that is solely responsible for its neural HRP epitope deficiency. EXPERIMENTAL PROCEDURES Genomic DNA homozygous flies were obtained from the Bloomington Drosophila Stock Center (Indiana University) and maintained at 28°C. Genomic DNA was extracted from a single adult fly, as previously described (36). Briefly, the fly was homogenized in a lysis buffer containing RNAse A and the homogenate was incubated at 55°C for 1 h. The lysate was briefly centrifuged to remove debris and the DNA was precipitated. The DNA was dissolved in TE buffer, extracted once with phenol-chloroform, and 1 µl of the resulting DNA preparation was used as the template for a PCR with primers that flanked the Gfr gene transcript (AAGGGATGGGGCCAAGAAGC AATCCACCCCCGCACTCAAC). All PCRs were performed using Phusion™ DNA polymerase (New England Biolabs). electrophoresis showed that the PCR yielded a single major amplification product of the expected size, which was gel-purified using the Qiaquick gel purification kit (Qiagen) and directly sequenced with the primers used for the PCR. Expression plasmids and baculoviruses—All plasmid constructs derived directly from PCRs were sequence verified and amplimers for TOPO cloning were gel purified and then treated with Taq DNA polymerase (NEB). Total RNA was isolated from the Drosophila wt Canton-S strain using TRIzol® reagent (Invitrogen) and cDNA was synthesized using SuperScript® III RT (Invitrogen) and oligo-dT. The cDNA was used as the template to amplify the wt Gfr ORF (primers TCAGGCCTTCTGGGTGGCGGTGCT CACCATGTACAAGAATCTGGAGGAGCAC), which was cloned into pcDNA™3.1/V5-His-TOPO® (Invitrogen), and a sequence-verified clone was designated pcDNA-DmGFR-wt. This plasmid was used as the template for PCR mutagenesis with the GTGCACCTTGATATTGACGGTATTCG GTCAATATCAAGGTGCACCAGTAGAGC to produce the nac1 Gfr ORF by overlap PCR. The amplimer was cloned into pcDNA™3.1/V5-His-Analysis—nac1 and Agarose gel and additional primers and at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 3
Drosophila neurally altered carbohydrate mutant 3 TOPO®, and a clone encoding the nac1 mutant Gfr was designated pcDNA-DmGFR-nac. Baculovirus transfer produced by cloning the BamHI-NotI fragment encoding the wt or nac1 mutant Gfr gene from pcDNA-DmGFR-wt and pcDNA-DmGFR-nac-, respectively, into the pAcp(+)IE1TV3 (37), resulting in production of the pAcp(+)DmGFR-wt and pAcp(+)DmGFR-nac transfer plasmids, respectively. Plasmids encoding C-terminally eGFP-tagged GFR proteins were constructed by PCR overlap extension. The wt or nac1 mutant Gfr ORFs minus their stop codons were PCR amplified using the respective pcDNA plasmids as the template, respectively, with the primers CACCATGTACAAGAATCTGGAGGAGCAC and GCTCACCATGGCCTTCTGGGTGGCGGT. The eGFP ORF was PCR amplified using peGFP-N1 (Clontech) as the template with the primers CAGAAGGCCATGGTGAGCAAGGGCGAG and CTACTTGTACAGCTCGTCCATGC. The reaction products were gel-purified and used as the template in PCR overlap extension reactions. The reaction products pcDNA™3.1/V5-His-TOPO®, encoding the C-terminally GFP-tagged wt and nac1 Gfr were designated pcDNA-DmGFR-wt-GFP and pcDNA-DmGFR-nac-GFP, respectively. The fused ORFs were excised from these plasmids using BamHI and NotI and cloned into the BglII-NotI sites of pAcp(+)IE1TV3 (37), yielding pAcp(+)IE1TV3-DmGFR-wt-GFP pAcp(+)IE1TV3-DmGFR-nac-GFP, respectively. Transfer plasmids were used to produce recombinant baculoviruses by a standard allelic transplacement method (38,39) with BestBac viral DNA (Expression Systems ) as the target for homologous recombination. All recombinant baculoviruses were amplified in Sf9 cells, and titered by plaque assay on Sf9 cells, as described previously (39). Autographa californica (AcMNPV) strain E2 served as a negative control for some of the experiments included in this study. Transient expression in CDG-IIc (LAD-II) cells, lectin blotting—Primary fibroblast cells from a CDG-IIc (LAD-II) patient were maintained essentially as described (40) in αMEM medium supplemented with 15% FBS in 5% CO2 at 37°C. For transfections, cells were seeded in a 75 cm2 plasmids were BglII-NotI sites of were cloned and into clones and plaque-purified once, nucleopolyhedrovirus culture flask, grown to confluence and transfected with pcDNA, pcDNA-DmGFR-wt, or pcDNA-DmGFR-nac using Lipofectamine™ (Invitrogen). Briefly, cells were transfected using 30 µg of DNA and 75 µl of transfection reagent for 3 h using serum-free αMEM according to the manufacturer's protocol. Cells were subsequently incubated for 24 h in αMEM with 15% FBS, after which cells were collected by trypsinization, washed twice in PBS and lysed in 500 µl lysis buffer (20 mM Tris-HCl, pH 7.4, 1.0% NP-40, 150 mM NaCl, 0.5 mM PMSF, 1 mM EDTA, one Complete Mini™ protease inhibitor cocktail tablet (Roche) / 10 ml buffer). After vigorous vortexing, the lysate was centrifuged for 10 minutes at 13,000 g, after which the supernatant was collected and assayed for soluble protein concentration using a commercial BCA assay (Pierce). CHO cell lysates were prepared as described above from CHO cells cultured as described before (41). Aliquots containing 50 µg of total protein were separated by SDS-PAGE (42) and transferred to an Immobilon-P PVDF membrane (Millipore). The membrane was blocked, probed and developed essentially as described before (43), except that biotin-conjugated AAL lectin (Vector labs) was used as the probe. Subcellular localization—Sf9 Drosophila S2 cells were transfected with expression plasmids encoding RFP-tagged S. frugiperda MGAT1 (44) and GFP-tagged wt or nac1 mutant GFR proteins [pAcP(+)DmGFR-wt-GFP or pAcP(+)DmGFR-nac-GFP], plated on Concanavalin A coated dishes and photographed essentially as described before (44). An Olympus FSX100 microscope magnification and the manufacturer's FSX-BSW version 03.01 software was used for image capture at 1360x1024 pixels. Images were processed with Photoshop CS3 to reduce background and to provide similar signal intensities for the red and green channels. GDP-fucose transport assays—Fifty ml of Sf9 cells were seeded at a density of 5 x 105 cells/ml in complete TNM-FH medium, grown overnight to 1 × 106 cells/ml and infected with the appropriate viral stock at a multiplicity of infection of about 2 plaque-forming units/cell. After 22 h, infected cells were pelleted at 500 g for 2000 and was used at 80X at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 4
Drosophila neurally altered carbohydrate mutant 4 5 min, resuspended in 25 ml of ice-cold PBS (pH 7.4) and re-pelleted. The pellet was resuspended in 6.5 ml of lysis buffer (250 mM sucrose, 5 mM imidazole, 0.5 mM mercaptoethanol; pH 7.0, one Complete Mini™ protease inhibitor cocktail tablet (Roche) / 10 ml buffer). The cells were subsequently homogenized on ice in a Dounce homogenizer with pestle A, after which nuclei and remaining intact cells were pelleted at 4°C at 1000 g for 10 min. The crude microsomal preparation was then layered onto a sucrose cushion (1.3 M sucrose, 5 mM imidazole, 0.1 mM EDTA; pH 7.0), covered with sucrose overlay (125 mM sucrose, 5 mM imidazole, 0.1 mM EDTA; pH 7.0), and then centrifuged in a Beckman SW28 rotor at 100,000 gav for 40 min at 4°C. Subsequently, the microsomal band was harvested, diluted with sucrose overlay and re-centrifuged in a SW28 rotor at 110,000 gav for 20 min at 4°C. The microsomal pellet was resuspended in 600 µl of STM buffer (250 mM sucrose, 10 mM Tris HCl, 1 mM MgCl2, 1 mM DDT; pH 7.5), divided into aliquots, and stored at -80°C for up to one week. To determine total protein concentrations, aliquots were thawed on ice, briefly vortexed and solubilized by the addition of an equal volume of water containing 1.0 % (v/v) NP-40. These mixtures were then centrifuged at 13,000 g for 10 minutes and the concentration of solubilized protein in the supernatants were determined using a commercial BCA assay (Pierce). For transport assays, aliquots of the microsomal preparations were thawed on ice, thoroughly vortexed, and transport assay mixtures were prepared by adding 10 µl of the microsomal preparation to 80 µl STM buffer, cooling the mixture in an ethanol-ice bath (approx. -5°C), and then adding 10 µl STM buffer containing 30 nCi 3H-GDP-fucose, (Fucose-2-3H(N), Perkin-Elmer Life Sciences; 15-35Ci/mmol). The mixture was briefly vortexed and quickly returned to the ethanol-ice bath. The mixture was then transferred to a water bath at 18°, 25° or 32°C for precisely 1 min, returned to the ethanol-ice bath and quenched by the addition of 900 µl ice-cold STM buffer. The mixtures were then filtered through water-wetted 0.45 µm mixed cellulose esters filters (Type HA; Millipore) using a 1225 Sampling Manifold (Millipore). The disks were washed three times with 5 ml of ice-cold STM buffer, air-dried, placed in 7 ml of Ultima Gold F scintillation Company), and counted for 10 min in a Model LS-6500 liquid scintillation spectrometer (Beckman Coulter). Background counts were determined by counting an unused filter as described above. All samples were assayed at least three times in triplicate (n=9). Raw counts were corrected for background and normalized to 30 µg of soluble protein content. Significant differences were determined by one-way Microsoft® Excel. Mass-spectrometry and HPLC—For nac1 and wt Canton S flies, pepsin glycopeptides were enriched and N-glycans were released with PNGase A prior to pyridylamination and RP-HPLC, MALDI-TOF MS or ESI-MS analysis (45). As a first step, linear MALDI-TOF mass spectra of unlabeled N-glycans were obtained prior to pyridylamination using a Thermo Bioanalysis Dynamo mass spectrometer in linear mode with 2,5-dihydroxybenzoic acid as matrix. For ESI-MS of the pyridylaminated glycans with a Micromass Q-TOF Ultima Global mass spectrometer, the [M+H]+ ions were calculated by applying the MassLynx MaxEnt3 software to the raw multiply-charged ion data. For reversed-phase HPLC analysis of pyridylaminated N-glycans, an ODS Hypersil column (250 x 4 mm) with a gradient of 0.3% methanol per minute was used with an oligohexose series (3-11 glucose units) as a calibration standard; elution times in terms of glucose units can be compared to previous data on wt fly N-glycans (24). Individual RP-HPLC fractions were also analyzed by MALDI-TOF MS and MS/MS using a Bruker autoflex™ speed instrument in reflectron mode and 6-aza-2-thiothymine as matrix. Drosophila transgenesis and rescue—All Drosophila strains (OreR, w1118, elav-GAL4 inserted on the X chromosome, nac1, and balancer stocks) were obtained from the Bloomington Drosophila Stock Center. The full wt Canton S Gfr ORF was isolated by PCR using the primers GGAATTCCGAAATGTACAAGAATCTG and GGGGTACCTCAGGCCTTCTGGGTGG. amplimer was purified and cut with EcoRI and KpnI and ligated into the same sites of the pUAST transgenesis plasmid (46). Transgenic stocks carrying UAS-Gfr elements chromosomes were generated by injection of cocktail (Packard Instrument ANOVA using The on all three at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 5
Drosophila neurally altered carbohydrate mutant 5 pUAST-Gfr into pre-cellularized embryos using standard methods (46). Drosophila embryo anti-HRP staining—Embryos were dechorionated, devitellinized, stained with antibodies and staged according to standard methods (47,48). Antibody dilutions were 1:5000 for rabbit anti-HRP (Jackson Immunoresearch) and 1:2000 for peroxidase-conjugated (Jackson Immunoresearch). All embryos were processed identically and in parallel (same antibody dilutions, same development time, same day) to facilitate objective comparison of HRP epitope levels in all genotypes. RESULTS Gfr Gene Sequence in the nac1 Mutant—The sequence of a PCR amplimer from a genomic region that includes the Gfr transcript revealed that the Gfr gene in the nac1 mutant has a single mutation consisting of a cytosine to thymidine transition at position 86 (C86T) of the ORF. This mutation was independently identified in the Jarvis and Wilson labs in nac1 stocks obtained at different times and from different sources. The nac1 C86T transition results in the substitution of a leucine for a serine residue at position 29 (S29L) in the predicted GFR amino acid sequence. The wild type (wt) serine residue is fully conserved among all known and putative GDP-fucose transporters throughout the animal kingdom (Fig. 1B) and is located in the first predicted transmembrane region (Fig. 1C). As production of the HRP epitope requires GDP-fucose in the Golgi apparatus, where it serves as the donor substrate, these observations were consistent with the idea that nac1 flies might have a defective Golgi GDP-fucose transporter. Thus, we examined the impact of the nac1 S29L mutation on the GDP-fucose transport function of the mutant Gfr gene product. GDP-fucose Transport by the nac1 mutant GFR product—Our transport assays measured the amount of GDP-fucose microsomes, analogous to previously described nucleotide-sugar transport assays employing Golgi-enriched microsomes from cells expressing heterologous NSTs expression vectors were used to express the wt and nac1 mutant GFRs in Sf9 insect cells and then Golgi-enriched microsomes were isolated from those cells and used for GDP-fucose transport fixed, secondary antibodies transported into (35,49). Baculovirus assays. Microsomes from cells infected with the empty baculovirus vector background controls and each assay was performed at different temperatures to determine if there were any temperature-dependent differences that could explain nac1 cold sensitive phenotypes (34). As compared to the background controls, microsomes containing wt GFR imported more and those containing the nac1 mutant GFR imported less GDP-fucose at all temperatures examined (Fig. 2A). Increasing the assay temperature from 18°C to 25°C did not produce a statistically significant increase in GDP-fucose import in either the background controls or wt GFR samples. However, this temperature shift significantly increased GDP-fucose import in the nac1 GFR samples. These experiments were extended by performing additional assays at 32°C to determine if the nac1 mutant transporter gained even more function at this higher temperature. Indeed, GDP-fucose import with background control, wt GFR, and nac1 GFR microsomes was increased further at 32°C (Fig. 2A). As with the previous temperature shift, the increase in GDP-fucose import activity was highest in the nac1 GFR samples, confirming that the nac1 mutant GFR is more cold sensitive than wt GFR, which potentially contributes to the cold sensitive nac1 phenotypes (34). We also assessed the function of nac1 GFR in vivo by using cells from a CDG-IIc (LAD-II) patient (40). These cells cannot produce fucosylated N-glycans because they have a defective GFR, but their fucosylation-negative phenotype can be rescued by transfection with a wt human Gfr gene (28,30,31). Thus, we transfected CDG-IIc (LAD-II) cells with plasmids encoding the wt or nac1 Drosophila Gfr genes, prepared total cell lysates and CHO cell lysates as a positive control, and probed them with the fucose-specific lectin AAL (50). AAL bound strongly to multiple proteins in the CHO cell lysate, but not to any proteins in the empty vector-transfected CDG-IIc (LAD-II) cell lysate, as expected (Fig. 2B). AAL also bound to multiple proteins in the wt Gfr-transfected CDG-IIc (LAD-II) cell lysate, but not to any proteins in the nac1 Gfr-transfected CDG-IIc (LAD-II) cell lysate (Fig. 2B). The higher level of AAL binding observed with the CHO cell lysate as compared to the wt Gfr-transfected CDG-IIc (LAD-II) cell lysate were used as at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 6
Drosophila neurally altered carbohydrate mutant 6 probably reflects cell toxicity associated with the transfection, as we observed significant cell death at later time points. Alternatively, it might reflect the inefficiencies inherent in the transfection process, or the differences between the CHO and LAD-II cell types. Regardless, these results showed that the nac1 Gfr gene failed to rescue the fucosylation-negative phenotype in CDG-IIc (LAD-II) cells, indicating that the nac1 mutant gene product is defective in vivo. The nac1 Mutant GFR Localizes to the Golgi Apparatus—GDP-fucose typically localize to the Golgi apparatus (28,40). Hence, the transport defect observed with the nac1 mutant gene product could have resulted from a direct impact of the mutation on its biochemical function or an indirect impact on its intracellular trafficking. To distinguish possibilities, we expressed GFP-tagged forms of the wt and nac1 GFRs in Drosophila S2 cells, as well as in Sf9 cells, which had been used for the in vitro GDP-fucose transport assays. We used an RFP-tagged acetylglucosaminyltransferase I (MGAT1) as a Golgi marker, as this enzyme acts immediately upstream of HRP epitope synthesis by producing the FucTA acceptor substrate (44,51-53). The red and green fluorescence patterns observed in these experiments each had punctate, cytoplasmic distributions typical of the multiple Golgi apparati found in lepidopteran insect cells (Fig. 2C, 54,55). Furthermore, there was a close overlap between the GFR and MGAT1 fluorescence patterns in all cases, indicating that these two proteins reside in the same subcellular compartment. The similarity in the fluorescence patterns observed with the wt and nac1 mutant GFRs and their close overlap with the Golgi marker indicated that the nac1 mutation does not impact the intracellular trafficking of GFR, which was consistent with the presence of only a single amino acid substitution in the mutant protein. These data also indicated that this mutation does not dramatically reduce GFR stability, though it is possible that the mutant protein was stabilized by being fused to GFP. Core α1,3- and α1,6-Fucosylation are Both Reduced in nac1 Flies—Golgi-localized GDP-fucose is required as the donor substrate for both core α1,3- and α1,6-fucosylation. Thus, one might expect a functional knockout of the Gfr gene to reduce both types of core fucosylation in transporters between these insect N-nac1 flies. To test this expectation, we determined the relative levels of core fucosylated N-glycans in wt and nac1 flies using ESI-MS. The results showed that 21% and 10% of the N-glycans from wt (Fig. 3A) and nac1 mutant (Fig. 3B) adult flies, respectively, were mono-fucosylated glycans with the structure Hex3HexNAc2Fuc. Similarly, the prevalence of mono-fucosylated N-glycans with the structure Hex2HexNAc2Fuc was 5% in wt, but only 1.4% in nac1 mutant adults (Fig. 3B). Low levels of difucosylated N-glycans bearing both the HRP epitope and core α1,6-linked fucose residues also were detected in wt, but not in nac1 mutant flies (Fig. 3C and D). We further assessed the levels of mono-fucosylated N-glycans in nac1 mutant and wt flies by reverse phase HPLC (Fig. 3E) and MALDI-TOF (Fig. 3F and G); analysis of individual RP-HPLC fractions by MALDI-TOF MS revealed only trace amounts of the difucosylated glycans Hex2-3HexNAc2Fuc2 in nac1 (co-eluting with compared to wt flies (data not shown). The results obtained using both of these analytical methods confirmed that nac1 flies have lower levels of mono-fucosylated N-glycans. independent methods indicated that the nac1 mutation reduced both α1,3 and α1,6 linked core fucosylation, as would be expected from the loss of GFR function. In addition, all three methods also revealed a relative increase in the levels of the non-fucosylated N-glycan corresponding to the decreased levels of fucosylated N-glycans, further confirming the lack of fucosylation. wt Gfr Expression Rescues the Neural HRP Epitope in Immunohistochemistry with an anti-HRP antibody confirmed that HRP epitope expression in the ventral nerve cord was much lower in nac1 than in wt embryos (Fig. 4A, B, E, and F), as shown previously (21,33). In order to determine if the Gfr C86T mutation was solely responsible for this change, we generated transgenic Drosophila stocks designed to express the wt Gfr coding sequence in nac1 embryos using the GAL4/UAS system (46). A second chromosome UAS-Gfr transgenic strain and an X chromosome elav-GAL4 driver strain were both crossed into the third chromosome nac1 background, resulting in stocks that were homozygous for nac1 and either the UAS-Gfr or elav-GAL4 element. These stocks Hex3HexNAc2) as Thus, three Hex3HexNAc2 nac1 Embryos— at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 7
Drosophila neurally altered carbohydrate mutant 7 were crossed to generate embryo collections, which were then stained with the anti-HRP antibody to assess whether transgenic Gfr expression could rescue the nac1 core α1,3-fucosylation defect. As expected for the neural specificity of the elav-GAL4 element (56), expression of the HRP-epitope was rescued in differentiating neurons of elav-GAL4; UAS-Gfr; nac1/nac1 progeny (Fig. 4C and D). Thus, reduced HRP epitope expression in nac1 flies is due solely to a defect in their Gfr gene. Staining with anti-HRP antibody could be detected in late stage 10 rescued embryos, which is substantially earlier than in wild-type embryos, where staining first appeared in early stage 12. This is consistent with the time course of elav expression (56), suggesting that Gfr expression at least partially limits core α1,3-fucosylation in Drosophila. Surprisingly, elav-driven expression of Gfr resulted in embryonic lethality at mid-embryogenesis. This is likely a result of our use of the very strong elav-GAL4 driver, which typically provides highly efficient expression of UAS-transgenes in the embryonic nervous system. In the course of generating UAS-Gfr transgenic stocks, we identified a line that exhibited partially expression in the ventral nerve cord and peripheral nervous system of nac1 mutant embryos without crossing to a GAL4 driver line (data not shown). This leaky expression line (UAS-Gfrvk2) was homozygous viable and fertile in both nac1 and wt backgrounds, suggesting a significantly lower Gfr expression level than was obtained by crossing UAS-Gfr lines to the elav-GAL4 driver line. Interestingly, the UAS-Gfrvk2 leaky expression line rescued temperature-sensitive lethality associated with the nac1 mutant; only 6% of nac1/nac1 adults survived after shift to 18°C, while 82% of UAS-Gfrvk2/UAS-Gfrvk2; nac1/nac1 adults survived and reproduced at 18°C. Thus, while overexpression of Gfr proved to be embryonic lethal, moderate expression was well tolerated and rescued both HRP-epitope expression and developmental arrest defects associated with the nac1 mutation. S29 is Conserved in Other GDP-sugar Transporters—Finally, we compared the amino acid sequences of the Drosophila GFR and other known Golgi nucleotide sugar transporters (NSTs) to more generally assess the potential functional relevance of S29 (Fig. 5). We found that plant, rescued HRP-epitope fungal, transporters have clear homology to an amino acid sequence in the N-terminal region of the Drosophila GFR. Several fungal Golgi GDP-mannose transporters (57-62), a Leishmania Golgi GDP-mannose, -fucose, and -arabinose transporter (63,64), and the Arabidopsis and Volvox Golgi GDP-mannose transporters (65-67) are clearly similar to Drosophila GFR in this region and each has a serine residue in positions corresponding to Drosophila GFR S29. Other multi-substrate transporters that also could transport a GDP-sugar, including human HFRC1 (68), Drosophila FRC (69), human UGTrel7 (70), and nematode SQV-7 (71) are similar to Drosophila GFR, as well, and each has a serine residue at a position corresponding to Drosophila GFR S29. In contrast to these GDP-sugar transporters, other Golgi NSTs including the human UDP-galactose (72), CMP-sialic acid (73), UDP-N-acetylglucosamine (74), and UDP-xylose transporters (75) lack significant homology to GFR. Of these, the human UDP-galactose and UDP-N-acetylglucosamine transporters have a serine residue at a position corresponding to Drosophila GFR S29. However, these serine residues are not conserved in the homologous transporters from most other species, indicating that, unlike Drosophila GFR S29, they are probably not essential for functionality. Based on these data, we suggest that a serine residue at a position corresponding to Drosophila GFR S29 in Golgi NSTs that also have similarity to an amino acid sequence in the first transmembrane domain of Drosophila GFR might predict GDP-sugar transport capacity. DISCUSSION Congenital disorders of glycosylation (CDG's) are a diverse group of heritable diseases caused by mutations in genes involved in glycosylation. The study of CDG's has been facilitated by the availability of animal models, as much of the glycosylation evolutionarily conserved (76,77). Since the description of the nac1 mutant in 1988 by Katz and coworkers (33), Drosophila established as a model organism for the study of human genetic disorders, including CDG's (78,79). However, the genetic defect underlying the nac1 mutation had not yet been elucidated. protozoan, and animal GDP-sugar machinery is has become at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 8
Drosophila neurally altered carbohydrate mutant 8 In this study, we identified a single nucleotide transition (C86T) that produces a leucine for serine substitution at position 29 of the Golgi GDP-fucose transporter encoded by the nac1 Gfr gene. The mutagen originally used to isolate the nac1 mutant was ethyl methane sulfonate (EMS, 33). Mechanistically, EMS is expected to produce G/C to A/T transitions (80,81), and this expectation has been confirmed in mutagenesis studies (82-84). Thus, the C86T transition in the nac1 Gfr gene is fully consistent with the use of EMS mutagenesis in producing the nac1 strain. There are three currently known missense mutations in the human GDP-fucose transporter that cause CDG-IIc (LAD-II; T308R, R147C, Y337C, 30,85). In each case, these mutations alter a residue analogous to Drosophila GFR S29, which is fully conserved among animal GDP-fucose transporters and is located in a predicted transmembrane helix. Like these human GFR missense mutations, the nac1 S29L mutation also abolishes GDP-fucose transport function in vitro and in vivo. Serine residues corresponding to Drosophila GFR S29 are conserved in GDP-sugar transporters from a wide variety of species, but not in other types of NSTs. Thus, we speculate that a serine residue at a position corresponding to Drosophila GFR S29 might predict GDP-sugar transporting capacity in Golgi NSTs that are also otherwise similar to Interestingly, the first TM domains of human HFRC1 and UGTrel7, like that of Drosophila FRC, are similar to GFR and each has a conserved serine corresponding to GFR S29, unlike any other human or Drosophila Golgi NSTs. Thus, it is possible that the low level of fucosylation in nac1 flies is due to GDP-fucose transport by the Drosophila FRC gene product. Similarly, it is possible that the alternative GDP-fucose transport activity observed in CDG-IIc (LAD-II) cells supplemented with fucose is due to this same function of the human HFRC1 or UGTrel7 gene products. For our in vitro GDP-transport assays, we used microsomes from Sf9 cells (86), which have endogenous Golgi GDP-fucose transport activity, as these cells typically produce α1,6 core fucosylated N-glycans (87). Despite the presence of this endogenous activity, we were able to demonstrate a >6-fold increase in transport activity in microsomes from cells infected with a Drosophila GFR. baculovirus encoding wt GFR, as compared to background controls. Surprisingly, microsomes from cells infected with a baculovirus encoding the nac1 GFR samples had reduced GDP-fucose import activity compared to the controls, indicating a possible dominant negative effect. Considering that GFR dimerization might be necessary to produce a functional transporter (29), co-expression of the nac1 GFR could have produced a subpopulation of heterodimers consisting of endogenous transporter molecules and recombinant nac1 GFR molecules, which were less functional than the endogenous transporter homodimers. A similar dominant negative phenotype in which co-expression of a mutant transporter negatively affects transport has been observed with the yeast Golgi GDP-mannose transporter (88), which also functions as a homodimer. Alternatively, it is possible that overexpression of the nac1 GFR altered the subcellular distribution transporter, thereby reducing the number of transporter molecules in those microsomal preparations. Non-functional, mutant NSTs that fail to exit the ER typically have frameshift mutations that eliminate one or more transmembrane domains and the C-terminal domain. Two such mutations have been identified in the human GDP-fucose transporter (40,89). On the other hand, point mutations that change single amino acids typically do not alter the Golgi localization of NSTs, including the GDP-fucose transporter (31,40,90). Like the mutations in human GFR, the nac1 S29L mutation did not affect subcellular distribution, as both wt and nac1 GFR were Golgi localized. ESI-MS, RP-HPLC and MALDI-TOF analyses demonstrated that nac1 flies have reduced levels of core α1,3-fucosylated and only trace levels of core α1,6-/α1,3-difucosylated N-glycans, which is consistent with the original observation that nac1 flies have significantly reduced levels of the HRP epitope. We also discovered that these flies had reduced levels of core α1,6-fucosylated N-glycans, which is requirement of a Golgi GDP-fucose transporter for both core α1,6- and α1,3-fucosylation. The residual levels of monofucosylated N-glycans indicate that nac1 flies are still able to transport some GDP-fucose into the Golgi. This suggests of the endogenous inactivating missense consistent with the at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 9
Drosophila neurally altered carbohydrate mutant 9 the presence of an alternative, functionally redundant GDP-fucose transport mechanism, a notion that is supported by the results of another study, in which faint anti-HRP and AAL staining could still be detected in flies with a large deletion in the Gfr gene (91). This redundant transport mechanism is not provided by the ER-localized GDP-fucose transporter encoded by the Efr gene, as flies lacking Gfr alone or both Gfr and Efr have comparable amounts of residual core fucosylated N-glycans (91). Similarly, humans also have an alternative, but less efficient Golgi GDP-fucose import mechanism, supplementation can restore N-glycan core fucosylation in CDG-IIc (LAD-II) patients that are homozygous for a completely non-functional Golgi GDP-fucose transporter (40,89). The precise nature of the redundant GDP-fucose transport mechanism remains to be determined in both humans and flies, however, its low-affinity and non-saturable character suggests it is not provided by another specific GDP-fucose transporter (92,93). Interestingly, nac1 embryos rescued with elav-driven wt Gfr expressed the HRP epitope at earlier developmental stages than wt embryos, suggesting that N-glycan fucosylation is at least partially limited by transport of GDP-fucose into the Golgi apparatus. The notion that Gfr expression limits fucosylation corroborated by the demonstration that increased N-glycan fucosylation in cancer cells correlates with increased Gfr fucosylation can be increased directly by overexpressing Gfr (94). A surprising observation was that elav-driven wt Gfr overexpression triggered embryonic lethality. This is likely a pleiotropic phenotype arising from the increased availability of GDP-fucose in the Golgi apparatus for a variety of N-linked and O-linked fucosylation reactions. Intriguingly, the nac1 phenotypes and the embryonic lethality observed in elav-GAL4; UAS-Gfr rescued nac1 flies suggest that it is biologically necessary fucosylation within a certain range. Furthermore, the relation between Gfr expression levels, embryonic lethality, and HRP-epitope production suggests that Gfr is part of the regulatory system that maintains Golgi GDP-fucose levels within a physiologically acceptable range. as dietary fucose in vivo is expression, and that to maintain protein Finally, the observation that transgenic wt expression can restore production in nac1 flies indicates that the Gfr C86T transition is the only genetic defect responsible for the neurally altered carbohydrate phenotype. Hence, the defective Golgi GDP-fucose transporter and the resulting fucosylation deficit in nac1 flies is analogous to human CDG-IIc (LAD-II). Coupled with our observation that the nac1 mutant GFR is more cold-sensitive than its wt counterpart, we suggest that the nac1 fly is a useful model of human CDG-IIc (LAD-II) that could be effectively exploited in a variety of creative ways, such as using its cold-sensitive phenotypes to titrate fucosylation. Acknowledgments—We thank Dr. M. Wild (Max Planck Institute for Molecular Biomedicine, Münster, Germany) and Dr. P. Robinson (Royal Hospital for Sick Children, Glasgow, UK) for providing the CDG-IIc cell line, D. Kerner for assistance with glycan preparations, and Dr. F. Altmann for access to mass spectrometers. Gfr HRP epitope N-glycoprotein core at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 10
Drosophila neurally altered carbohydrate mutant 10 REFERENCES 1. Freeze, H. H., Eklund, E. A., Ng, B. G., and Patterson, M. C. (2012) Neurology of inherited glycosylation disorders. The Lancet Neurology 11, 453-466 2. Morava, E., Lefeber, D. J., and Wevers, R. A. (2011) Protein glysosylation and congenital disorders of glycosylation. in Post-translational modifications in health and disease (Vidal, C. J. ed.), Springer, New York. pp 97-117 3. Jaeken, J. (2010) Congenital disorders of glycosylation. Annals of the New York Academy of Sciences 1214, 190-198 4. Fukuda, M. N., Papayannopoulou, T., Gordon-Smith, E. C., Rochant, H., and Testa, U. (1984) Defect in glycosylation of erythrocyte membrane proteins in congenital dyserythropoietic anaemia type II (HEMPAS). Br. J. Haematol. 56, 55-68 5. Jaeken, J., van Eijk, H. G., van der Heul, C., Corbeel, L., Eeckels, R., and Eggermont, E. (1984) Sialic acid-deficient serum and cerebrospinal fluid transferrin in a newly recognized genetic syndrome. Clin. Chim. Acta 144, 245-247 6. Bach, G., Bargal, R., and Cantz, M. (1979) I-cell disease: Deficiency of extracellular hydrolase phosphorylation. Biochemical and Biophysical Research Communications 91, 976-981 7. Hasilik, A., and Neufeld, E. F. (1980) Biosynthesis of lysosomal enzymes in fibroblasts. Phosphorylation of mannose residues. Journal of Biological Chemistry 255, 4946-4950 8. Fukuda, M. N., Dell, A., and Scartezzini, P. (1987) Primary defect of congenital dyserythropoietic anemia type II. Failure in glycosylation of erythrocyte lactosaminoglycan proteins caused by lowered N-acetylglucosaminyltransferase II. J. Biol. Chem. 262, 7195-7206 9. Van Schaftingen, E., and Jaeken, J. (1995) Phosphomannomutase deficiency is a cause of carbohydrate-deficient glycoprotein syndrome type I. FEBS Lett. 377, 318-320 10. Reitman, M. L., Varki, A., and Kornfeld, S. (1981) Fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy are deficient in uridine 5'-diphosphate-N-acetylglucosamine: glycoprotein N-acetylglucosaminylphosphotransferase activity. J Clin Invest 67, 1574-1579 11. Hasilik, A., Waheed, A., and von Figura, K. (1981) Enzymatic phosphorylation of lysosomal enzymes in the presence of UDP-N-acetylglucosamine. Absence of the activity in I-cell fibroblasts. Biochemical and Biophysical Research Communications 98, 761-767 12. Schwarz, K., Iolascon, A., Verissimo, F., Trede, N. S., Horsley, W., Chen, W., Paw, B. H., Hopfner, K. P., Holzmann, K., Russo, R., Esposito, M. R., Spano, D., De Falco, L., Heinrich, K., Joggerst, B., Rojewski, M. T., Perrotta, S., Denecke, J., Pannicke, U., Delaunay, J., Pepperkok, R., and Heimpel, H. (2009) Mutations affecting the secretory COPII coat component SEC23B cause congenital dyserythropoietic anemia type II. Nat Genet 41, 936-940 13. Matthijs, G., Schollen, E., Pardon, E., Veiga-Da-Cunha, M., Jaeken, J., Cassiman, J. J., and Van Schaftingen, E. (1997) Mutations in PMM2, a phosphomannomutase gene on chromosome 16p13, in carbohydrate-deficient glycoprotein type I syndrome (Jaeken syndrome). Nat Genet 16, 88-92 14. Tiede, S., Storch, S., Lübke, T., Henrissat, B., Bargal, R., Raas-Rothschild, A., and Braulke, T. (2005) Mucolipidosis II is caused by mutations in GNPTA encoding the / GlcNAc-1-phosphotransferase. Nat Med 11, 1109-1112 at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 11
Drosophila neurally altered carbohydrate mutant 11 15. Kudo, M., Brem, M. S., and Canfield, W. M. (2006) Mucolipidosis II (I-Cell disease) and mucolipidosis IIIA (classical pseudo-Hurler polydystrophy) are caused by mutations in the GlcNAc-phosphotransferase α/β–subunits precursor gene. The American Journal of Human Genetics 78, 451-463 Kubelka, V., Altmann, F., Staudacher, E., Tretter, V., Marz, L., Hard, K., Kamerling, J. P., and Vliegenthart, J. F. (1993) Primary structures of the N-linked carbohydrate chains from honeybee venom phospholipase A2. European Journal of Biochemistry 213, 1193-1204 Prenner, C., Mach, L., Glossl, J., and März, L. (1992) The antigenicity of the carbohydrate moiety of an insect glycoprotein, honey-bee (Apis mellifera) venom phospholipase A2. The role of 1,3-fucosylation of the asparagine-bound N-acetylglucosamine. Biochem. J. 284 (Pt 2), 377-380 Tretter, V., Altmann, F., Kubelka, V., März, L., and Becker, W. M. (1993) Fucose 1,3-linked to the core region of glycoprotein N-glycans creates an important epitope for IgE from honeybee venom allergic individuals. International Archives of Allergy and Immunology 102, 259-266 Paschinger, K., Rendić, D., and Wilson, I. (2009) Revealing the anti-HRP epitope in Drosophila and Caenorhabditis. Glycoconj. J. 26, 385-395 Wilson, I. B., Harthill, J. E., Mullin, N. P., Ashford, D. A., and Altmann, F. (1998) Core 1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts. Glycobiology 8, 651-661 Jan, L. Y., and Jan, Y. N. (1982) Antibodies to horseradish peroxidase as specific neuronal markers in Drosophila and in grasshopper embryos. Proc. Natl. Acad. Sci. U. S. A. 79, 2700-2704 Kurosaka, A., Yano, A., Itoh, N., Kuroda, Y., Nakagawa, T., and Kawasaki, T. (1991) The structure of a neural specific carbohydrate epitope of horseradish peroxidase recognized by anti-horseradish peroxidase antiserum. J. Biol. Chem. 266, 4168-4172 Snow, P. M., Patel, N. H., Harrelson, A. L., and Goodman, C. S. (1987) Neural-specific carbohydrate moiety shared by many surface glycoproteins in Drosophila and grasshopper embryos. J. Neurosci. 7, 4137-4144 Fabini, G., Freilinger, A., Altmann, F., and Wilson, I. B. (2001) Identification of core α1,3-fucosylated glycans and cloning of the requisite fucosyltransferase cDNA from Drosophila melanogaster. Potential basis of the neural anti-horseradish peroxidase epitope. J. Biol. Chem. 276, 28058-28067 Rendić, D., Linder, A., Paschinger, K., Borth, N., Wilson, I. B., and Fabini, G. (2006) Modulation of neural carbohydrate epitope expression in Drosophila melanogaster cells. J. Biol. Chem. 281, 3343-3353 Rendić, D., Sharrow, M., Katoh, T., Overcarsh, B., Nguyen, K., Kapurch, J., Aoki, K., Wilson, I. B., and Tiemeyer, M. (2010) Neural-specific α3-fucosylation of N-linked glycans in the Drosophila embryo requires fucosyltransferase A and influences developmental signaling associated with O-glycosylation. Glycobiology 20, 1353-1365 Roos, C., Kolmer, M., Mattila, P., and Renkonen, R. (2002) Composition of Drosophila melanogaster proteome involved in fucosylated glycan metabolism. J. Biol. Chem. 277, 3168-3175 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 12
Drosophila neurally altered carbohydrate mutant 12 28. Lühn, K., Laskowska, A., Pielage, J., Klambt, C., Ipe, U., Vestweber, D., and Wild, M. K. (2004) Identification and molecular cloning of a functional GDP-fucose transporter in Drosophila melanogaster. Exp. Cell Res. 301, 242-250 Puglielli, L., and Hirschberg, C. B. (1999) Reconstitution, identification, and purification of the rat liver Golgi membrane GDP-fucose transporter. J. Biol. Chem. 274, 35596-35600 Lübke, T., Marquardt, T., Etzioni, A., Hartmann, E., von Figura, K., and Körner, C. (2001) Complementation cloning identifies CDG-IIc, a new type of congenital disorders of glycosylation, as a GDP-fucose transporter deficiency. Nat. Genet. 28, 73-76 Lühn, K., Wild, M. K., Eckhardt, M., Gerardy-Schahn, R., and Vestweber, D. (2001) The gene defective in leukocyte adhesion deficiency II encodes a putative GDP-fucose transporter. Nat. Genet. 28, 69-72 Jaeken, J., Hennet, T., Matthijs, G., and Freeze, H. H. (2009) CDG nomenclature: time for a change! Biochim. Biophys. Acta 1792, 825-826 Katz, F., Moats, W., and Jan, Y. N. (1988) A carbohydrate epitope expressed uniquely on the cell surface of Drosophila neurons is altered in the mutant nac (neurally altered carbohydrate). EMBO J. 7, 3471-3477 Whitlock, K. E. (1993) Development of Drosophila wing sensory neurons in mutants with missing or modified cell surface molecules. Development 117, 1251-1260 Ishikawa, H. O., Higashi, S., Ayukawa, T., Sasamura, T., Kitagawa, M., Harigaya, K., Aoki, K., Ishida, N., Sanai, Y., and Matsuno, K. (2005) Notch deficiency implicated in the pathogenesis of congenital disorder of glycosylation IIc. Proc. Natl. Acad. Sci. U. S. A. 102, 18532-18537 Laird, P. W., Zijderveld, A., Linders, K., Rudnicki, M. A., Jaenisch, R., and Berns, A. (1991) Simplified mammalian DNA isolation procedure. Nucleic Acids Res. 19, 4293 Jarvis, D. L., Weinkauf, C., and Guarino, L. A. (1996) Immediate-early baculovirus vectors for foreign gene expression in transformed or infected insect cells. Protein Expression and Purification 8, 191-203 O'Reilly, D. R., Miller, L. K., and Luckow, V. A. (1992) Baculovirus expression vectors, W.H. Freeman and Company, New York Summers, M. D., and Smith, G. E. (1987) A manual of methods for baculovirus vectors and insect cell culture procedures. Tx. Ag. Expt. Stn. Bull. No. 1555 Helmus, Y., Denecke, J., Yakubenia, S., Robinson, P., Lühn, K., Watson, D. L., McGrogan, P. J., Vestweber, D., Marquardt, T., and Wild, M. K. (2006) Leukocyte adhesion deficiency II patients with a dual defect of the GDP-fucose transporter. Blood 107, 3959-3966 Aumiller, J. J., and Jarvis, D. L. (2002) Expression and functional characterization of a nucleotide sugar transporter from Drosophila melanogaster: relevance to protein glycosylation in insect cell expression systems. Protein Expr. Purif. 26, 438-448 Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 Geisler, C., and Jarvis, D. L. (2011) Effective glycoanalysis with Maackia amurensis lectins requires a clear understanding of their binding specificities. Glycobiology 21, 988-993 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 13
Drosophila neurally altered carbohydrate mutant 13 44. Geisler, C., and Jarvis, D. L. (2012) Substrate specificities and intracellular distributions of three N-glycan processing enzymes functioning at a key branch point in the insect N-glycosylation pathway. J. Biol. Chem. 287, 7084-7097 Pöltl, G., Kerner, D., Paschinger, K., and Wilson, I. B. H. (2007) N-Glycans of the porcine nematode parasite Ascaris suum are modified with phosphorylcholine and core fucose residues. FEBS J. 274, 714-726 Brand, A. H., and Perrimon, N. (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401-415 Campos-Ortega, J. A., and Hartenstein, V. (1985) The embryonic development of Drosophila melanogaster, Springer Verlag, Berlin Patel, N. H. (1994) Imaging neuronal subsets and other cell types in whole-mount Drosophila embryos and larvae using antibody probes. Methods Cell Biol. 44, 445-487 Berninsone, P., Eckhardt, M., Gerardy-Schahn, R., and Hirschberg, C. B. (1997) Functional expression of the murine Golgi CMP-sialic acid transporter in Saccharomyces cerevisiae. J. Biol. Chem. 272, 12616-12619 Kochibe, N., and Furukawa, K. (1980) Purification and properties of a novel fucose-specific hemagglutinin of Aleuria aurantia. Biochemistry 19, 2841-2846 Altmann, F., Kornfeld, G., Dalik, T., Staudacher, E., and Glössl, J. (1993) Processing of asparagine-linked oligosaccharides in insect cells. N-Acetylglucosaminyltransferase I and II activities in cultured lepidopteran cells. Glycobiology 3, 619-625 Rabouille, C., Hui, N., Hunte, F., Kieckbusch, R., Berger, E. G., Warren, G., and Nilsson, T. (1995) Mapping the distribution of Golgi enzymes involved in the construction of complex oligosaccharides. Journal of Cell Science 108, 1617-1627 Sarkar, M., and Schachter, H. (2001) Cloning and expression of Drosophila melanogaster UDP-GlcNAc:-3-D-mannoside 1,2-N-acetylglucosaminyltransferase I. Biol. Chem. 382, 209-217 Kawar, Z., and Jarvis, D. L. (2001) Biosynthesis and subcellular localization of a lepidopteran insect alpha 1,2-mannosidase. Insect Biochem. Mol. Biol. 31, 289-297 Vadaie, N., and Jarvis, D. L. (2004) Molecular cloning and functional characterization of a lepidopteran insect 4-N-acetylgalactosaminyltransferase with broad substrate specificity, a functional role in glycoprotein biosynthesis, and a potential functional role in glycolipid biosynthesis. J. Biol. Chem. 279, 33501-33518 Robinow, S., and White, K. (1988) The locus elav of Drosophila melanogaster is expressed in neurons at all developmental stages. Developmental Biology 126, 294-303 Arakawa, K., Abe, M., Noda, Y., Adachi, H., and Yoda, K. (2006) Molecular cloning and characterization of a Pichia pastoris ortholog of the yeast Golgi GDP-mannose transporter gene. J Gen Appl Microbiol 52, 137-145 Cottrell, T. R., Griffith, C. L., Liu, H., Nenninger, A. A., and Doering, T. L. (2007) The pathogenic fungus Cryptococcus neoformans expresses two functional GDP-mannose transporters with distinct expression patterns and roles in capsule synthesis. Eukaryot. Cell 6, 776-785 Dean, N., Zhang, Y. B., and Poster, J. B. (1997) The VRG4 gene is required for GDP-mannose transport into the lumen of the Golgi in the yeast, Saccharomyces cerevisiae. J. Biol. Chem. 272, 31908-31914 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 14
Drosophila neurally altered carbohydrate mutant 14 60. Jackson-Hayes, L., Hill, T. W., Loprete, D. M., Fay, L. M., Gordon, B. S., Nkashama, S. A., Patel, R. K., and Sartain, C. V. (2008) Two GDP-mannose transporters contribute to hyphal form and cell wall integrity in Aspergillus nidulans. Microbiology 154, 2037-2047 Nishikawa, A., Mendez, B., Jigami, Y., and Dean, N. (2002) Identification of a Candida glabrata homologue of the S. cerevisiae VRG4 gene, encoding the Golgi GDP-mannose transporter. Yeast 19, 691-698 Nishikawa, A., Poster, J. B., Jigami, Y., and Dean, N. (2002) Molecular and phenotypic analysis of CaVRG4, encoding an essential Golgi apparatus GDP-mannose transporter. J. Bacteriol. 184, 29-42 Hong, K., Ma, D., Beverley, S. M., and Turco, S. J. (2000) The Leishmania GDP-mannose transporter is an autonomous, multi-specific, hexameric complex of LPG2 subunits. Biochemistry 39, 2013-2022 Ma, D., Russell, D. G., Beverley, S. M., and Turco, S. J. (1997) Golgi GDP-mannose uptake requires Leishmania LPG2. A member of a eukaryotic family of putative nucleotide-sugar transporters. J. Biol. Chem. 272, 3799-3805 Baldwin, T. C., Handford, M. G., Yuseff, M. I., Orellana, A., and Dupree, P. (2001) Identification and characterization of GONST1, a Golgi-localized GDP-mannose transporter in Arabidopsis. Plant Cell 13, 2283-2295 Handford, M. G., Sicilia, F., Brandizzi, F., Chung, J. H., and Dupree, P. (2004) Arabidopsis thaliana expresses multiple Golgi-localised nucleotide-sugar transporters related to GONST1. Mol. Genet. Genomics 272, 397-410 Ueki, N., and Nishii, I. (2009) Controlled enlargement of the glycoprotein vesicle surrounding a Volvox embryo requires the InvB nucleotide-sugar transporter and is required for normal morphogenesis. Plant Cell 21, 1166-1181 Suda, T., Kamiyama, S., Suzuki, M., Kikuchi, N., Nakayama, K., Narimatsu, H., Jigami, Y., Aoki, T., and Nishihara, S. (2004) Molecular cloning and characterization of a human multisubstrate specific nucleotide-sugar transporter homologous to Drosophila fringe connection. J. Biol. Chem. 279, 26469-26474 Selva, E. M., Hong, K., Baeg, G. H., Beverley, S. M., Turco, S. J., Perrimon, N., and Hacker, U. (2001) Dual role of the fringe connection gene in both heparan sulphate and fringe-dependent signalling events. Nat. Cell Biol. 3, 809-815 Muraoka, M., Kawakita, M., and Ishida, N. (2001) Molecular characterization of human UDP-glucuronic acid/UDP-N-acetylgalactosamine transporter, a novel nucleotide sugar transporter with dual substrate specificity. FEBS Lett. 495, 87-93 Berninsone, P., Hwang, H. Y., Zemtseva, I., Horvitz, H. R., and Hirschberg, C. B. (2001) SQV-7, a protein involved in Caenorhabditis elegans epithelial invagination and early embryogenesis, transports UDP-glucuronic acid, UDP-N-acetylgalactosamine, and UDP-galactose. Proc. Natl. Acad. Sci. U. S. A. 98, 3738-3743 Miura, N., Ishida, N., Hoshino, M., Yamauchi, M., Hara, T., Ayusawa, D., and Kawakita, M. (1996) Human UDP-galactose translocator: molecular cloning of a complementary DNA that complements the genetic defect of a mutant cell line deficient in UDP-galactose translocator. J Biochem 120, 236-241 Ishida, N., Miura, N., Yoshioka, S., and Kawakita, M. (1996) Molecular cloning and characterization of a novel isoform of the human UDP-galactose transporter, and of related complementary DNAs belonging to the nucleotide-sugar transporter gene family. J. Biochem. (Tokyo) 120, 1074-1078 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 15
Drosophila neurally altered carbohydrate mutant 15 74. Ishida, N., Yoshioka, S., Chiba, Y., Takeuchi, M., and Kawakita, M. (1999) Molecular cloning and functional expression of the human Golgi UDP-N-acetylglucosamine transporter. J. Biochem. (Tokyo) 126, 68-77 Ashikov, A., Routier, F., Fuhlrott, J., Helmus, Y., Wild, M., Gerardy-Schahn, R., and Bakker, H. (2005) The human solute carrier gene SLC35B4 encodes a bifunctional nucleotide sugar transporter with specificity for UDP-xylose and UDP-N-acetylglucosamine. J. Biol. Chem. 280, 27230-27235 Freeze, H. H., and Sharma, V. (2010) Metabolic manipulation of glycosylation disorders in humans and animal models. Semin. Cell Dev. Biol. 21, 655-662 Thiel, C., and Körner, C. (2011) Mouse models for congenital disorders of glycosylation. J. Inherit. Metab. Dis. 34, 879-889 Bier, E. (2005) Drosophila, the golden bug, emerges as a tool for human genetics. Nat. Rev. Genet. 6, 9-23 Ten Hagen, K. G., Zhang, L., Tian, E., and Zhang, Y. (2009) Glycobiology on the fly: Developmental and mechanistic insights from Drosophila. Glycobiology 19, 102-111 Krieg, D. R. (1963) Ethyl methanesulfonate-induced reversion of bacteriophage T4rII mutants. Genetics 48, 561-580 Lawley, P. D., and Brookes, P. (1963) Further studies on the alkylation of nucleic acids and their constituent nucleotides. Biochem. J. 89, 127-138 Bentley, A., MacLennan, B., Calvo, J., and Dearolf, C. R. (2000) Targeted recovery of mutations in Drosophila. Genetics 156, 1169-1173 Greene, E. A., Codomo, C. A., Taylor, N. E., Henikoff, J. G., Till, B. J., Reynolds, S. H., Enns, L. C., Burtner, C., Johnson, J. E., Odden, A. R., Comai, L., and Henikoff, S. (2003) Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics 164, 731-740 Vidal, A., Abril, N., and Pueyo, C. (1995) DNA repair by Ogt alkyltransferase influences EMS mutational specificity. Carcinogenesis 16, 817-821 Gazit, Y., Mory, A., Etzioni, A., Frydman, M., Scheuerman, O., Gershoni-Baruch, R., and Garty, B. Z. (2010) Leukocyte adhesion deficiency type II: long-term follow-up and review of the literature. J. Clin. Immunol. 30, 308-313 Vaughn, J. L., Goodwin, R. H., Tompkins, G. J., and McCawley, P. (1977) The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro 13, 213-217 Staudacher, E., Kubelka, V., and März, L. (1992) Distinct N-glycan fucosylation potentials of three lepidopteran cell lines. Eur. J. Biochem. 207, 987-993 Gao, X. D., and Dean, N. (2000) Distinct protein domains of the yeast Golgi GDP-mannose transporter mediate oligomer assembly and export from the endoplasmic reticulum. J. Biol. Chem. 275, 17718-17727 Hidalgo, A., Ma, S., Peired, A. J., Weiss, L. A., Cunningham-Rundles, C., and Frenette, P. S. (2003) Insights into leukocyte adhesion deficiency type 2 from a novel mutation in the GDP-fucose transporter gene. Blood 101, 1705-1712 Etzioni, A., Sturla, L., Antonellis, A., Green, E. D., Gershoni-Baruch, R., Berninsone, P. M., Hirschberg, C. B., and Tonetti, M. (2002) Leukocyte adhesion deficiency (LAD) type II/carbohydrate deficient glycoprotein (CDG) IIc founder effect and genotype/phenotype correlation. Am. J. Med. Genet. 110, 131-135 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 16
Drosophila neurally altered carbohydrate mutant 16 91. Ishikawa, H. O., Ayukawa, T., Nakayama, M., Higashi, S., Kamiyama, S., Nishihara, S., Aoki, K., Ishida, N., Sanai, Y., and Matsuno, K. (2010) Two pathways for importing GDP-fucose into the endoplasmic reticulum lumen function redundantly in the O-fucosylation of Notch in Drosophila. J. Biol. Chem. 285, 4122-4129 Lübke, T., Marquardt, T., von Figura, K., and Korner, C. (1999) A new type of carbohydrate-deficient glycoprotein syndrome due to a decreased import of GDP-fucose into the Golgi. J. Biol. Chem. 274, 25986-25989 Sturla, L., Puglielli, L., Tonetti, M., Berninsone, P., Hirschberg, C. B., De Flora, A., and Etzioni, A. (2001) Impairment of the Golgi GDP-L-fucose transport and unresponsiveness to fucose replacement therapy in LAD II patients. Pediatr. Res. 49, 537-542 Moriwaki, K., Noda, K., Nakagawa, T., Asahi, M., Yoshihara, H., Taniguchi, N., Hayashi, N., and Miyoshi, E. (2007) A high expression of GDP-fucose transporter in hepatocellular carcinoma is a key factor for increases in fucosylation. Glycobiology 17, 1311-1320 92. 93. 94. FOOTNOTES *This work was supported by grants from the Austrian Fonds zur Förderung der wissenschaftlichen Forschung [P17681] to I.B.H.W. and from NIH/NIGMS (R01GM072839) to M.T. and (R01GM49734) to D.L.J. 1To whom correspondence should be addressed. Tel.: +1-307-766-4282; Fax: +1-307-766-5098; e-mail: email@example.com 2The abbreviations used are: AAL, Aleuria aurantia lectin; CNS, Central nervous system; ER, Endoplasmic reticulum; ESI-MS, Electrospray ionization mass spectrometry; NST, Nucleotide sugar transporter; RFP, red fluorescent protein; RP-HPLC, reverse phase HPLC; TM, Transmembrane. at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 17
Drosophila neurally altered carbohydrate mutant 17 FIGURE LEGENDS FIGURE 1. Production of the HRP epitope and the Drosophila nac1 GFR mutation. A, GDP-fucose is produced in the cytoplasm and transported into the Golgi lumen by the GFR transporter in exchange for GMP. FucTA uses GDP-fucose in the Golgi lumen to produce the HRP epitope consisting of core 1,3-fucosylated N-glycans. Squares: N-acetylglucosamine; circles: mannose; triangles: fucose. B, Amino acid sequence comparison of known and predicted GDP-fucose transporters. The arrow indicates the conserved serine residue that is mutated to a leucine in Drosophila nac1 GFR. Amino acid residue numbering is according to the Drosophila gene product. Genbank accession numbers are: Drosophila: NP_649782.1; Mosquito: XP_312562.2; Honeybee: XP_623632.1; Flour beetle: XP_967192.1; Human: NP_001138737.1; Chinese hamster: BAE16173.1; Zebrafish: NP_001008590.1; Sea urchin: XP_798515.1; Nematode: XP_002637574.1. C, S29 is located in the middle of the first predicted transmembrane domain of Drosophila GFR. FIGURE 2. Wt, but not nac1 GFR can transport GDP-fucose in vitro and in vivo, and both are Golgi localized. A, 3H-GDP-fucose import activity of Golgi-enriched microsomes from Sf9 cells infected with baculovirus vectors encoding wt GFR, nac1 GFR, or no exogenous transporter (-) at 18°, 25° or 32°C. Background import at 18°C (1.5 fmol GDP-fucose µg total protein-1 minute-1) is set at 100% (-), error bars: 95% CI. p-values for different samples at the same temperatures were all <0.01. *: p<0.05; **: p<0.01. B, AAL lectin blot of CHO cells or CDG-IIc (LAD-II) cells transfected with expression plasmids encoding wt GFR, nac1 GFR, or nothing (-). C, Subcellular distribution of wt and nac1 GFR in Sf9 and Drosophila S2 cells. Columns: phase contrast, GFP-tagged GFR, RFP-tagged MGAT1 (insect Golgi marker), GFP and RFP merge, overlay. Scale bar, 10 µm. FIGURE 3. Core di-, 1,3- and 1,6-fucosylated N-glycan levels are strongly reduced in nac1 flies. PNGase A-released N-glycans from wt Canton S (A, C, E, F) and nac1 adults (B, D, E, G) were subjected to analysis by ESI-MS (A-D), RP-HPLC (E) and MALDI-TOF MS (F,G). Abbreviations: F, fucose; G, glucose; M, mannose; N, N-acetylhexosamine. Symbols: red triangles, fucose; green circles, mannose; blue squares, N-acetylglucosamine. The late elution time of M3N2F (E) indicates it is a core α1,6-fucosylated glycan and its reduced relative intensity in nac1 flies is shown by all three methods, MALDI-TOF MS analysis (not shown) of individual RP-HPLC fractions indicated trace levels of difucosylated glycans co-eluting with M3N2 in nac1 flies. The exploded views of the ESI-MS spectra (C, D; m/z 1265-1335) are set to the same ion count (y-axis; 1.5 x 105) and show the almost complete absence in nac1 flies of the difucosylated HRP epitope MMF3F6 glycan in its [M+H]+ and [M+Na]+ forms. FIGURE 4. Reduced HRP epitope expression in nac1 homozygous embryos is rescued by transgenic expression of wt Gfr. A,C,E, lateral view. B,D,F, ventral view. All embryos are late stage 12 to early stage 13. In nac1/nac1 embryos (A,B), HRP epitope expression is reduced in comparison to wt embryos (E,F). A wt Gfr transgene driven by the neural-specific elav promoter rescues neural HRP epitope expression (C,D). Scale bar, 70 µm. FIGURE 5. Drosophila GFR S29 is conserved in other GDP-sugar transporters. Alignment of Drosophila GFR with other characterized GDP-sugar transporters from L. donovani (GDP-SugarT; 64), S. cerevisiae (Yeast GDP-ManT; 59), P. pastoris (Pichia GDP-ManT; 57), C. albicans (C. albicans GDP-ManT; 62), C. glabrata (C. glabrata GDP-ManT; 61), C. neoformans (Cryptococcus GDP-ManT-1 and -2; 58), A. nidulans (Aspergillus GMT-1 and -2; 60), V. carteri (Volvox GDP-ManT; 67), A. thaliana (Arabidopsis GONST-1 and -2; 65,66), and similar NSTs of C. elegans (Nematode SQV-7; 71), D. melanogaster (Drosophila FRC; 69), and humans (Human FRC1 and hUGTrel7; 68,70). at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 18
Drosophila neurally altered carbohydrate mutant 18 Figure 1 at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 19
Drosophila neurally altered carbohydrate mutant 19 Figure 2 at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 20
Drosophila neurally altered carbohydrate mutant 20 Figure 3 at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 21
Drosophila neurally altered carbohydrate mutant 21 Figure 4 at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from
Page 22
Drosophila neurally altered carbohydrate mutant 22 Figure 5 at Univ. of Wyoming Libraries, on June 29, 2012www.jbc.orgDownloaded from


text: Interview with Bassam Haddad on Syria by Jadaliyya published on 2011-04-22T04:45:43Z Interview with Bassam Haddad on Syria, conducted by Shahram Aghamir of Pacifica Radio's Voices of the Middle East and North Africa A wave of protests have been shaking Syria since nearly the middle of March. The protests that began by calling for political reforms, escalated after the security forces opened fired on demonstrators in the southern city of Daraa. Shahram Aghamir of Pacifaca's Voices of the Middle East and North Africa spoke with professor Bassam Haddad about the nature of these protests, the response of the Syrian Regime to them, and their regional implications,

subset: commoncrawl


text:
Competition winner Mark Harrison, with Simon Lewis, Purity's head of sales and distribution, and Mr Harrison's weight in beer.
/ News
A MAN from Redditch has won his own body weight in beer after a winning a competition organised by the Alcester based Purity brewery.
33-year-old Mark Harrison from Lodge Park entered the prize draw, and found his name was selected, meaning that since he weighs 85kg, he's been able to take home 16 crates of Purity's Mad Goose, Pure UBU and Pure Gold ales.
He said: "This is a fantastic prize to win. I've been drinking Purity's beers since the brewery opened in 2005 and I'm a big fan. You always enter competitions hoping to win but never actually expect to do so."
Paul Halsey, Purity's managing director, added: "Congratulations to Mark. No doubt he'll need some help from his mates to drink all that beer.
"We wanted to organise a special competition to mark the launch of our new website, puritybrewing.com. We knew the chance to win your weight in beer would be popular, but we were genuinely overwhelmed to receive over 3,000 entries."
Promoted Stories
Competition winner takes home his weight in beer
Jammy so and so !!!! i salute you
Jammy so and so !!!! i salute you


text:
Unknown. In YMCA trail. Lily?
It's actually more closely related to skunk cabbage and peace plant than the true lilies.
Is this observation inappropriate, spam, or offensive? Flag this observation
If you think this observation is inaccurate, please add an ID, participate in the quality assessment above, or describe the inaccuracy in a comment.


text:
This profile was last updated on 6/14/13 and contains information from public web pages.
Mary MunroWrong Mary Munro?
Board Member
Scottish Official Board of Highland Dance Adjudicator Panel
A highly respected Highland dance judge Mary Munro from Burnaby, B.C. will provide for a high level dance competition.
New this year is the Kids' Fun Run. It is open to all kids, ages 3-12.
ProPub_Iteration_NoTests.312[01] SEIDXWK-7


text:
GREEN BAY, Wis. -- The outlook for injured Green Bay Packers cornerback Al Harris is encouraging.
Packers coach Mike McCarthy said Friday that Harris, who suffered a serious spleen injury Sunday night, could return to the team before the midpoint of the season.
"I would say it's going to be at least three to four weeks," McCarthy said of a timetable for when Harris might be ready to return to the field.
Harris has been ruled out for Sunday, when the Packers will play at Tampa Bay, putting an end to a streak of 83 straight starts since Green Bay acquired him in a trade with Philadelphia in 2003.
Harris' injury occurred in the first quarter of the Packers' loss to Dallas when he collided with linebacker A.J. Hawk on a running play. Harris left the game but returned two plays later.
He left the game for good, however, after tackling Cowboys running back Marion Barber. Harris went to the locker room in the second quarter with what team officials described as cramping.
McCarthy said after the game that Harris had blood in his urine and was not allowed to return by the team's medical staff.
Initial fears that Harris would miss the rest of the season and his playing career could be in jeopardy were tempered earlier this week. Harris underwent tests and sought multiple opinions from specialists.
McCarthy said Friday it was his understanding Harris' spleen wouldn't need to be surgically removed.
"Injuries like this go back to a personal nature," McCarthy said. "You're just glad it's not anything that could be life-threatening or as serious as it possibly could be."
Instead, Harris will have a chance to let the spleen heal on its own.
"The thing that we know for sure is we've had to totally shut him down for seven days," McCarthy said. "So, he'll start probably Monday morning with some form of conditioning, and it'll just progress as we move forward. There will be some tests that are done along the way. I don't have an exact time frame for you."
Teammates welcomed the news that Harris could be playing again soon.
"Oh, that's a break because Al brings so much to the defense," safety Atari Bigby said. "He's a lockdown corner. So, anytime you have a corner like that in the game, it definitely takes the pressure off of everybody else."
Bigby spoke with Harris a day after he sustained the injury. Bigby said Harris had mixed emotions.
"Of course he doesn't want to be out, but he felt like he's not going to be out the whole season," Bigby said. "He feels good. He knows his body. So, hopefully, he's not out the whole season."
The Packers will try to compensate for the absence of Harris, who was selected to the Pro Bowl for the first time last season, with a trio of young players.
Tramon Williams will start Sunday opposite veteran cornerback Charles Woodson. Will Blackmon will assume Williams' former spot at nickel back.
Pat Lee, a second-round draft pick this year, likely will be activated for the first time to serve as the dime back.
"You don't ever want to have players injured, especially you don't want to have your star players injured. But it's also part of the game," McCarthy said.
"It's important for the younger players to realize they have an opportunity. They've had their opportunity to learn from two veterans that have played a lot of football, that play that style of football very effectively. It's their time to put it to use because we need them as a team to perform."
Following a course of action the previous two weeks, Woodson didn't practice the entire week to rest a broken right toe. He is probable to play Sunday.
McCarthy said Woodson's insistence on playing every week with the injury, which he suffered in the Sept. 8 opener, interferes with the healing process.
"As far as the bone improving, it's obviously going to take a lot longer than a normal fracture because he keeps playing on it," McCarthy said. "I don't know if he's staying about the same or slightly improving, but I know he's really uncomfortable [after a game] until at least Wednesday."
Besides Harris, the Packers also are expected to be without Bigby on Sunday. He is listed as doubtful on the injury report.
Bigby didn't practice this week as he recovers from a left hamstring injury that kept him out of the previous game.
"It's disappointing. I'm a safety, and I love to play the game," Bigby said. "Whenever I can't play, I'm feeling pretty down."
Aaron Rouse is set to start again in Bigby's place.
Nick Collins, the Packers' other starting safety, is probable for Sunday after he left the last game with a bruised back.


text:
About this listing
Space where guests can cook their own meals
Towels, bed sheets, soap, and toilet paper
The Space
Villa Christa is a very homey villa located in the heart of Seminyak. Just a walking distance away from famous restaurants, shops and boutiques the villa, embedded in a beautiful tropical garden, is perfectly located in a very quiet neighborhood.
The interior of the villa reflects the typical Balinese artwork and ambience and you feel home "far away from home" right from the beginning of your vacation and upon your departure. Sit back and relax in the garden or take a dip in the georgeous private pool.
The open living room area, which incorporates local rattan furniture as well as traditional timber ceiling ensures a place in the shade, but can also be closed at night. Use the fully equipped kitchen to prepare your own food and drinks. Both bedrooms have ensuite bathrooms and king size beds. The upper bedroom has its own balcony and the lower bedroom comes with a specious bathroom with a big bathtub.
Your private shuttle service will pick you up at Denpasar airport, which is only 20 minutes away from "Villa Christa". If you want, our friendly maid will prepare and serve you a breakfast every morning to kick-off your new beautiful day on Bali. In-house massage and catering service is available too. For car rental, do the shopping or any other service, my staff is there to serve only your needs.
We are also completely equipped if you want to stay with your baby. We have baby chairs, baby cots, a stroller, toys and even a pool fence if needed.
Our services:
- Free airport transfer to the villa
- Welcome drinks
- Complementary fruit basket
- Complementary snacks, juices, soft drinks and beers
- Drinking water throughout your stay
- Free WIFI Internet
- Laundry delivery service
- Daily Maid service
- Concierge Service (Car Rental, Tour Organizing, Cooking Lesson, Massage, Chef,etc)


text:
5 Prong V1 by sedWatch
©2010-2016 sed
Started as a 5 point star in Bryce... But I kept adding and bending into this abZtract thingy!!
4 sizes in zip 1920 and down
4 sizes in zip 1920 and down
Damn you're doing some fine work!
lovel those reflections
Wow awesome work
This is a great job sed! Love it
super cool job
well done sed
well done sed
This is wacky. Reminds me of when the T1000 died in the end of Terminator 2. Good movie as well as this equally good art piece. Awesome!
Beautiful piece my friend...
Whoooooooooooooooooo,,, Dam Sed..This is nice!!! 5 point star? WOW. Love the colors...
Oh...Did you know we are only a hundred miles apart...I am in western PA. near the ohio line....Just off I-80. LOL.
Oh...Did you know we are only a hundred miles apart...I am in western PA. near the ohio line....Just off I-80. LOL.
Whoa! This is awesome
excellent work!
Really an awesome piece Seddy, this started out as a star? Amazing! Really awesome!
Great work here !
Great stuff!
Crazy cool!
Great work!
Nice work sed..
its really amazin art & its lovely colors
"To learn how to use your head, you have to go out of your mind."
pretty cool.
Awesome render Sed Bravo once again.
Nice work sed...nice palette!
wow~~~ glad you went nuts! LOVE IT
you need it after this one Sed lol awesome


text:
Massena—Patricia A. Guay 75, recently of County Route 46 Massena, passed away early Sunday morning at the St. Regis Nursing home surrounded by her family. Arrangements are under the care and supervision of the Phillips Memorial Home Massena. www.... Read More >>
Massena—Marvin A. Beaudette 86, passed away peacefully early Saturday morning at the St. Regis Nursing Home. Marvin is survived by his two children, Robert and wife Janet of Hershey, PA; Barbara and husband Scott Anderson of Massena. Arrangements... Read More >>
Massena—Gale Castleman 67, recently of Allen Street Massena passed away Sunday morning at the Massena Memorial Home surrounded by her family and Hospice. Arrangements are under the care and direction of the Phillips Memorial Home, Massena. Read More >>
Graveside services for Mrs. Marion Kot, 84, formerly of Massena, will be held Tuesday 10:00 AM at Calvary Cemetery, Massena with Rev. Donald Manfred, officiating.
Mrs. Kot passed away March 8, 2013 in Naples, Florida.
Arrangements are with the... Read More >>
Funeral services for Thelma Leona Langley Carmody of 232 Washington St., Ogdensburg will be held on Tuesday, June 25, 2013 at 10:00 a.m. at St. Mary's Cathedral with Rev. Joseph Morgan officiating. Burial will be in Ogdensburgh Cemetery. Calling... Read More >>
Sanfordville- Marjorie Genevieve (Bronson) White, 89, passed away after a long illness, on Friday, June 21, 2013 at the home of her daughter, Ann, in Brasher Falls. Arrangements are entrusted to the Hammill Funeral Home in Winthrop.
Marge was born... Read More >>
Graveside services for Marion H. Milsap, age 81, of Hammond will be held 11:00 a.m. on Friday, June 28, 2013 at Pleasant Valley Cemetery with Rev. Tom Nichols officiating. Mrs. Milsap passed away January 20, 2013 Arrangements are with the Fox... Read More >>
Graveside services for 87-year-old Mr. Samuel H. Clarke of Clifton Park, NY and formerly of Potsdam will be held on Monday, June 24th at 10 a.m. at Bayside Cemetery with Rev. Dr. Christopher Brown celebrant. Mr. Clarke passed away on the 17th in... Read More >>
Graveside services for Rita Graveline, age 98, and James Manley Silver Sr., age 67, will be held at Notre Dame Cemetery on June 24, 2013 at 1:00pm with Rev. F. James Shurtleff officiating. Mrs. Graveline passed away on January 3, 2013 & Mr.... Read More >>
Dorothy Codkind, 85, of Potsdam, passed away at her home on June 11, 2013.
Dorothy was a retired Professor Emeritus of Psychology from SUNY Potsdam. As per Dorothy's wishes, no funeral services will be held. Burial has taken place in New York... Read More >>
Thomas J. O'Connor, 52, formerly Winthrop, passed away unexpectedly Saturday, May 25, 2013 at his home. Tom was born in Potsdam on December 3, 1960, son of Robert and Rita (Huston) O'Connor.
He graduated from St. Lawrence Central School in 1979,... Read More >>
Graveside service for Elmer C. Dana, 91, of Russell, will be held on Saturday, June 22, 2013 at 9:30 a.m. at the South Russell Cemetery. Elmer died on Thursday, June 20, 2013 at the Canton-Potsdam Hospital after being admitted on June 18.
There will... Read More >>


text:
With 1.7 million freelancers, we'll match you with the perfect talent.
The client has made the following changes to the job.
Job awarded.
Client prefers freelancers from:
You are still able to submit a proposal for this job.
The client prefers freelancers from
a different location.
We need a design for our upcoming exhibition booth. The exhibition booth must reflect the dynamism and stature of our company as key development agency and corporate image. The design concept has to be contemporary and innovative, at the same time welcoming and functional. The size of our booth is 3m (L) x 12m (W) x 4.5m (max H).
Our corporate colour is Burgandy red and light gray. The booth colour shemes should have a combination of our corporate colour and other appropriate vibrant colours.
Furnitures that will be required in the booth are:
1) information counter & seats
2) display stands/shelves
3) small modern sofa with small table for discussion
4) corporate signage
5) carpeted platform, and
6) 42" flat screen TV
The selected design must be presented in 3d perspective and overall layout, with some details on the suggested material for construction.
For selection purposes, please submit previous design work on exhibition booth of contemporary and/or modern concept only....
Sign in or Register to see more


text:
11 comments
Haiku CornerThe Tricycle Community Poetry Club is pleased to present our new Haiku Corner, led by Gary Gach, haiku teacher and author of The Complete Idiot's Guide to Buddhism. You can read and comment on others efforts, offer your own haiku, or get guidance from Gary and the others there. You need to be a member of the Tricycle Community to visit, but it's easy to sign up, and best of all it's free. More »
Without Compassion, What Hope?Q: Is there any cause for optimism? A: Well, personally, yeah. Everybody's got a life to lead and they've got a bodhisattva tendency, everybody wants to do good, so I just think on a personal level, yeah. On a larger scale, there doesn't seem to be any hope unless compassion becomes a more widespread important teaching on how to live. Compassion to self and others. –Allen Ginsberg, from "Spontaneous Intelligence: An Interview with Allen Ginsberg," Tricycle, Fall 1995 Read the complete article. Follow us on Twitter and Facebook. Sign up for the Daily Dharma or Tricycle Community Newsletter More »
2 comments
More tools to navigate your way through the rich world of Tibetan artYesterday I linked to a page that shows you how to identify and understand the deities of the Tibetan pantheon. More »
3 comments
How to Identify A Tibetan DeityJeff Watt at Himalayan Art Resources knows everything about Tibetan Buddhist iconography. I sometimes call him and ask questions like, "Who is that odd-looking deity?" Or, with urgent requests like, "We need an image, and we need it now." I sometimes try his patience, too, so I have to make sure I sort of know what I'm talking about before I call him, and still, it's hard to sound halfway intelligent: the complex landscape of Tibetan iconography is no easy thing to navigate. But those days may be over. More »
China & Tibet in happier timesChina and Tibet have a very long history together (take a look here) but it hasn't always been so inharmonious. For instance, the artistic synergy between the two peoples has been impressive, and a new exhibit at the Museum of Fine Art proves it: As each tradition interacted with the other, Chinese painters took from their Tibetan counterparts a fearlessness in use of color and composition, pushing at the boundaries of their more reserved styles," Scheier-Dolberg said. More »
Buddhawatch: A thousand Buddhas appearWow! Hundreds of Buddha carvings appeared on three cliffs bordering a reservoir in Mei Shan City, Sichuan province. Apparently, they're well enough preserved to discern their different expressions. The reservoir that submerged the Buddhas was apparently built against the wishes of "cultural sectors," according to the People's Daily, which also asserts that water served as a better preservative than air. Buddhatwatch: Post your own favorite Buddhas as comments below and we'll pick a few and put them up. More »


text:
NEW! Visit DocsAdda , the smartest social network for medical professionals
Retina is world's leading search engine for medical students and doctors.
Keep us with you, always
Become our marketing partner
Bidding is now open to reach thousands of medical professionals across the globe. An unmatched offer starting from unbelievable low price!
Drop your message


text:
In Year of Crash, These Bank Stocks Soared
(FFIN - Get Report), Tompkins Financial (TMP - Get Report), NBT Bancorp (NBTB - Get Report), SY Bancorp (SYBT - Get Report) and Arrow Financial (AROW - Get Report) have outperformed larger financial companies such as Goldman Sachs (GS - Get Report) and BlackRock (BLK - Get Report).First Financial, which carries a "buy" recommendation from TheStreet.com Ratings, is a top performer, based not only on stock performance but also on fundamentals. Focusing on the regional market in Texas, the bank has carved out a profitable niche. Although third-quarter revenue declined 5.57%, the company managed to boost earnings per share by 8.5%, outperforming the industry average and the S&P 500. Gross margins widened to 81.39% from 71.97% a year earlier, and operating margins stretched to 46.06% from 40.71%. Regional, national and international banks have reported massive losses as a result of exposure to subprime mortgages. First Financial, which has a market value of about $1 billion, has avoided common pitfalls by staying true to the fundamentals of sound lending, maintaining regional focus and providing outstanding customer service. Investors have recognized the bank's potential, fueling a one-year price appreciation of 34.6%. Consequently, the stock is trading at a premium to its industry peers based on price to earnings, price to book and price to sales. However, the stock is below its 52-week high of $67. Other regional banks demonstrated a similar resistance to the pessimism that plagued financial stocks in 2008. The aforementioned stocks aren't cheap on an absolute basis, but there may be upside potential for people looking to play a trodden sector.When it comes to banking shares, smaller is better. First Financial Bankshares
There are numerous risks facing small-cap financials. Many have a high institutionally owned percentage and thin trading volume. Also, while large banks have sustained heavy losses from subprime-related investments, most have survived and will live to fight another day. However, these regional players are susceptible to immediate bankruptcy if bets turn against them. Rescuing small-cap financials doesn't top the list of Treasury priorities.
Check Out Our Best Services for Investors
Action Alerts PLUS
Portfolio Manager Jim Cramer and Director of Research Jack Mohr reveal their investment tactics while giving advanced notice before every trade.
Product Features:
- $2.5+ million portfolio
- Large-cap and dividend focus
- Intraday trade alerts from Cramer
Quant Ratings
Access the tool that DOMINATES the Russell 2000 and the S&P 500.
Product Features:
- Buy, hold, or sell recommendations for over 4,300 stocks
- Unlimited research reports on your favorite stocks
- A custom stock screener
Stocks Under $10
David Peltier uncovers low dollar stocks with serious upside potential that are flying under Wall Street's radar.
Product Features:
- Model portfolio
- Stocks trading below $10
- Intraday trade alerts
Dividend Stock Advisor
David Peltier identifies the best of breed dividend stocks that will pay a reliable AND significant income stream.
Product Features:
- Diversified model portfolio of dividend stocks
- Updates with exact steps to take - BUY, HOLD, SELL
Trifecta Stocks
Every recommendation goes through 3 layers of intense scrutinyquantitative, fundamental and technical analysisto maximize profit potential and minimize risk.
Product Features:
- Model Portfolio
- Intra Day Trade alerts
- Access to Quant Ratings
Real Money
More than 30 investing pros with skin in the game give you actionable insight and investment ideas.
Product Features:
- Access to Jim Cramer's daily blog
- Intraday commentary and news
- Real-time trading forums


text:
My son just got out of the hospital with rota last night. He had been in there since Tuesday, but had been sick since last Saturday. He's great now, but now me and my husband have it. OMG it feels like death. Literally, water goes in and comes right back out. I'm crazy dehydrated and the diarrhea is causing tons of contractions. I guess I need to go in for fluids but ugh we JUST freaking left the hospital :( And we're talking about a 45 minute car ride there... I wouldn't be able to make it without stopping.
Also, I have felt very very little movement today... like I can only recall 3 kicks. Do babies usually rest too when mama is sick?


text:
Social care is essential for anyone with sight loss.
This Social care campaign is focused on legislation in England, but you can find out how you can get involved in the rest of the UK by visiting our campaigning in your area section.
What should I expect from my council?
We're campaigning to ensure that blind and partially sighted people have access to rehabilitation and social care services as well as ensuring that blind and partially sighted people's needs are met by the Care Act.Find out what we're doing on Social care


text:
Just thought I would post this here as I remember going to the post thrift store A LOT when we lived on-post prior to recruiting duty. Even now, being in Los Angeles I go to thrift stores because they have some nice stuff especially the ones that the rich people donate or re/sale stuff to.
I personally have never had an issue with buying kids stuff at yard sales or thrift stores - they grow so fast and can be so hard on things and with DH in the military we aren't millionaires. Not to mention I make home-made clothes that I sell and I buy them too. Argh!
I understand the whole lead and pthalate thing - but I think the law is a little too broad.


text:
Who contradicted whom over Greek exit from the euro?
Much is being made of the apparently irreconcilable difference between the statements made by Richard Corbett, an adviser to the president of the European Council, and Christine Lagarde, director of the IMF, on the hugely contentious possibility of a Greek exit from the euro.
Ms Lagarde has seemed to state quite unequivocally that such contingency planning was well under way because, as she put it, the eurozone had to be "technically prepared for anything". (Surely an unimpeachably practical view?) But Mr Corbett, speaking on the Today programme, was of an apparently contradictory opinion: "No one is planning for a Greek exit," he said. (No one? Not even the French and German banks which have been extricating themselves as much as possible from vulnerability to Greek default? Or did he mean "no one" in official, EU institutions?) Just to underline the point, he repeated it in different forms: "We're not planning for a Greek exit..nobody is planning for a Greek exit, etc, etc."
Could this be true? Could the EU authorities really be in such deep denial that they are refusing even to contemplate (and hence, plan for) the possible repercussions of an event which most commentators now regard as virtually inevitable?Hopefully not. I think what Mr Corbett was trying to say hinged not just on the meaning of the word "planning" but on the force of the preposition that followed it. What he probably meant, to give a charitable interpretation, is that the EU was not planning ON a Greek exit: that is, was not expecting it, or running on the assumption that it would occur. Which is rather different from not planning FOR it – which could simply mean anticipating the problems that could follow and trying to guard against the worst effects. In other words, he was trying to talk down the sense of inevitability that has begun to dominate the scene, and thus fend off further falls in the markets.
What a state we have reached when the economic stability of Europe can be thought to stand or fall on the force of a preposition. Our future is now so fragile that it can be undermined by semantics.
Recent Posts
A story of two by-elections
October 10th, 2014 3:23
David Cameron: he said everything we might have hoped for
October 1st, 2014 13:31
George Osborne's conference speech was a less-than-inspiring invitation to vote Tory
September 29th, 2014 16:51
Ed Miliband on the margin of reality
September 23rd, 2014 17:08
Alex Salmond should be praying for a narrow No
September 17th, 2014 11:04


text:
View History
|"Dungeness Lighthouse" changed to "South Foreland Lighthouse."
|Jan 28, 2014
|Deleted photograph.
|Nov 20, 2012
|Add new Web site: How Stuff Works - Adventure - How Lighthouses Work.
|May 01, 2012
|Add new Web site: ThinkQuest - History of Lighthouses.
|May 01, 2012
|Add new Web site: Buzzle.com - What are Lighthouses and How Do They Work?
|May 01, 2012
|Add new Web site: Fact Monster - History - lighthouse.
|Aug 18, 2011
|Added a photo of Portland Head Light, a historic lighthouse at Cape Elizabeth, Maine, U.S.
|Jun 02, 2011
|Modified date of dioptric system from 1828 to 1821. Added date of 1824 to production of several beams. Added date of 1836 to production of drum lens. Modified producer of drum lens from "Fresnel" to "English glassmaker William Cookson."
|Jan 13, 2011
|Added image of lightship at the entrance to New York Harbour.
|May 25, 2010
|Media added.
|Oct 08, 2007


text:
Deviant Art
Shit shit shit...That is my life as of right now, total poo.
I have so much I have to do it's not even funny.
Theres the contest prizes I have to draw and submit for the winners, then all the questions I really need to get to for all of my accounts, oh and my very late art trades.
Cant forget those.
With all of this stuff I have, and the accounts that Im sharing with people and working with, I feel like Im just going to dissapoint people.
I'm just so tired after I get home from school, it seems the only time I ever get a decent time of drawing done is during the weekend and that is just not good enough.
I ask for too much from people, especially from these oc's I pull out of thin air and throw at people. I think I just need a break on submitting any new character designs...
I havent even really been giving Rico any attention at all, and he was practically my one responsibility at the time. Now look at all this other stuff I got myself into.
The point is that I think I can handle myself and I
|Sta.sh Submit